Fc epsilon receptor, a specific differentiation marker transiently expressed on mature B cells before isotype switching.

The expression of Fc epsilon R on human lymphocytes was studied with the anti-Fc epsilon R mAbs. Fc epsilon R was expressed on most mu+,delta+ circulating B cells, whereas T cells did not express Fc epsilon R even in patients with hyper-IgE syndrome. B cells with gamma, alpha, or epsilon phenotype did not express Fc epsilon R, moreover its expression could not be induced, suggesting that the Fc epsilon R expression was correlated with isotype switching. mu+delta+ B cells in bone marrow did not express Fc epsilon R, but PHA-sup (supernatant from PHA-stimulated cell cultures) could induce its expression, and the addition of IgE augmented this induction. Recombinant IL-2, IL-1, IFN-gamma or -beta, or purified B cell differentiation factor (BSF-2 B cell-stimulatory factor 2) could not induce Fc epsilon R expression in bone marrow B cells. IFN-gamma inhibited the Fc epsilon R expression induced by PHA-sup, suggesting that the human counterpart of BSF-1 may be responsible for Fc epsilon R expression in bone marrow B cells. B cells from patients with common variable immunodeficiency and ataxia telangiectasia did not express Fc epsilon R, but PHA-sup could induce its expression, indicating that circulating B cells of these patients are at a differentiation stage similar to B cells in bone marrow. The study showed that Fc epsilon R is a B cell-specific differentiation marker, the expression of which is restricted to a defined stage of B cell differentiation.

An erythrocyte-sensitising antigen from Vibrio cholerae.

Erythrocyte-sensitising antigens (AE) were prepared from Vibrio cholerae serotypes, from EL-Tor vibrio, Escherichia coli and Salmonella enteritidis by digesting the organisms with NaOH followed by precipitation with alcohol. When AE was used in indirect haemagglutination (IHA) tests, the results in a number of cases were somewhat more sensitive and more specific than those obtained in classical agglutination tests. No cross reactions occurred between V. cholerae serotypes and E. coli and S. enteritidis. Much of the reactive part of the AE was not sedimentable at 100,000 g for 1 h. The eluant from the B. cholerae AE on Sephadex G-200 yielded three fractions, one of which was the most active in IHA tests. Treatment of the AE with trypsin resulted in an appreciable increase in the heterotypic serum titres in IHA tests. The spectrophotometric absorption of the AE at 260 nm showed a hump that may have been indicative of the presence of nucleic acid. Treatment of the antigen with ribonuclease reduced its nucleic acid content but did not change to any significant extent the reactivity of the preparation. The AE antigen of V. cholerae was Molisch-positive and was capable of sensitising untanned erythrocytes in IHA tests. It is suggested that the reactive part of the AE antigen is a carbohydrate complex.

Generation of leukotrienes by antigen and calcium ionophore A23187 stimuli from a mouse mastocytoma cell line, MMC-16.

The generation of leukotrienes and histamine release by the mouse mastocytoma cell line MMC-16 was investigated. These cells produced leukotriene C4 (LTC4) and released histamine upon calcium ionophore A23187 and antigen stimulation. The ionophore also stimulated the biosynthesis of leukotriene B4 (LTB4) by MMC-16. Generation of LTC4 was confirmed by its characteristic UV absorption spectrum, fast atom bombardment-MS, equivalent HPLC retention time with an authentic standard and radioimmunoassay. Leukotriene B4 was characterized by its distinctive UV spectrum and HPLC retention time compared with synthetic material. IgE-mediated LTC4 generation was also observed in a dose dependent fashion with MMC-16 cells passively sensitized with monoclonal IgE specific for ovalbumin. LTC4 biosynthesis was effectively inhibited by the lipoxygenase inhibitor NDGA.

Nonspecific and specific immunological mitogenicity by group A streptococcal pyrogenic exotoxins.

Group A streptococcal pyrogenic exotoxin (SPE) types A, B, and C induced lymphocyte proliferation both specifically and nonspecifically, and the responses showed characteristics associated with both types of stimulation. Guinea pig lymphocytes from animals presensitized to SPE A displayed immunologically specific proliferation in response to SPE A; control lymphocytes showed little activity in the presence of SPE A. Lymphocytes from guinea pigs not presensitized to SPE responded nonspecifically to SPE types B and C. Guinea pig lymphocytes from SPE A-presensitized animals showed enhanced proliferation over controls when treated with SPE B, suggesting that a degree of cross-reactivity between SPE types may exist, though they are serologically distinct. Mouse splenic lymphocytes exhibited low-level responsiveness to all SPE types, as would be expected for an antigen-specific proliferative response. Unlike mouse splenic lymphocytes, rabbit spleen cells and human cord blood, lymphocytes responded nonspecifically to all SPE types. Although rabbit spleen cells and human cord blood lymphocytes responded nonspecifically, the maximum response occurred at day 4 or 5, comparable to an antigen-specific system rather than a day 2 or 3 such as that with the nonspecific thymus-derived cell mitogen, concanavalin A.

Further purification of group A streptococcal pyrogenic exotoxin and characterization of the purified toxin.

Streptococcal pyrogenic exotoxin (SPE) isolated from culture filtrates of strain NY-5 (type 10), and separated from other extracellular by differential solubility in ethanol and acetate-buffered saline, has previously been shown to exhibit a wide range of biological activities including erythrogenic activity, pyrogenicity, enhancement of susceptibility to endotoxin shock, blockage of the reticuloendothelial system immmunosuppression, and lymphocyte mitogenicity. Toxin prepared in this way was found to consist of hyaluronic acid and several proteins which could be distinguished by thin-layer polyacrylamide isoelectric focusing (IEF), SPE has been further purified by ion exchange chromatography on QAE-Sephadex columns. One of the fractions isolated from QAE-Sephadex, and shown to be a homogenous protein by thin-layer IEF and Ouchterlony with hyperimmune serum, was highly active erythrogenically, pyrogenically, and in enhancing susceptibility to endotoxin. This fraction was identified as exotoxin A. A second, less active fraction identified as SPE B showed similar activities, but differed from the other fraction antigenically and in net charge and molecular weight. These findings indicate that a single highly purified protein can mediate at least three of the biological activities attributed to SPE and NY-5 produces pyrogenic exotoxins A and B in vitro as well as in vivo.

Heterogeneity of group A streptococcal pyrogenic exotoxin type B.

Streptococcal pyrogenic exotoxin type B purified from culture filtrates of either the NY-5 or T-19 strain of group A streptococcus was found to be heterogeneous in charge. Three protein fractions with isoelectric points of 8.0, 8.4, and 9.0 were isolated by differential solubility in ethanol and acetate-buffered saline followed by isoelectric focusing and shown to be antigenically identical to streptococcal pyrogenic exotoxin type B. The molecular weights of all three fractions were approximately 17,500, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with aggregates forming in the presence of hyaluronic acid. Only the pI 8.4 fraction showed the characteristic activities of streptococcal pyrogenic exotoxin in rabbits: pyrogenicity and ability to enhance susceptibility to lethal endotoxin shock. The pI 8.0 and pI 9.0 fractions were not pyrogenic, but could be used to immunize against pyrogenicity. These two fractions failed either to enhance lethal endotoxin shock or to immunize against enhancement activity. When the isolated fractions were electrofocused again they appeared heterogeneous, suggesting an instability of the B toxin molecular forms.

Establishment of four mouse mastocytoma cell lines.

Four mastocytoma cell lines were isolated from four different mouse mastocytoma tumors. The tumors had been induced in mice treated with tetramethylpentadecane (pristane) and infected with Abelson murine leukemia virus. The cell lines have been carried in culture for over a year and can induce tumors when injected into the mouse strain in which the tumor originated. The cells contain histamine, have high affinity IgE receptors and release histamine by IgE, immune complex or ionophore A23187-induced reactions. This histamine release reaction requires Ca2+, is optimal at 37 degrees C, and is blocked by a number of metabolic inhibitors. There is no requirement for phosphatidylserine. Cloned sublines have been obtained which will be useful for Fc epsilon R, Fc gamma R; and histamine release studies.

IgE-induced histamine release from rat basophilic leukemia cell lines: isolation of releasing and nonreleasing clones.

The rat basophilic leukemia (RBL) cell lines were cloned and the various sublines compared for their chromosome number, IgE-mediated histamine release and for IgE surface receptors. It was found that cell lines started from tumors at different times vary in both their chromosome number and their ability to release histamine by an IgE-mediated reaction. RBL-I and III have approximately 44 chromosomes and did not respond to an IgE-mediated reaction. RBL-II and RBL-IV have 68-73 chromosomes and showed moderate levels of histamine release (percent release mean = 5 +/- 2 and 10 +/- 4, respectively). The cloning of the RBL-IV line resulted in some sublines which were excellent histamine releasers (range 39-100%) and some which were relatively refractory (less than 10%) to IgE-mediated histamine release. These clones did not differ significantly in chromosome number. Recloning the releasing lines gave rise to poor releasers, whereas the recloning of poor releasers did not produce good releasers indicating that the mutational drift in culture is toward loss of histamine-releasing capacity. The number of IgE receptors and the rate of IgE association and dissociation were similar for the different cell lines. The study failed to disclose significant molecular weight differences in the IgE receptor from the various clones and sublines indicating that the failure to release probably does not reside in the receptor. The various cloned sublines are phenotypically stable, and the isolation of excellent histamine-releasing sublines are useful for studies of the complex phenomenon of the histamine release.

Two species of human Fc epsilon receptor II (Fc epsilon RII/CD23): tissue-specific and IL-4-specific regulation of gene expression.

The Fc epsilon receptor II (Fc epsilon RII, CD23) functions in B cell growth and differentiation and in IgE-mediated immunity. The Fc epsilon RII structure expressed on various cell types has been analyzed identifying two species, Fc epsilon RIIa and Fc epsilon RIIb. Sequence analysis of the cloned cDNAs revealed that they differ only at the N-terminal cytoplasmic region, but share the same C-terminal extracellular region. These Fc epsilon RII species appear to be generated utilizing different transcriptional initiation sites and alternative RNA splicing. Fc epsilon RIIa is constitutively expressed only in normal B cells and B cell lines, whereas Fc epsilon RIIb expression is detectable in various cell types, such as monocytes and eosinophils. Normally, Fc epsilon RIIb is undetectable in B cells and monocytes, and can be induced by interleukin-4. Moreover, Fc epsilon RIIb is expressed on peripheral blood lymphocytes in atopic individuals. These findings may explain the difference in Fc epsilon RIIa and Fc epsilon RIIb function in B cells and the effector phase of IgE-mediated immunity.

Detection and localization of a lectin on Actinomyces viscosus T14V by monoclonal antibodies.

A cell-associated lectin activity that mediates lactose-inhibitable adherence of Actinomyces viscosus T14V has been localized to a specific population of fimbriae by the use of monoclonal antibodies. Nine monoclonal antibodies were produced that reacted with only 1 of 2 immunoelectrophoretically distinct fimbrial components on T14V. The fibrillar morphology of this component was revealed by the immunoelectronmicroscopic examination of bacteria incubated with the monoclonal antibodies. The lectin activity associated with these structures was detected when isolated fimbriae were cross-linked with monoclonal antibodies to form immune complexes with agglutination activity for neuraminidase-treated human erythrocytes, a reaction that was inhibited by lactose. Although the 9 monoclonal antibodies differed in their fine specificities, they reacted only with strains of A. viscosus and A. naeslundii that exhibited lactose-inhibitable adherence. These findings indicate that the lectin activity common to these bacteria resides on fimbriae that are antigenically related to those of T14V.

Variants of the rat basophilic leukemia cell line for the study of histamine release.

Cloning of the rat basophilic leukemia (RBL) cell lines demonstrates variability in cell chromosome number (approximately 44-70) and in their capacity to release histamine following an IgE- or Ca2+-ionophore stimulus. After IgE activation there is increased phospholipid methylation, Ca2+ influx, arachidonic acid, and histamine release. On Ca2+ ionophore A23187 stimulation, phospholipid methylation is not increased, but Ca2+ influx, arachidonic acid, and histamine release occur. Variants of the RBL-cloned sublines defective at different stages in the release process were obtained and used to sequence the different events in the release process: IgE activation is followed by methylation, Ca2+ influx, arachidonic acid, and histamine release. However, there are other variants with defects in intermediate steps in the pathway, e.g., increased phospholipid methylation that is not followed by Ca2+ influx or arachidonic acid release not followed by histamine release. Isolating variants carrying drug-resistance markers made hybridization (reconstitution) experiments possible. Two variants were recognized, each of which was deficient in one of the two phospholipid methyltransferase enzymes. Neither of these two variants released histamine; hybrids formed by fusion of these two cell lines have both phospholipid methyltransferase enzymes and release histamine. By other complementation experiments, groups of variants with defects at different steps in the histamine release sequence were recognized. Clearly, these basophilic leukemia cell lines provide a unique system for the study of the mechanism of histamine release.

Significance of soluble Fc epsilon receptor II (sFc epsilon RII/CD23) in serum and possible application of sFc epsilon RII for the prevention of allergic reactions.

The significance of sFc epsilon RII in IgE-mediated allergic disease was examined. sFc epsilon RII in serum was found to decrease following clinical improvement, suggesting sFc epsilon RII in serum may be an indicator of allergic diseases. Significant proportions of sFc epsilon RII in serum were present as complexes with IgE in normals as well as in atopic patients, and these complexes were more prominent in the former than in the latter group. From these observations, attempts were made to inhibit IgE-mediated allergic reactions in vitro employing recombinant sFc epsilon RII. sFc epsilon RII inhibited IgE-binding as well as IgE-mediated release of chemical mediators from Fc epsilon RI and Fc epsilon RII expressing cells. These results show the functional significance of sFc epsilon RII in the negative regulation of IgE-mediated allergic reactions.

Recombinant soluble Fc epsilon receptor II (Fc epsilon RII/CD23) has IgE binding activity but no B cell growth promoting activity.

We have undertaken the production of recombinant soluble Fc epsilon receptor II (Fc epsilon RII) as a secretory protein, but not as a cleavage product of membrane-bound receptor. Several plasmid constructs containing soluble receptor sequence were prepared. Only a chimeric gene containing the sequences encoding IL-6 signal peptide and the soluble moiety of Fc epsilon RII could be expressed in Xenopus laevis oocytes and CHO cells, resulting in the secretion of soluble Fc epsilon RII. The recombinant soluble Fc epsilon RII was also produced in the yeast expression system. The NH2-terminal sequence analysis of the chimeric gene product generated by oocytes demonstrated the correct cleavage of IL-6 leader sequence by a signal peptidase. Moreover, most of CHO cell and all of the yeast-derived recombinant molecules were products identical with the native B cell-derived soluble Fc epsilon RII. These recombinant products as well as the natural soluble receptor derived from a human B cell line could bind both human IgE and two different anti-Fc epsilon RII mAb and could competitively inhibit the binding of IgE to Fc epsilon RII-expressing cells. However, the recombinant soluble Fc epsilon RII and highly purified native molecules did not display any B cell growth-promoting activity.

Molecular structure of human lymphocyte receptor for immunoglobulin E.

We have isolated and sequenced a cDNA clone encoding the human lymphocyte receptor for IgE (Fc epsilon R). The deduced protein sequence reveals that Fc epsilon R consists of 321 amino acids, without any signal sequence, and is oriented with its N-terminus on the cytoplasmic side and its C-terminus on the outside of the cell. This molecule shows striking sequence homology with chicken asialoglycoprotein receptor (hepatic lectin), suggesting a possible role for Fc epsilon R in endocytosis. Fc epsilon R mRNA is expressed in B cells, B cell lines, and macrophage cell lines. It is not expressed in T cells or T cell lines, with the exception of an HTLV-transformed T cell line. mRNAs expressed in a macrophage line and in the latter T cell line differ in size from mRNA expressed in B cells. Human BSF-1 (or IL-4) induces the expression of Fc epsilon R mRNA in B cells, but not in T cells.

A B cell-specific differentiation antigen, CD23, is a receptor for IgE (Fc epsilon R) on lymphocytes.

Two independent L cell transformants expressing human lymphocyte Fc epsilon R were established by using cellular DNA from RPMI 8866 cells. The surface expression of the receptor was confirmed on the basis of the binding of a panel of anti-Fc epsilon R antibodies and its ability to bind IgE. Anti-CD23 antibodies strongly stained the transformants, indicating possible identity or antigenic relationship between Fc epsilon R and CD23. This interesting observation warrants additional clarification as to the role of CD23 and Fc epsilon R in B cell differentiation.

Structure and function of Fc epsilon receptor II (Fc epsilon RII/CD23): a point of contact between the effector phase of allergy and B cell differentiation.

The Fc epsilon receptor II (Fc epsilon RII/CD23) has been proposed to have multiple functions as a membrane-bound or soluble molecule: a function in B cell growth and differentiation and a role in the effector phase of IgE-mediated immunity. We recently demonstrated the presence of two forms of Fc epsilon RII (Fc epsilon RIIa and Fc epsilon RIIb) whose structures differ only at their N-terminal cytoplasmic regions. The regulatory mechanisms of their expression strongly suggest that Fc epsilon RIIa and Fc epsilon RIIb function in B cells and in the effector cells of IgE-mediated immunity, respectively. To elucidate the function of soluble Fc epsilon RII/CD23 (sFc epsilon RII) the recombinant soluble molecule was produced. This recombinant receptor could competitively block the IgE binding of eosinophils, monocytes and even basophils and could inhibit the IgE-mediated function of effector cells such as monocytes. These findings suggested that sFc epsilon RII could competitively regulate the function of effector cells in IgE-mediated immunity and that the recombinant sFc epsilon RII could be applied clinically for the control of allergic reactions. The expression of Fc epsilon RII on Fc epsilon RII-negative B and T cell lines by cDNA transfection resulted in homocytic aggregation. The function of Fc epsilon RII on B cells as an adhesion molecule was also demonstrated.

Monoclonal anti-Fc epsilon receptor antibodies with different specificities and studies on the expression of Fc epsilon receptors on human B and T cells.

Three monoclonal antibodies, 1-7 (gamma 2b), 3-5 (gamma 1), and 8-30 (mu), specific to Fc epsilon receptors (Fc epsilon R) on human B cells were established. The two monoclonals (1-7 and 8-30) could inhibit the binding of IgE to Fc epsilon R in rosette formation assays, as well as FACS analysis, and were shown to recognize the same epitope of Fc epsilon R. The other monoclonal antibody (3-5) recognized the same molecule but a different epitope, and marginally inhibited the IgE binding. The molecules on RPMI 8866 cells recognized by these monoclonal antibodies had Mr of 46,000 and 25,000 to 30,000 daltons as determined by immunoprecipitation and SDS-PAGE analysis. By employing these monoclonal antibodies, the expression of Fc epsilon R on circulating lymphocytes was studied. Approximately 50% of B cells from normal, nonatopic individuals were found to express Fc epsilon R, and a remarkable increase in the expression of Fc epsilon R was observed in B cells of atopic patients. The expression of Fc epsilon R was not detected in T cells from atopic patients (including hyper IgE syndrome) as well as normal individuals. Incubation of B cells with PHA-conditioned medium plus IgE augmented the expression of Fc epsilon R in the Fc epsilon R+ B cell population but not in Fc epsilon R- population. PHA-conditioned medium plus IgE did not induce Fc epsilon R expression on T cells.

Immunochemical and functional studies of Actinomyces viscosus T14V type 1 fimbriae with monoclonal and polyclonal antibodies directed against the fimbrial subunit.

Each of five monoclonal antibodies (mAbs) prepared against the type 1 fimbriae of Actinomyces viscosus T14V reacted with a 54 kDa cloned protein previously identified as a fimbrial subunit. This purified protein completely inhibited the reaction of a specific anti-type-1-fimbria rabbit antibody with A. viscosus whole cells. Maximum values for the number of antibody molecules bound per bacterial cell ranged from 7 x 10(3) to 1.2 x 10(4) for the different 125I-labelled mAbs and was approximately 7 x 10(4) for 125I-labelled rabbit IgG or Fab against either type 1 fimbriae or the 54 kDa cloned protein. Although the different mAbs, either individually or as a mixture, failed to inhibit the type-1-fimbria-mediated adherence of A. viscosus T14V to saliva-treated hydroxyapatite, each rabbit antibody gave 50% inhibition of adherence when approximately 5 x 10(4) molecules of IgG were bound per cell. However, binding of each corresponding rabbit Fab had no significant effect on bacterial attachment unless much higher concentrations were used. These findings suggest that antibodies directed solely against the 54 kDa fimbrial subunit do not react with the putative receptor binding sites of A. viscosus T14V type 1 fimbriae. Instead, inhibition of attachment by the polyclonal antibodies may depend on an indirect effect of antibody binding that prevents the fimbria-receptor interaction.