The regulating effects of IL-4 and pokeweed mitogen on IgE synthesis in vitro by human peripheral blood leucocytes has been compared with the corresponding effect of these regulators on the expression of IgE mRNA. The latter was measured by dot blot hybridization with an oligonucleotide coding for a unique six amino acid region of the CH-epsilon-2 domain. Specificity of the oligonucleotide probe was established by its inability to hybridize with RNA extracted from HMY-2 (IgG) and XQ-15 (IgM) secreting cell lines whilst producing intense signals with RNA extracted from the IgE secreting cell line U266. Whilst IgE mRNA was detected in RNA extracted from PBL of both atopic and control subjects, spontaneous IgE synthesis was restricted to atopic PBL. IL-4 increased both IgE mRNA and IgE synthesis in all PBL samples but PWM, while significantly increasing IgE mRNA expression either failed to modify IgE synthesis or actively suppressed it. The assay system employed to quantitate IgE synthesis in vitro was shown to be inhibited by both IgE binding factors and IgG anti-IgE autoantibodies which are produced in PBL cultures. IgE mRNA levels might therefore more accurately monitor the regulatory effects of IL-4 and PWM on IgE synthesis than quantitation of the IgE by radioimmunoassay.

The regulating effects of IL-4 and pokeweed mitogen on IgE synthesis in vitro by human peripheral blood leucocytes has been compared with the corresponding effect of these regulators on the expression of IgE mRNA. The latter was measured by dot blot hybridization with an oligonucleotide coding for a unique six amino acid region of the CH epsilon 2 domain. Specificity of the oligonucleotide probe was established by its inability to hybridize with RNA extracted from HMY-2 (IgG) and XQ-15 (IgM) secreting cell lines whilst producing intense signals with RNA extracted from the IgE secreting cell line U266. Whilst IgE mRNA was detected in RNA extracted from PBL of both atopic and control subjects, spontaneous IgE synthesis was restricted to atopic PBL. IL-4 increased both IgE mRNA and IgE synthesis in all PBL samples but PWM, while significantly increasing IgE mRNA expression either failed to modify IgE synthesis or actively suppressed it. The assay system employed to quantitate IgE synthesis in vitro was shown to be inhibited by both IgE binding factors and IgG anti-IgE autoantibodies which are produced in PBL cultures. IgE mRNA levels might therefore more accurately monitor the regulatory effects of IL-4 and PWM on IgE synthesis than quantitation of the IgE by radioimmunoassay.

SummaryFractionation by Percoll density cetrifugation of peripheral blood leucocyte cells, from atopic subjects with seasonal hay fever, unmasked IgE‐B cell populations whose individual capacities to synthesize IgE in vitro were obscured in cultures of unfractionated B cells. B cell cultures from all six subjects in the study released rye pollen‐specific IgE during the 6 days of culture, but actual synthesis was significant only in October, the pollen season. Synthesis in October occurred most frequently in cultures of mature, low density B cells, which generally responded to the addition of autologous T cells with enhanced synthesis (T‐help). T‐help was also found for high density B ceils in the mid‐winter (July) cultures. Total IgE synthesis in vitro demonstrated a less seasonal relationship, although it tended to be maximal for low density B cell cultures in October and for high density B cells in May. All B cell cultures contained preformed total and rye‐specific IgE antibody which persisted throughout the pre‐ and post‐pollen seasons, particularly in the low density B cell fractions, even in the absence of de novo synthesis. Moreover, the intracellular levels of rye pollen‐specific IgE antibody were often higher in the winter than in the peak of the pollen season. The relevance of this preformed IgE remains to be established.

Monocyte-enriched preparations derived from peripheral blood leukocytes of atopics were probed via a cocktail comprising peroxidase-conjugated (Fab1)2 fragments of two monoclonal antibodies against human IgE. Reaction product indicative of intracellular IgE was identified by electron microscopy in both large and small vacuoles, and at high magnification exhibited a characteristic granular deposition pattern consistent with highly concentrated (perhaps insolublized) material. IgE-containing vacuoles were observed with comparable frequency to those containing IgG, despite the greater than 10,000-fold relative excess of the latter in serum suggesting highly selective uptake of IgE by the monocytes. These results are similar to those reported recently for IgA in human milk macrophages.

Peripheral blood mononuclear B cells from 6 rye pollen-allergic patients, with no consistent perennial symptoms, were isolated before (July), during (October) and after (February and May) the pollen season. The T-depleted cells were fractionated on a discontinuous percoll density gradient and the B cell fractions, together with unfractionated B cells, incubated in vitro for quantitation of spontaneous synthesis of rye pollen-specific IgE. Markedly higher levels of IgE were synthesised by the fractions, as opposed to unfractionated B cells. The low-density fraction (B5) contributed most towards synthesis in the pollen season and the denser B6 cells in the pre- and post-pollen season. All low-density B cell fractions (B1-3-B5) and some high-density fractions contained large but variable amounts of preformed specific IgE which was retained, even in the absence of de novo synthesis in vitro, during the post- and pre-pollen season. Since only a fraction of this preformed IgE escaped into culture supernatants the contribution of preformed IgE to in vitro IgE synthesis in general may require reappraisal.

Fractionation of human peripheral blood leukocytes (PBL) B cells by differential sedimentation on percoll gradients separates B cell subpopulations which vary markedly in rates of spontaneous IgE synthesis, in their response to identical populations of autologous T cells and in their response to soluble T cell factors. This B cell fractionation technique often reveals the presence of active IgE-secreting cells which are totally suppressed within unfractionated PBL B cell preparations and greatly improves the efficiency of detection of T cell-responsive, IgE-producing B cells in PBL of atopics.

A new system is described for the enumeration of human immunoglobulin-secreting cells (ISC), based upon the ELISA methodology. In principle, putative ISC are incubated over a solid phase containing bound anti-Ig of the isotype being tested. Secreted Ig is immobilized at or near the point of release from the ISC, and the resulting Ig fingerprint of the ISC is then visualized by the sequential application of an anti-Ig-alkaline phosphatase conjugate, followed by a substrate-agarose overlay. The system is capable of detecting IgE-secreting cells, and pokeweed mitogen-stimulated IgG-secreting cells with sensitivity at least equivalent to the protein A hemolytic plaque assay.