Arnold Froese

Isolation and characterization of IgM from bovine colostral whey

Abstract Bovine IgM was isolated from colostral whey, by molecular sieve and DEAE-cellulose chromatography. It was characterized using physico-chemical and immuno-chemical techniques. The molecular weight of the intact molecule was found to be 1,030,000, while that of the computer μ and light chains was 76,000 and 22,500, respectively.

Characterization of the target cell receptor for IgE—I. Solubilization of IgE-receptor complexes from rat mast cells and rat basophilic leukemia cells

Abstract Purified rat monoclonal IgE was labeled with 125I and allowed to interact with either purified rat mast cells (RMC) or rat basophilic leukemia cells (RBL). Virtually 100% of the cell bound 125I-IgE was solubilized by treating the cells with buffer containing 0.5% Nonider P-40. When this solubilized IgE was subjected to gel filtration it was found to elute ahead of free IgE or IgE which had been allowed to interact with control (non-IgE binding) cells, indicating that at least a portion of the cellular IgE receptor remained attached to the IgE. The IgE-receptor complexes isolated from either cell type eluted at almost identical volumes, and a comparison with polymeric IgA suggested that they had a mol. wt of 3·5–5·5 × 105 daltons. Complex formation also occured if cells were solubilized prior to the addition of IgE. Soluble cell extracts from both RMC and RBL cells caused inhibition of passive cutaneous anaphylaxis, however, the inhibition was less efficient than that given by comparable numbers of intact cells.

Kinetic studies on antibody-hapten reactions-I: Reactions with antibodies and their univalent Fab′ fragments☆

Abstract Rate constants and equilibrium constants were determined for reactions of antibodies specific to the 2,4-dinitrophenyl determinant and their univalent Fab′ fragments with the haptens, ϵ-DNP-lysine and 1-hydroxy-4-(2,4-dinitrophenylazo)-2,5 napthalene disulfonate. Binding constants were established by spectrophotometric and fluorescence quenching techniques, and the rate constants with the temperature-jump relaxation and stopped-flow methods. The Fab′ fragments had a slightly higher affinity for the haptens than the intact antibodies, which was primarily a consequence of an increase in the rate constants, by a factor of almost two, for the association reactions. This increase in rate constants for the univalent fragments was attributed to a greater accessibility of their combining sites.

The N-linked oligosaccharides of the Fcϵ receptors of rat basophilic leukemia cells☆

Abstract This study was undertaken to investigate the nature and microheterogeneity of the carbohydrate moiety of the Fcϵ receptors of RBL-CA10 and RBL-CA10.7 cells. Treatment using the glycosylation processing inhibitors, castanospermine (CN), 1-deoxymannojirimycin (DMJ), and swainsonine (SW) resulted in a decrease of the relative molecular mass (Mr) of both the α-chain of the high affinity receptor for IgE, FcϵRI(α), and the low affinity receptor for IgE, FcϵRL. Exposure to DMJ had the greatest effect on the Mr, while CN seemed to lead to a decreased cell surface expression of FcϵRI. Both receptors are largely resistant to endoglycosidase H as their Mr decreased only by approximately 2 kDa. These results suggest that both receptors are composed primarily of complex oligosaccharides with a single high mannose, N-glycosylated site. Both Fcϵ receptors become endoglycosidase H sensitive if first exposed to DMJ which indicates that the carbohydrate composition is indeed altered by this processing inhibitor presumably by blocking the formation of complex structures. When the Fcϵ receptors were reduced and hydrolyzed by N-glycanase, the Mr values for FcϵRI(α) and FcϵRL decreased to approximately 28 and 34–38 kDa respectively. In the case of FcϵRI(α), this implies the presence of only a small amount of O-linked oligosaccharides.

Binding of the Receptors for IgE by Various Lectins

Two receptors for IgE, having apparent molecular weights of 55,000 and 45,000 daltons were isolated from rat basophilic leukaemia cells by means of IgE-Sepharose. Both molecules were bound by concanavalin A, wheat germ agglutinin and Ricinus communis agglutinin (castor bean lectin). The lectins of pea and gorse origin only bound the 45,000 dalton receptor.

Establishment and characterization of hybrid rat mast cells

Rat peritoneal mast cells (RPMC) and rat basophilic leukemia (RBL) cells are representative of connective tissue-type (CTMC) and mucosal-type (MMC) mast cells, respectively. Using polyethylene glycol, we have fused RPMC with 6-thioguanine resistant, HAT (hypoxanthine, aminopterin, thymidine) sensitive RBL-CA10.7 or RBL-CK2 cells, yielding several hybrid rat mast cell lines (HRMC). The hybridomas exhibited different size and cytoplasmic granularity when compared with parental cell lines. Analysis of both high (Fc epsilon RI) and low affinity (Fc epsilon RL) receptors for IgE revealed that the hybrid lines had more variable receptor patterns than the parent lines. Three hybridoma lines were chosen for further study. Differential histochemical staining with alcian blue and safranin O dyes indicated the hybrids to be predominantly of the MMC type: however, a few cells of one of these uncloned hybridomas were found to be of the CTMC type. Attempts to isolate the CTMC hybridomas yielded one culture which was predominantly of the CTMC phenotype and in a number of other cultures, cells were found expressing simultaneously both the CTMC and the MMC phenotype. After 3 weeks in culture, however, all hybridomas, including those which were cloned further, expressed only the MMC histochemical phenotype. This was found to correlate with the presence of rat mast cell protease II (RMCPII) and the absence of RMCPI in all hybridomas, as detected by Western blot analysis. In addition, the histamine content of all cells was significantly lower than that of the parent RPMC. Most hybrid mast cells expressed both Fc epsilon RI and Fc epsilon RL which in some cases exhibited significant variations in the Mr. These results indicate that somatic cell hybrids expressing the MMC and CTMC phenotype can be produced by the fusion of RBL and RPMC. The CTMC phenotype, however, is unstable, and possible reasons for this are discussed.

Characterization of the target cell receptor for IgE—IV.: ISolation of IgE-receptor complexes

Abstract Complexes, consisting of IgE and its receptor from rat basophilic leukemia (RBL) cells, were isolated by allowing RBL cells to interact with DNP-IgE, solubilizing the cells with Nonidet P-40 and by allowing the extract to bind to anti-DNP-Sepharose. Of the RBL surface material eluted with 2,4-dinitrophenolate, 51–65% was precipitable by anti-IgE. Polyacrylamide gel analysis of eluates in SDS. using 10% gels, revealed a single surface component with an apparent molecular weight of about 45.000 daltons.

Properties of bovine colostral IgG1 anti-DNP antibodies--I. Isolation and binding studies.

Abstract Bovine IgG 1 anti-DNP antibodies were isolated from the colostrum of two animals using a combination of DEAE-cellulose and Bio-Gel chromatography as well as an immunosorbent. Binding studies with ϵ-DNP-lysine yielded conventional results while those with the dye-hapten 1-hydroxy-4-(2,4-dinitrophenylazo)-2,5-napththalene disulfonate (1N-2,5S-4DNP), revealed the presence of two kinds of combining site present in about equal concentration, as determined by computer analysis. Titration of one antibody preparation with 1N-2,5S-4DNP detected only a maximum of 1.2 combining sites per intact IgG 1 molecule. However, upon removal of the Fc component by pepsin, the expected 1.8 binding sites per F(ab′) 2 were found.

Factor-independent tissue cultured mast cells: Establishment from rat peritoneal mast cells

We report here that extended culture of purified rat peritoneal mast cells (RpMC), typical of the connective tissue-type (CTMC), gives rise to continuously proliferative cell lines without the requirement of exogenous growth factors such as IL-3 and IL-4 or accessory cells. Two of the cell lines established, RCMC1 and RCMC2, are described here. Both cell lines have been maintained in continuous culture in vitro for over a year. Although these cell lines were derived from CTMC, they exhibit phenotypic characteristics of mucosal-type mast cells, i.e., they contain rat mast cell protease II (RMCP II), low levels of histamine and stain alcian blue+/safranin-. Previous studies have identified both high and low affinity receptors for IgE, designated Fc epsilon RI and Rc epsilon RII, respectively, on RpMC and rat basophilic leukemia (RBL) cells. At the early stages of cell culture, RCMC1 expressed predominantly Fc epsilon RI and a gradual increase in the expression of Fc epsilon RII has been observed with time in culture. By comparison, RCMC2 expressed predominantly Fc epsilon RII throughout its entire period of cell culture.

Lentil-Lectin Affinity Chromatography of Surface Glycoproteins and the Receptor for IgE from Rat Basophilic Leukemia Cells

Surface proteins and glycoproteins of RBL cells were labelled enzymatically with 125I and then solubilized with Nonidet P-40. Analysis by polyacrylamide-gel electrophoresis in SDS on 10% gel revealed 10 distinctive peaks ranging in molecular weights from 17,000 to 200,000 daltons. Mainly components of higher molecular weights were bound by lentil-lectin Sepharose and could be eluted with alpha-methyl mannoside. The receptor for IgE was clearly shown to bind to the lentil-lectin. A second cell surface component which previously had been shown to bind to IgE-Sepharose as well, was found to bind only slightly to lentil-lectin. Thus, it can be concluded that the receptor for IgE is a glycoprotein with mannose and/or N-acetylglucosamine in the carbohydrate moiety(s).