A. Coutinho

Thymus-Independent B-Cell Induction and Paralysis

Publisher Summary Paralysis is seriously considered only in the “two specific signal” hypothesis by postulating that the first signal, generated by the combination of antigen with the specific receptor, results in tolerance rather than induction, except if the cell is simultaneously exposed to the second specific signal, generated by the recognition of the antigen by associative antibody. This model of associative recognition is designed in order to explain self-nonself discrimination and denies the existence of truly thymus-independent (TI) antigens as well as the possibility of triggering B cells by any mechanism not involving the first specific signal. The main general conclusion concerning induction of paralysis by TI antigens is that specific B cells are turned off at higher concentrations of the TI antigen than those causing activation. TI antigens do induce specific B cells directly and one cannot exclude that at least some of them also activate other cell types, including T cells, nor that some or possibly all of them interact with macrophages. It is suggested that the effects on other cell types represent trivial or optimizing events on top of the basic, fundamentally effective mechanism of direct B-cell activation. All TI antigens are competent to activate B cells polyclonally and this nonspecific property seems to be responsible for induction of specific cells. The inactivation of competent specific cells is also a result of the interaction between nonspecific surface receptors and the competent ligand.

Inhibition of In Vitro Immune Responses by a Fraction from Seminal Plasma

We studied the inhibitory effects of a fraction of bovine seminal plasma on in vitro lymphocyte responses to specific and nonspecific ligands. Seminal plasma was fractionated by Sephadex G‐100 chromatography, and the inhibitory component was found to have a molecular weight of above 100,000 daltons. This fraction was devoid of lymphocytotoxity and could inhibit, in a dose‐dependent mode, both the primary antibody response of normal mouse spleen cells to the thymus‐dependent antigen sheep erythrocytes and the one‐way mixed lymphocyte reactions. Concanavalin A‐induced proliferate T‐cell responses were also inhibited by the same fraction. The inhibition of the T‐lymphocyte responses to Con A was complete at suboptimal concentrations of the mitogen and could only partially be overcome at supraoptimal concentrations. Four other fractions in seminal plasma were also studied in this particular system, three being without effect and one being stimulatory. The inhibitory fraction was also found to inhibit the polyclonal B‐cell responses induced by fetal calf serum. The importance of the present findings for the immunophysiology of reproduction are discussed.

Functional Analysis of B Cell Heterogeneity

Polyclonal B cell activators (PBAs^) induce resting B cells to proliferation and antibody secretion (G. Moller 1972, Melchers & Andersson 1974a, b). However, this does not imply that all activated cells both proliferate and secrete antibodies upon activation, nor that the pattern of activation achieved with different PBAs will always be the same. In view of the great heterogeneity of the B cell population, in terms of surface markers, homing properties and redrculation pattern, etc. (see this volume), it is to be expected that in the functional activity, as measured by PBA responses, the heterogeneity should also be reflected. This suggestion is supported by the fact that increasing numbers of substances with PBA activity, each inducing a particular pattern of response, to different extent, have been demonstrated lately (Coutinho & Moller 1973, Diamantstein et al. 1973, Sultzer & Nilsson 1972, Bona et al. 1974, Nakashima & Kato 1974). Most of these substances induce B cells to go through a pathway which most likely does not

Heterogeneity of B cells: direct evidence of selective triggering of distinct subpopulations by polyclonal activators.

This investigation was undertaken to assess directly the previous postulate that distinct subsets of B cells in the adult mouse spleen are selectively triggered by different polyclonal B‐cell activators (PBA). Several strains of mice and lipopolysaccharide (LPS), purified protein derivative of tuberculin (PPD), and dextran sulphate (DS) were used in this study. Two experimental approaches were used: stimulation after addition of two PBAs simultaneously to cell cultures and eliminating the responding population to one PBA, by a hot pulse of radioactive thymidine, on a later response to another PBA. The results of these experiments indicated that DS stimulated a cell population completely different from that stimulated by the LPS‐ and PPD‐sensitive cells. When LPS and PPD Stimulations were compared, it was found that the cells responding to these PBAs were largely distinct, although some cells were sensitive to both these PBAs. The extension of the overlaps in these subsets (the number of cells that could be activated by either PBA) was found to vary from one strain to another. These experiments gave direct evidence of the existence of subsets that cm Inactivated by different PBAs. The present results also provided indications of the functional performance of distinct subsets of B cells on activation.

Responsiveness of Lymphoid Precursors to Polyclonal B‐Cell Activators

We have shown previously that in the differentiation of fetal liver cells to mature B cells in irradiated hosts, these cells sequentially gain responsiveness to the polyclonal B‐cell activators dextran‐sulphate (DxS), lipopolysaccharide (LPS)and purified protein derivative from tuberculin (PPD). in that order. In this paper we show that both fetal liver cells and adult bone marrow cells responded with proliferation to DxS. but not to LPS or PPD. However, neither fetal liver nor bone marrow cells gave rise to detectable numbers of high‐rate antibody‐secreting cells on short‐term stimulation by polyclonal B‐cell activators The lack of LPS and PPD responses of fetal liver and bone marrow cells could not be ascribed to the presence of inhibitory cells, and the DxS‐induced response in these cell populations was not dependent on adherent cells. However, LPS could inhibit the DxS response of fetal liver cells, possibly indicating that DxS‐responsive cells are precursors to B cells. Direct evidence was provided that DxS activated B‐Cell precursors in bone marrow. Thus, this cell population became responsive to LPS after DxS prestimulation. as measured by DNA synthesis. Bone marrow cells, sequentially stimulated with DxS and LPS. contained increased numbers of cells with surface immunoglobulin, although no significant increase in numbers of antibody‐secreting cells was obtained. These data indicate that DxS had the capacity to increase the rate of differentiation of B‐cell precursors. Finally, we show that the sequential appearance of responsiveness in B‐cell differentiation to polyclonal B‐cell activators is not due to lack of accessory cells during early stages in maturation

The theory of the 'one nonspecific signal' model for b cell activation.

The extensive discussion of the model and the various competing concepts or statements, as well as the exhaustive presentation of the experimental facts which support it, have been dealt with in detail elsewhere (Coutinho & Moller 1974, Coutinho et al. 1974, Coutinho & Moller 1975). I will now restrict myself to the outline of the theory behind the model, and to some of its implications. It is useless to say that the model was built up together with Dr. Goran Moller, and that I shared with him and with Dr. Eva Gronowicz the elaboration of many of the concepts presented here.

Mechanism of B-cell activation and paralysis by thymus-independent antigens. Additive effects between NNP-LPS and LPS in the specific response to the hapten.

Normal spleen cells showed a bell‐shaped dose response profile when stimulated in vitro with die thymus independent antigen (4 ‐hydroxy‐3,5 dinitiophenyl)acetyl (NNP)‐lipopolysaccharide (LPS) with regard to the development of high‐avidity plaque‐forming cells to NNP The addition of suboptimal concentrations of LPS to cultures stimulated by suboptimal concentrations of NNP LPS resulted in optimal induction of B cells in that affinity fraction Addition of LPS to cultures optimally stimulated by NNP‐LPS resulted in paralysis of the specific cells These results are interpreted in terms of the additive effects between the mitogenicity LPS and the mitogenicity of NNP LPS the latter being selectively focused on the specific cells, thus providing further evidence for the ‘one nonspecific signal’ hypothesis for immune activation of B cells

FACTORS INFLUENCING ACTIVATION OF B‐CELLS IN IMMUNITY*

The hypotheses so far advanced for B-cell activation by antigen are based on the assumption that the interaction between antigen and the Ig receptors delivers at least the initial triggering signal. There are few, if any, experimental findings to support this assumption. On the contrary, a variety of findings indicate that the Ig receptors do not deliver any signal to the cells, whereas activation can be regularly achieved without participation ofIg receptors. The available evidence forces us to suggest that antigen-induced B-cell triggering is always caused by one nonspecific signal, which is delivered to the cells by surface structures, which are not the Ig receptors. For distinctive features of this hypothesis see One Nonspecific Triggering Signal. Various competing hypotheses for B-cell activation have been analyzed, but none of them appears to satisfy the experimental findings.

Regulation of in vitro immunocyte activation: origin of and targets for cell-released inhibitors.

Supernatants from incubated normal mouse spleen cells suppressed the DNA synthetic response induced by polyclonal B‐ and T‐cell activators and the primary immune response to sheep erythrocytes in normal spleen cells in vitro. The inhibitory effects of the supernatants were found to be dependent on the culture system used. The main cell population producing or releasing the inhibitory factors was nonadherent spleen cells, but macrophages and bone marrow cells could also give supernatants with inhibitory effects. Thymocytes did nut release any inhibitors under the same conditions. Separation of the supernatants showed that there were it least two factors with inhibitory effects on proliferation in lymphocytes. One of these factors had a molecular weight of less than 10,000 (not degradable by protease) and suppressed activation by all tested polyclonal B‐ and T‐cell activators. Another factor (a protein with a molecular weight of more than 30, 000) inhibited activation by some polyclonal B‐cell activators (lipopolysaccharide, type III pneumococcal polysaccharide) whereas the proliferation induced by dextran sulfate Was less or not at all affected under the same culture conditions. Trivial mechanisms for inhibition by supernatant factors, such as medium depletion, general toxicity, and accumulation of free thymidine in the medium, were excluded.