Biomedical subjects
Publications and source records attributed to F Uher.
Activation of mature B cells to proliferation and terminal differentiation is a multistep process controlled mainly by macrophages and T cells. However, there is growing evidence that B cells and other factors can also regulate the humoral immune response. Here, Ferenc Uher, Eva Rajnavölgyi and Anna Erdei describe their work on the emerging role of regulatory interactions between subsets of B cells and their soluble products.
Culture supernatants of LPS-stimulated murine B lymphocytes are able to inhibit the growth of freshly isolated splenic B cells via an IgM antibody. The binding specificity of this IgM is not yet defined, but appears to be a B lymphocyte surface structure distinct from membrane immunoglobulin, MHC class II antigen, transferrin and Fc gamma receptors, and B220. The regulatory autoantibody allows the normal progression of early, but not late steps in the cycle of polyclonally-stimulated B lymphocytes and does not affect the increased antigen-presenting capacity of activated B cells. Therefore, this autoregulatory cycle is apparently ubiquitous and may be a major component of B lymphocyte homeostasis under physiological, as well as pathological conditions. Moreover, these findings bring into focus a possible regulating role of B lymphocytes in the humoral immune response.
The results of this study demonstrate that the culture supernatant of lipopolysaccharide (LPS)-stimulated murine spleen cells is able to inhibit the growth of freshly isolated B lymphocytes. The inhibition is specific for B cells because the suppression of the LPS, Fc-fragment of human IgG, dextran sulfate, and anti-mu induced proliferation of B, but not the Concanavalin A response of T lymphocytes could be shown. The cells producing the inhibitor do not adhere to plastic, and are Thy-1 negative but surface Ig positive, i.e. they are B lymphocytes. The regulatory substance is heat resistant, sensitive to trypsin treatment, and has a high molecular weight of approximately 1000 kDa. Moreover, it is specifically adsorbed on, and can be eluted from, anti-mu and anti-Ig immunoaffinity columns. Thus, it seems to be an IgM antibody. Non-specific effects were excluded by the ineffectiveness of poly- and monoclonal IgM proteins. IgM-IgG complexes were also excluded. Thus, these results suggest the existence of a novel IgM antibody mediated control mechanism regulating B cell growth during polyclonal activation.
Mouse B lymphocytes were fractionated from normal T lymphocyte-depleted spleen cell populations using discontinuous percoll gradients and were stimulated with rabbit F(ab')2 anti-mouse mu-specific antibodies (anti-mu) plus the supernatant of Con A-stimulated rat spleen cells (SN) as a source of lymphokines. The responses of small (mean volume 120 mu 3), dense (greater than 1.087 specific gravity), resting (least spontaneous thymidine incorporation) B lymphocytes were augmented by irradiated (4000 rad), larger (mean volume greater than 170 mu 3), less dense (less than 1.081 specific gravity), activated (greater spontaneous thymidine incorporation) B lymphocytes. Proliferation was augmented 2- to 4-fold and polyclonal antibody-forming cell responses three- to sixfold. Maximal augmentation of the responses of 5 X 10(4) resting B cells was obtained with 10(4) activated B cells. Augmenting activity was specific for activated B lymphocytes in that responses were not augmented by irradiated thymocytes, T lymphoblasts, macrophages, or additional supernatant. B lymphocytes activated in vitro by LPS or anti-mu also had augmenting activity. Augmentation of responses was maximal only when activated B lymphocytes were added simultaneously with anti-mu. The interaction between activated and resting B lymphocytes did not appear to be genetically restricted. Interestingly, the augmenting activity of activated B cells could be reconstituted by a combination of supernatant and cell membranes from these cells but not by either alone, suggesting that two components are required, one soluble and the other membrane-bound. Thus, a functional interaction has been demonstrated between B lymphocyte subpopulations which differ in their state of activation, and this interaction appears to involve a novel mechanism of action.
The expression of Fc mu receptors was investigated using spleen cells from (LOU/M/Wsl X CFY) F1 rats bearing the IR202 tumour, an IgM-producing immunocytoma. It was found that the progressively growing solid tumour was accompanied by an extraordinary expansion of host splenocytes with Fc mu receptors. These cells were nylon-wool non-adherent, Thy-1-positive and sIg-negative: features they have in common with T lymphocytes. In addition, the expansion of Fc mu receptor-bearing T cells in IR202 immunocytoma is correlated to the high level of serum IgM. These observations provide further insight into the mechanism of isotype-specific T-lymphocyte Fc receptor expression, and identify a potential model with which to analyse the immunoregulatory role of Fc mu receptor-bearing T cells.
IgG antibody-antigen complexes bound to B lymphocyte Fc gamma receptor (Fc gamma R) but not surface immunoglobulin inhibit the antibody-forming cell response but not proliferation of these cells in response to F(ab')2 anti-mu and lymphokines. The role of B lymphocyte antigen receptors in B lymphocyte Fc gamma R-mediated inhibition was evaluated. With the use of several different antigen receptor-dependent plaque-forming cell (PFC) responses, it was found that inhibition occurred irrespective of the type of stimulatory signal used, or whether antigen receptors were bound by antibody or occupied by nominal antigen. The degree of inhibition appeared to be directly related to the extent of antigen receptor cross-linking. Maximal inhibition only occurred if both antigen receptors and Fc gamma R were occupied by their respective ligands simultaneously during the early hours of activation. In contrast, antigen receptor-independent PFC responses to the mitogen lipopolysaccharide (LPS) were not inhibited by complexes. However, the antigen-independent TNP-specific PFC response to LPS was inhibited by the combination of IgG antibody-antigen complexes and the hapten, but by neither alone. These results suggest that a down-regulatory signal is generated by functional cooperation between Fc gamma R and antigen receptors. Generation of this inhibiting signal could be mediated by the previously described physical interaction between these two receptors, and interactions between membrane receptors may be a general mechanism utilized by lymphocytes for the integration of multiple signals.
When two distinct B-lymphocyte membrane receptors (Fc gamma R and sIg) are independently occupied by their respective multivalent ligands, inhibition of the antibody-forming cell response occurs but proliferation is not inhibited. This regulatory signal was examined in various B-lymphocyte populations. Unprimed B lymphocytes from immune deficient CBA/N and autoimmune MRL/l mice were responsive to this regulatory signal. In contrast, unprimed B lymphocytes from autoimmune NZB mice and antigen-primed B lymphocytes from normal DBA/2 mice were not. Together with previous results, these data suggest that resting B lymphocytes which have not encountered antigen are most susceptible to this regulatory signal. Lack of responsiveness to this downregulatory signal may contribute to the hyper-responsiveness of NZB B lymphocytes.
We studied the effect of soluble antigen-antibody complexes on the responses of polyclonally activated murine B lymphocytes. For this, normal B lymphocytes were stimulated with rabbit F(ab')2 anti-mu and lymphokines. IgG complexes, particularly in antigen excess, inhibited the plaque-forming cell response (55-70%), while proliferation was unaffected. Maximal inhibition was obtained with small amounts (0.2-1.0 microgram/ml) of complexes. Neither antigen or antibody alone was inhibitory. Inhibition was mediated via binding of the IgG complexes to Fc gamma receptors of B lymphocytes: (1) neither T lymphocytes or adherent accessory cells were required; (2) IgM complexes did not inhibit; and (3) inhibition was not seen when monoclonal anti-Fc gamma receptor antibodies prevented binding of the IgG complexes to these receptors. Kinetic experiments showed that B lymphocytes are susceptible to this inhibitory signal for only a short time after stimulation. We conclude that IgG complexes bound to the Fc gamma receptors of B lymphocytes regulate B-lymphocyte differentiation.
Some effector functions of antigen-antibody and antibody-antibody (idiotype-anti-idiotype) complexes were analyzed. As a model system a monoclonal IgM antibody specific for the hapten NP (antibody B1-8) was reacted either with hapten and hapten-carrier conjugates or with monoclonal anti-idiotope antibodies with specificity for B1-8 idiotopes. The precipitating, C1q-binding, complement-activating and Fc receptor binding properties of these complexes were compared. Binding of both hapten-carrier conjugates and anti-idiotope antibodies to B1-8 results in formation of complexes which depending on the B1-8:ligand ratio precipitate, activate complement, bind C1q and exhibit increased avidity for Fc mu and Fc gamma receptors of mouse spleen cells. In both types of complexes cross-linking of IgM molecules is essential for triggering these Fc-dependent functions, and a functional heterogeneity if idiotype-anti-idiotope complexes based on different idiotype-anti-idiotope ratios could also be observed. The functional similarity of B1-8-hapten-carrier and B1-8-anti-idiotope complexes suggests that regulatory functions so far assigned to antigen-antibody complexes could be carried out also by idiotype-anti-idiotope complexes.
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The primary receptor ligand interaction between rat leucocyte membrane receptors for IgM and their ligands were examined. We found that the incubation time for optimal IgM binding is different on the two types of IgM-Fc receptor-bearing spleen cells and peritoneal macrophages. The investigation of cytophilic activity of polyclonal or monoclonal IgM proteins and their fragments from various species indicated the fine specificity of receptors. Finally, data are presented which suggest a multiple point co-operative binding between IgM-coated erythrocytes and IgM-Fc receptor-bearing cells.
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The features and function of IgM-FcR of rat peritoneal macrophages were studied. Macrophages specifically bind and phagocytose ox red blood cells coated with rat IgM (EA-IgM) through a specific receptor. This receptor is trypsin sensitive and its activity requires Ca++ ions. Both sodium azide and low temperature (4 degrees) inhibit the bindings as well as ingestion of EA-IgM by macrophages, suggesting the metabolically dependent character of the interaction between EA-IgM and macrophages. Colchicine inhibits the binding of EA-IgM by macrophages. Similarly, the ingestion of EA-IgM was also inhibited when peritoneal exudate cells (PEC) were pre-treated with colchicine or vinblastine or cytochalasin B. It is suggested that cytoskeletal elements of macrophages play an important role both in the binding of EA-IgM to their receptors and in the subsequent internalization of the receptor-ligand complexes. Ingestion of soluble IgM antibodies containing immune complexes (IC) resulted in a release of beta-glucuronidase from macrophages.