Polyclonal B-cell activator with esterolytic activity and polyclonal gammopathy induced by allogeneic cells in rabbits

A factor associated with α-macroglobulin (αM) which behaves in an Ig-turnover assay as any T-independent antigen or polyclonal B-cell activator (PBA) can be induced in vivo by inoculating rabbits with allogeneic lymph node cells (A1-LNC) or with sheep red blood cells (SRBC). Before inoculation and 3 days after inoculation, the PBA activity was not detected in the serum. PBA appeared and reached a maximum on day 4, decreased gradually after 1 week, and became undetectable at 6 to 7 weeks. The PBA titre for rabbits inoculated with A1-LNC was 100 to 1000 times higher than for rabbits inoculated with SRBC. The PBA activity was associated entirely with the αM fraction. The αM separated from normal rabbit serum had no PBA activity but when normal αM was complexed with trypsin, a high PBA titre was obtained. Trypsin had PBA, proteolytic and esterolytic activity but when it was bound to αM, it lost its proteolytic activity and maintained its esterolytic and PBA activity. The PBA activity of the normal αM-trypsin complex and of the αM from A1-LNC injected rabbits were inhibited by serine protease inhibitors of low molecular weight, such as aprotinin and phenylmethylsulphonyl fluoride, but not by an inhibitor of high molecular weight such as soybean trypsin inhibitor. The inhibition of PBA activity correlated with the loss of esterolytic activity. For the rabbits inoculated with A1-LNC, the IgG concentration increased in the serum to a four-fold maximum at 3–5 week. We concluded that the PBA induced in vivo by allogeneic stimulation is an αM-serine protease complex which appears in the serum and that this is followed by a transitory polyclonal gammopathy.     

Identification of a polyclonal B-cell activator in Plasmodium falciparum

Polyclonal B-cell activation and hypergammaglobulinemia are prominent features of human malaria. We report here that Plasmodium falciparum-infected erythrocytes directly adhere to and activate peripheral blood B cells from nonimmune donors. The infected erythrocytes employ the cysteine-rich interdomain region 1alpha (CIDR1alpha) of P. falciparum erythrocyte membrane protein 1 (PfEMP1) to interact with the B cells. Stimulation with recombinant CIDR1alpha induces proliferation, an increase in B-cell size, expression of activation molecules, and secretion of immunoglobulins (immunoglobulin M) and cytokines (tumor necrosis factor alpha and interleukin-6). Furthermore, CIDR1alpha binds to Fab and Fc fragments of human immunoglobulins and to immunoglobulins purified from the sera of different animal species. This binding pattern is similar to that of the polyclonal B-cell activator Staphylococcus aureus protein A. Our findings shed light on the understanding of the molecular basis of polyclonal B-cell activation during malaria infections. The results suggest that the var gene family encoding PfEMP1 has evolved not only to mediate the sequestration of infected erythrocytes but also to manipulate the immune system to enhance the survival of the parasite.     

Comparative studies on the actions of antigen and polyclonal B-cell activator in differentiation and proliferation of B-cells and B memory cells.

Using the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) as a polyclonal B-cell activator (PBA) and sheep red blood cells (SRBC) as a T cell-dependent antigen, we compared the ability of PBA and antigen to differentiate (generate antibody-forming cells, AFC) and proliferate (generate immunological memory) virgin B cells and B memory cells. In vitro CPS-K induced the differentiation of IgM virgin B cells, IgM B memory cells and IgG B memory cells to AFC, as well as or better than SRBC. The differentiation of B memory cells to AFC by CPS-K did not require the participation of macrophages or T cells, whereas the action of SRBC depended strictly upon the helper actions of these cells. The responsiveness to CPS-K and SRBC of normal and antigen-primed spleen cells as judged by anti-SRBC PFC responses in vitro was markedly decreased after stimulation of virgin B cells and B memory cells in vivo by CPS-K injection into normal or primed mice but greatly increased after the injection of SRBC. The decrease in the responsiveness to CPS-K of spleen cells from mice treated with CPS-K appeared principally due to exhaustion of the functions of B cells and B memory cells. From the present data it has been concluded that the signals required for the differentiation and proliferation of B cells of B memory cells are different from each other, the signal for differentiation being provided by either antigen (SRBC) or PBA (CPS-K), while the signal for proliferation only by antigen.     

Facb rosette-forming cells in mice: studies on their functional significance.

Lymphocytes bearing receptors for the Facb fragment of IgG have been shown previously to be elevated in the peripheral blood of patients with rheumatoid arthritis. The generation of these cells and their possible functional role in immune regulation have been investigated in mice. Facb rosette-forming (Facb-R+) lymphocytes were found to be elevated in the spleens of mice mounting a secondary plaque-forming cell (PFC) response to sheep erythrocytes but not during the primary response. Splenic Facb-R+ lymphocytes were also elevated when a cross-reacting antigen (goat erythrocytes) was used for the secondary immunization but not when a non-cross-reacting antigen (chicken erythrocytes) was used. Both primary and secondary immunization with bacterial lipopolysaccharide resulted in elevation of splenic Facb-R+ lymphocytes. Administration of antigen-specific Facb fragment in conjunction with antigen (calf erythrocytes) produced a suppression of the secondary PFC response. However, F(ab')2 fragments produced no such effect. This suppressive effect was shown to be antigen-specific since administration of Facb fragment of anti-calf erythrocyte IgG had no suppressive effect on the secondary PFC response to sheep erythrocytes. No change in splenic Facb-R+ lymphocytes was observed during delayed hypersensitivity responses to either sheep erythrocytes or the contact-sensitizing agent oxazolone. These results indicate that Facb-R+ lymphocytes are generated during secondary humoral responses but not cell-mediated immune responses, and suggest that these cells may exert a suppressive influence on antibody production. These findings are discussed in relation to the occurrence of these cells in patients with rheumatoid arthritis.     

X-ray enhancement of splenic rosette-forming cells in nonimmune mice

The formation of rosettes between spleen cells from nonimmune mice and sheep red blood cells (SRBC) has been investigated. The number of splenic rosette-forming cells (RFC) in the intact spleen was very low but greatly increased after sublethal whole body x-irradiation. These cells were inhibited by pretreatment of the cells with AKR anti-C3 H-Θ antigen antiserum, in the presence of fresh guinea pig serum, but not with rabbit anti-mouse immunoglobulin antiserum. The splenic RFC also increased after cortisone acetate administration into mice. These results suggest that the RFC are a T cell subpopulation having x-ray resistant and cortisone-insensitive characteristics. The x-irradiation of mice in vivo selectively increased the number of splenic RFC in consequence of reduction in the number of cells having non-T cell characteristics. These RFC are presumably antigen-specific sheep red blood cell rosettes, a quite different situation as in the case of human T lymphocyte rosette formation.     

The functions of immune T and B rosette-forming cells

From 7 to 35 days after CBA mice were primed with SRBC their spleens were removed and anti-SRBC rosettes were formed. The rosettes were purified from other spleen cells by velocity sedimentation at 4° and rosette-enriched, rosette-depleted and various control populations were injected into lethally irradiated CBA recipients. These were challenged with SRBC and their spleens analysed for direct (IgM) and enhanced (IgG) PFC 7 days later. Removal of RFC depleted the primed spleen cells of their capacity adoptively to transfer an immune response. This depletion was antigen-specific. Purified rosettes alone prepared 7–8 days after priming transferred significant (relative to controls) immune reactivity to the irradiated recipients. Both B and T RFC were present at this stage and the response was dependent upon cell collaboration between these two populations. Later in the primary response (35 days) purified rosettes transferred negligible immune reactivity. But these RFC (90 per cent B) collaborated with rosette-depleted cells to restore full reactivity. B memory lymphocytes (AFCP) form rosettes from 7 to 35 days after immunization but T memory cells only do so for a limited stage during the peak of the primary response. The majority of T memory cells probably never form rosettes in this system. It is suggested that most T RFC may be cells mediating delayed hypersensitivity or are `passive' rosettes.     

CD5-positive B cells in healthy elderly humans are a polyclonal B cell population

B cell chronic lymphocytic leukemia (B-CLL) is a disease of the elderly and is characterized by a malignant clone of CD5+ B cells. In old mice, clonal expansions of CD5+ B cells are a common feature, and these animals frequently develop B-CLL. To investigate whether clonal expansion of CD5+ B cells also occurs in elderly humans, predisposing for the development of B-CLL, we analyzed VH gene rearrangements of CD5+ B cells from blood samples of four healthy, 65-82-years-old volunteers as markers of clonality. CD5+ and CD5-B cells were obtained by cell sorting, CDRIII of rearranged VH genes were amplified by polymerase chain reaction, and fragment length analysis was performed. All samples demonstrated a polyclonal pattern of VH gene length distribution. In addition, VH gene rearrangements were amplified and sequenced from sorted single cells of two of the donors. No clonally related CD5+ or CD5- B cells were observed. Thus, development of dominant clones of CD5+ peripheral blood B cells is unlikely to be a common trait of elderly individuals.     

Induction of autoimmunity in normal mice by thymectomy and administration of polyclonal B cell activators: association with contrasuppressor function

In order to gain insight into the role of polyclonal B cell activation in the development of autoimmunity, non-autoimmune mice were given chronic injections of polyclonal B cell activators (PBA). In addition, to assess the contribution of T cell regulation of such PBA-induced B cell hyperactivity, the additional effect of postnatal thymectomy was studied. Mice that were postnatally thymectomized and given PBA (LPS +/- poly rI X rC) thrice weekly were found to have elevated levels of IgG and significantly increased serum concentrations of anti-ssDNA. This anti-DNA production was greater than that observed with PBA alone or with thymectomy alone. The entire experiment was repeated with different non-autoimmune mice with the same result. Numbers of proliferating cells in the spleens of the mice in the various groups were analysed by flow cytometry. The number of cells in S+G2+M phases of the cell cycle was significantly increased by PBA or thymectomy alone as well as by the combination. As a result, B cell proliferation was not sufficient to result in maximal anti-ssDNA; an additional T cell defect was required. This was further studied in an in vitro assay for suppressor and contrasuppressor activity. In three separate experiments, mice which were clinically autoimmune were found to have defective suppressor function and the presence of abnormal contrasuppressor activity, whereas non-autoimmune controls had normal suppressor function and no contrasuppressor function. These results indicate that the combination of PBA and thymectomy can most easily induce autoimmunity. The autoimmune state so induced in non-autoimmune strains was associated with a failure of normal suppressor function and the abnormal presence of contrasuppressor function. These results have important implications for spontaneously occurring autoimmune diseases.     

Suppression of the polyclonal B-cell response to lipopolysaccharide by concanavalin-A-treated non-T cells in vitro.

Concanavalin A (Con A), a T-cell mitogen, dose-dependent suppressed the polyclonal antibody response of a purified B-cell population to lipopolysaccharide (LPS) in vitro. This suppression can be attributed to a role of suppressor cells generated in B-cell cultures in response to relatively high doses of Con A. The suppressor cells can be produced in cultures of normal as well as athymic nude spleen cells deprived of plastic-adherent cells and Thy 1.2 positive cells. The suppressing activity of Con-A-treated cells was not abolished by pre-treatment of the cells with anti-Thy 1.2, anti-Lyt 1.1, anti-Lyt 1.2 or anti-Lyt 2.2 serum and complement, and was decreased partially by treatment with anti-Ig serum and complement. Moreover, the suppressing activity was partially decreased by using petri dishes coated with IgG of F(ab')2 fraction of anti-mouse immunoglobulins. Thus, the suppressor activity of Con-A-treated cells was mediated by a non-T cell, possibly a B cell. A role for macrophages was unlikely, but not formally ruled out. The suppressor cells retarded development of polyclonal antibody production by B cells to LPS only when added at the start of culture and was resistant to treatment with anti-mitotic doses of irradiation and mitomycin C. It is possible that the suppressor cells of a B-cell nature play a role in the regulation of excessive B-cell proliferation during antibody responses.     

X-ray enhancement of splenic rosette-forming cells in nonimmune mice.

The formation of rosettes between spleen cells from nonimmune mice and sheep red blood cells (SRBC) has been investigated. The number of splenic rosette-forming cells (RFC) in the intact spleen was very low but greatly increased after sublethal whole body x-irradiation. These cells were inhibited by pretreatment of the cells with AKR anti-C3H- antigen antiserum, in the presence of fresh guinea pig serum, but not with rabbit anti-mouse immunoglobulin antiserum. The splenic RFC also increased after cortisone acetate administration into mice. These results suggest that the RFC are a T cell sub-population having x-ray resistant and cortisone-insensitive characteristics. The x-irradiation of mice in vivo selectively increased the number of splenic RFC in consequence of reduction in the number of cells having non-T cell characteristics. These RFC are presumably antigen-specific sheep red blood cell rosettes, a quite different situation as in the case of human T lymphocyte rosette formation.     

Transglutaminase 2 modulates antigen-specific antibody response by suppressing Blimp-1 and AID expression of B cells in mice

Tansglutaminase 2 (TG2) mediates post-translational modifications of proteins that are involved in a variety of biological processes. Previous reports suggest an involvement of TG2 in adaptive immune responses. However, little has been elucidated in this regard. We explored, in this study, the role of TG2 in humoral immune response to keyhole limpet hemocyanin (KLH) using TG2(-/-) C57BL/6 mice. After primary and secondary immunization with KLH, the serum titer of the antigen-specific antibody was higher in the TG2(-/-) mice than in the wild-type mice. Not only the amount of the specific antibody was increased, but also the affinity of the antibody was estimated as higher in these mice. The TG2(-/-) spleen showed an enhanced germinal center response with higher percentages of GL7(+) germinal center B cells and B220(low) CD138(high) plasma cells. In addition, germinal center B cells from TG2(-/-) mice showed an increased expression of B lymphocyte induced maturation protein-1 (Blimp-1) as well as activation-induced cytidine deaminase (AID). Our results, in sum, indicate a regulatory role of TG2 in humoral immune response to a protein antigen, probably by way of modulating the expression level of proteins related to humoral immune reposes.     

Staphylococcal peptidoglycan: T-cell-dependent mitogen and relatively T-cell-independent polyclonal B-cell activator of human lymphocytes.

Staphylococcal cell wall products have been widely examined as probes for dissection of in vitro human immune responses. Mitogenic and polyclonal B-cell-activating properties have been attributed to intact cell walls or the protein A constituent thereof. We now report that staphylococcal peptidoglycan (PG), the major cell wall constituent, is not only a potent mitogen but also a polyclonal B-cell activator for human peripheral blood mononuclear cells (PBM). PG-induced proliferative responses of human PBM were comparable to that observed in pokeweed mitogen-stimulated cultures. As was true for pokeweed mitogen, PG-induced proliferation required the presence of T-cell help. Cultures of human PBM with PG also resulted in B-cell differentiation as reflected by an increase in numbers of immunoglobulin-secreting cells in stimulated cultures. In contrast to the proliferative response, PG-induced B-cell differentiation was relatively T-cell independent. This point became apparent when B-cell fractions were partially depleted of excessive numbers of monocytes before culture. Also, B-cell proliferation did not appear to be a major prerequisite for PG-induced B-cell differentiation responses. These data indicate that PG is a potent T-cell-dependent mitogen and relatively T-cell-independent polyclonal B-cell activator of human lymphocytes.     

Phenotypic and functional properties of B lymphocytes from aged mice

Phenotypic and functional properties of B lymphocytes from individual young and old mice of different inbred strains were studied. B lymphocyte subpopulations defined by the ratios of the densities of cell surface IgM and IgD were found to be altered with age. However, such alterations in B cell subsets were found only in 30-40% of the old mice. B cell mitogenic responses to anti-mu and anti-Lyb2 antibodies were decreased in a majority of DBA/2 mice. Proliferative responses to LPS and anti-mu were reduced only in a minority of CBA/Ca mice but there was a very good correlation in the responsiveness of the old mice to LPS and anti-mu. The anomalous properties of the individual old mice of these inbred strains may be due to a heterogeneity in the effects of aging or due to environmental influences.     

Effect of levamisole on autologous rosette-forming cells in nude mice.

Levamisole depresses the number of autologous rosette-forming cells (ARFC) in the spleen of nude (congenitally athymic) mice. Intravenous administration of 2.5 mg/kg of levamisole produces maximal depression. This effect appears 15 h after injection and is transient, partially disappearing after 48 hr. Dexamisole is devoid of this depressing activity. Thymopoietin, a thymic hormone, is also shown to lower the level of autologous rosette formation. These results suggest a stereospecific interference of levamisole with the maturation of immature T-cell precursors in a manner resembling the action of thymic hormones.     

Competition of the actions of antigen and polyclonal B-cell activator in the induction and amplification of B-memory cell function.

Using the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) as a polyclonal B-cell activator (PBA) and sheep red blood cells (SRBC) as a T-dependent antigen, the correlation of the actions of PBA and T-dependent antigen on B cells in induction and amplification of immunological memory was studied. B-memory cell function, as judged by anti-SRBC responsiveness in vitro of spleen cells of CPS-K, was amplified by the secondary injection of SRBC into SRBC-primed mice, whereas it was decreased markedly by injection of CPS-K. When CPS-K was injected simultaneously with, or 1 or 2 days before the secondary injection of SRBC, B-memory cell function was also decreased markedly. On the other hand, CPS-K did not inhibit induction of B-memory cell function when injected simultaneouly with the primary injection of SRBC. However, CPS-K inhibited induction of B-memory cell function when injected 3 days before the primary injection of SRBC. The inhibition by CPS-K of amplification of B-memory cell function in response to SRBC when CPS-K was injected simultaneously with the secondary injection of SRBC occurred markedly in mice primed with SRBC 8 days or longer before the secondary injection, whereas it was not detectable in mice primed 3 days before. It is concluded that the CPS-K-mediated signal and the SRBC-mediated signal act competitively on the same subpopulations of B cells in induction and amplification of memory, and that the susceptibility of B cells to the CPS-K-mediated negative signal changes correspondingly with their maturation stage.     

Activation and proliferation signals in mouse B cells. VII. Calcium ionophores are non-mitogenic polyclonal B-cell activators

Calcium ionophores cause polyclonal proliferation of lymphocytes from man, rabbit and pig, but are not mitogenic for mouse T or B lymphocytes. We show here that two Ca2+ ionophores (A23187 and ionomycin) nonetheless activate a substantial proportion of mouse B lymphocytes at concentrations which effectively inhibit DNA synthesis induced by conventional mitogens, such as anti-immunoglobulin antibodies. Activation of B cells was detected by (i) increased expression of Ia antigen after 24 hr culture with ionophores, and (ii) the accelerated onset of DNA synthesis in B cells primed with ionophores for 24 hr, washed and then rechallenged with anti-Ig. Unlike anti-Ig, the ionophores did not induce either the breakdown of inositol phospholipids, or RNA synthesis in B cells. Finally, activation of B cells by ionophores is highly susceptible to inhibition by cyclosporine. These results therefore suggest that elevation of intracellular Ca2+ induced by these ionophores is sufficient to cause B cells to leave Go, but not to enter the G1 phase of the cell cycle. Clearly, additional signals are required for B cells to progress further into cycle and eventually become committed to DNA synthesis.