Terminal maturation of resting B cells induced by macrophage factors

Mouse splenic macrophages from BALB/c nude mice (purified by plastic adherence) or cloned macrophage hybridomas stimulated with jacalin (12.5 microg/ml), a D-Gal binding lectin, produce one or more B-cell stimulatory factors which cause splenic B cells from BALB/c or C3H/HeJ mice to secrete immunoglobulin in a polyclonal manner as detected by reverse protein A plaque assays. Jacalin-stimulated macrophage supernatants (JacSup) activate both normal and Percoll gradient-purified small high-density (resting) B cells. Supernatants from total or resting BALB/c spleen cells cultured for 7 days in the presence of JacSup (derived from splenic BALB/c nude mice macrophages) were assayed for immunoglobulin isotypes by ELISA. Resting B cells produce only IgG3 and IgM, whereas total B cells secrete IgG3 and IgM as well as IgG1, IgG2a, IgG2b and IgA. Resting and total B cells from BALB/c nude mice are also stimulated by macrophage supernatants to secrete immunoglobulin, thus indicating that this activity is likely to be T cell independent. Moreover, jacalin-stimulated macrophage supernatants did not induce spleen cells or purified B cells to proliferate. Fractionation of factor-rich supernatants on a Sephacryl S-200 column revealed that the factor activity is located in fractions corresponding to a molecular mass of 25-27 kDa. Taken together, these results suggest that upon the action of a macrophage factor(s) resting B cells undergo terminal differentiation without proliferation in the absence of T cells.

Potent stimulation of murine B cells to proliferate and to secrete immunoglobulins by a lipoteichoic acid-like molecule produced by Clostridium botulinum C and D

Bacterial products have served as important immunological tools to study ly,phocyte activation. The lipopolysaccharides of the Gram-negative bacteria are well known to be potent activators of B lymphocytes. Several Gram-positive bacteria produce exotoxins that are superantigens for T cells. In the present study, we demonstrate that the Gram-positive bacteria Clostridium botulinum C and D produce a high molecular weight mitogen (Cb mitogen) that is a potent activator of murine B lymphocytes. The Cb mitogen was discovered as a consequence of our attempt to investigate a possible superantigen activity present in the botulinum exotoxins. We observed initially that mouse spleen cells were strongly stimulated to proliferate by culture supernatants of C. botulinum C and D. However, the characterization of the responding cell ruled out superantigen because only the B lymphocytes were stimulated to proliferate and to secrete immunoglobulins, and they did so independent of T cell help. In addition, the molecular characterization of the Cb mitogen demonstrated that the purified botulinum toxin was devoid of mitogenic activity. In contrast, the fractionation of the culture supernatant of C. botulinum C in an FPLC Superose 12 column indicated that the Cb mitogen was present in the void volume of the column (MW ò 300 kDa) which had no toxigenic activity. However, the fractions containing molecules of 150 KDa were highly toxic for mice and had no mitogenic activity...

Hydroxyurea before oral antigen blocks the induction of oral tolerance

1. Treatment with hydroxyurea (HU, 1 mg/g ip, 2 doses applied 7 h apart) eliminates the majority of cells undergoing mitosis (cycling cells) without affecting non-cycling cells. Oral tolerance, induced by a single gavage with 20 mg of ovalbumin, results in a drastic inhibition of anti-Ova antibody responses in young adult mice. Oral tolerance is actively maintained by the presence of specific suppressor T cells which may adoptively transfer the tolerance to naive syngeneic recipients. Under the clonal selection hypothesis, the induction of oral tolerance should be blocked by HU treatment applied soon after oral exposure to the antigen by the elimination of specific clones of lymphocytes activated by tolerogenic presentation of the antigen. 2. However, treatment with HU initiated 3, 6 or 24 h after oral exposure to ovalbumin had no effect on the induction of oral tolerance in B6D2F1 mice. However, treatment with HU 24 h before antigen exposure, totally blocked the induction of tolerance. Treatment with HU 72 h before ovalbumin had no effect. 3. In animals treated with HU 24 h before, the adoptive transfer of normal thymus, bone marrow or spleen cells partially restored the susceptibility to the induction of oral tolerance. 4. The results suggest that cycling cells, which may be totally regenerated within 72 h after treatment with HU, and are present in normal thymus, bone marrow and spleen, are crucially important for the induction of oral tolerance