Studies on in vitro IgE synthesis from lymph node cells of mice during infection with Nippostrongylus brasiliensis

Abstract

The effect of serum factors on Ig synthesis (IgE, IgG) in vitro was analyzed. Spleen and mesenteric lymph node cells were obtained from Nippostrongylus brasiliensis-infected and non-infected mice. Sera and ammonium sulphate precipitated serum fractions from mice of different genetic origin (Balb/c - H-2d, A.CA - H-2f, B10.G - H-2q) suppressed in vitro IgE synthesis whereas a pronounced enhancement of IgG antibody synthesis was obtained in several experiments. Our results obtained with sera from both high and low IgE responder strains demonstrated that no strain specificity exists as to the inhibitory efficacy of mouse sera for total IgE synthesis in vitro. The suppressive activity of the mouse sera was concentrated in a fraction precipitated with 20%-50% saturated ammonium sulphate. Amicon XM50 ultrafiltration suggested that this fraction had an apparent molecular weight greater than 50,000 daltons. Suppressive activity was removed by immunoadsorption of the 20-50% fraction with anti-IgE Sepharose. After exogenous addition of monoclonal IgE to an inactive fraction in vitro neither the fraction enriched in IgE nor monoclonal IgE alone were able to suppress IgE synthesis in the culture. Our results suggest that one or more serum factors in the presence of IgE are responsible for the suppression of total IgE synthesis in vitro.

Generation of Cell-Derived Factors in vitro Which Modulate IgE Synthesis

“…At days 4, 7 and 10 after rein fection, the mice were bled and the ¿/.¿.-specific IgE antibodies were determined by antibody titre (PCA). At days [7][8][9][10] As is demonstrated (table I), the various LMWCS generated in vitro did not modulate the total IgE syn thesis when added to cultured lymphocytes in vitro. It has been described that LMW factors which bind to IgE and which were generated by incubation of nor mal spleen cells with homologous IgE can be ob tained by immunoadsorption on IgE-Sepharose [5], Therefore, the LMWCS were absorbed with IgE-Sepharose; the columns were eluted with glycine/HCl, pH 2.7.…”

“…The cell-free culture superna tants were passed through an Amicon XM50 filter; high-molecular weight culture supernatants (HMWCS; > 50,000 daltons) and lowmolecular weight culture supernatant (LMWCS; <50,000 daltons) were obtained. After dialysis against phosphate-buffered saline (PBS) and concentration to one-fifth of the original volume, 0.1-ml aliquots of the supernatants were added for an additional 7 days to cultured Balb/c lymphoid cells (5 x lOVml), which were obtained from N.b.-reinfected mice and released pronounced quantities of IgE in vitro [6], The IgE and IgG synthesized in vitro was deter mined by a solid-phase radioimmunoassay [7] using monoclonal mouse IgE (DNP-specific) as antigen [8].…”

Generation of Cell‐Derived Factors Suppressing IgE Synthesis of Nippostrongylus brasiliensis‐Infected Mice in Vitro

“…Addition of monoclonal IgE to mouse lymphocytes in vitro led to increased IgE synthesis of the cells within 7 days of culture [28]. As previously described, this protocol leads to the generation of IgE-binding factors [32].…”

“…Anti-mouse IgG was raised in guinea pigs with monoclonal mouse IgGl used as antigen. The antiserum was purified as has been previously described [28].…”

“…Therefore, the results were expressed as percentages of modulation by comparing the ratio of cpm of test fraetion-treated eells xlOO epm of medium control = % modulation Radioimmunoassay for IgE and IgG. The IgE and IgG synthesized in vitro by lymphoid mouse cells was determined by a solid-phase RIA as was previously described [28]. The purified anti-mouse IgE and IgG antibodies were iodinated as performed by Klinman & Taylor [17].…”