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IgG-binding factors and polyclonal activation of human B cells
IgG-Binding Factors and Polyclonal Activation of Human B Cells Jean-Pierre Revillard, M.D. Le Thi Bich-Thuy, Ph.D. Laboratoire d'lmnnmologie H@ital E. Herriot Lyon. France The investigation of the regulation of human B-cell differentiation is an area of major interest for basic human immunobiology and for the study of immunologic disorders (28). Various methods have been set up in attempt to dissect the complex mechanisms of Bcell triggering as well as the multiple regulatory factors that control the maturation of B lymphocytes into secreting plasma ceils. Polyclonal B-cell activators (PBA), such as plant lectins or bacterial extracts, have been instrumental in deciphering cellular interactions and in the understanding of activating or suppressing signals that may control B-cell differentiation (4). For instance, distinct subsets of regulatory T cells were shown to either help or suppress PBA-induced B-cell proliferation and maturation. The requirement for helper T cells differs according to the type of PBA used in the assay. For example, with the pokeweed mitogen (PWM), the response is strictly T-dependent, whereas extracts from No. cardia opaca (NDCM) were shown to induce B-cell differentiation in the absence of helper T cells (12). However,
responses to either PBA are regulated by signals generated by other cell types, among which the most extensively investigated have been suppressor T cells and monocytes. By culturing relatively few peripheral blood mononuclear cells, it is now possible to assess subsequent steps of human B-cell maturation. The incorporation of 3H-thymidine into cell nuclei reflects the magnitude of the proliferative response to PBA. Also, with the help of fluorescent antibodies, it is possible to visualize and enumerate B cells synthesizing Ig (clg cells) of various isotypes. Furthermore, the quantitation of lg-secreting cells can be performed by a reverse hemolytic plaque forming cell (PFC) assay. The method initially described by Gronowicz, Coutinho, and Melchers (6) utilizes the capacity of staphylococcus protein A to bind to the Fc fragment of IgG molecules. Sheep erythrocytes are coated with protein A and mixed with Ig-secreting cells in an agarose gel. IgG fraction from a rabbit antihuman polyvalent or isotype-specific serum is added as a developing reagent and PFCs are formed after the addition of complement. This simple and reproducible method is now widely used. Finally, the total amount of Ig secreted in the culture supernatant, after appropriate culture periods, can be measured by RIA or by ELISA. Among the. many regulatory pathways that can be studied by employing human B cells cultured with PBA, those involving immune complexes
Figure 1. Polyclonal activation of B cells by Fc fragments (19.26).
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and F c receptors deserve special attention. Indeed, Fc receptors (FcR), which are expressed on a variety of effector or regulatory cells (23), as well as immune complexes, have been implicated as factors regulating the immune response. The effect of antigenIgG antibody complexes, especially their capacity to activate the complement system and to bind to Fc',/R on lymphocytes or monocytes, can be mimicked to some extent by chemically crosslinked or heat-aggregated IgG. It is shown below that IgG preparations, which are extensively used as immunomodulators in several immune disorders, often contain IgG aggregates that are responsible for major alterations of in vitro human B cell differentiation. Although in vitro models may not reflect the complex interactions that occur in vivo, such models may shed some light on the still poorly understood mode of action of gammaglobulin therapy.
Polyclonal Activation of B L y m p h o c y t e s
by Fc Fragments Fc fragments from human IgGl myeloma were shown by Weigle, Morgan, and Thomann to activate murine and human B cells by inducing their proliferation and maturation into Ig-secreting cells (Fig. I). Comparable activation can be achieved using aggregated Ig (20) or antigen-antibody complexes (21). Such activation requires the cooperation of macrophages and T cells. Fc fragment bound to adherent cell FcR are enzymatically degradated into a Fc peptide of 17 kd. This peptide triggers the proliferation of murine or human B cells in the absence of helper T cells. In addition, it stimulates inducer T ceils bearing the Ly 1 + 2 - 3 phenotype to secrete an interleukin named (Fc)TRF. This factor allows the differentiation of activated B lymphocytes int o Ig-secreting cells. (Fc)TRF has been partially characterized as two moieties of 3 5 - 4 0 kd and 5 8 - 6 0 kd, respectively, and it is distinct from IL-2 (26). The activation requires the Fc part of the antibody since antigen F(ab') 2 complexes are in-
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efficient. The mitogenic peptide bears Fc (19) determinants and acts directly on B cells (19). It is, thus, quite different from IL-I. The possibility of its binding to T-cell Fe receptors or to other structures has not been determined as yet, and its preferential triggering of one Ig isotype, although unlikely, remains to be investigated.
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Suppression of B-Cell Polyclonal A c t i v a t i o n by Aggregated IgG
Figure 2. Aggregated lgG-hlduced suppression mediated by monocytes and PGE,_ (2).
Monocytes and PGE2The addition of commercial preparations of human IgG (used for intramuscular injections) to peripheral blood lymphocyte cultures stimulated with PWM results in an altered proliferative response and in a marked decrease of the number of cells containing IgM, IgG, or IgA (2). Suppression requires the presence of IgG aggregates with intact Fc fragments and this is attributed to the release of prostaglandin PGE 2 from activated monocytes (Fig. 2). Indeed, the addition of indomethacin or antiserum to PGE2 was shown to inhibit this suppression. PGE 2 induces suppressor T cells, each having different membrane markers but all sharing the sensitivity to 2000 rad irradiation and to 24-hr incubation at 37°C. Such suppressor T cells could be detected among peripheral blood lymphocytes in children treated by intramuscular injections of gammaglobulins, up to 4 mo after the last injection (2). It was assumed, therefore, that PGE2 induces suppressor T cells that remain active long after the cessation of PGE2 secretion. Prevention of the induction of suppressor T cells by concomitant administration of indomethacin together with gammaglobulin in vivo might be attempted to further document the mechanisms of this suppression.
T Cells Bearing Fc',/Receptors (Tc) The presence of Fc~/R on a subset of T cells (TG) can be demonstrated by several techniques of which the most extensively used has been the formation of erythrocyte-IgG antibody (EA G) rosettes. Optimum binding of the Fc
© 1983by ElsevierSciencePublishingCo., inc.
part of aggregates or complexed IgG to T cell Fc3'R requires a short incubation at 37°C followed by centrifugation at 4°C (23). Conversely, the binding of IgG to monocytes is readily achieved at 37°C without centrifugation. Using the PWM, Moretta, Mingari, and Moretta (18) showed that positively selected TG cells suppressed B cell differentiation into Ig-containing cells without altering the proliferative response to the mitogen (Fig. 3). The suppression involves the release of soluble suppressor factor, which impairs the helper activity of T cells bearing receptors for IgM (TM) (18). Since only fluorescent antibodies to whole human Ig were used in this study, it is not known whether the three major Ig classes were equally suppressed.
We have investigated the suppressive effect of aggregated IgG on B cell stimulated by PWM or by NDCM. When assessed by 3H-thymidine incorporation, these two PBA act on distinct B cell subsets (1). The binding of aggregated IgG to rig cells was found to suppress the maturation of B cells and this suppression required intact Fc fragments since aggregated F(ab')2 fragments were inefficient. It was also abrogated by in vitro irradiation at 3000 rads or by T-cell depletion (13). The most striking feature of T G cellmediated suppression induced by lgG aggregates is its restriction to the IgG isotype: the number of IgG-containing cells and IgG-secreting cells averages 50% of control levels, whereas that of IgM and IgA cells are not modified
Figure 3. Stimulation of suppressor T6 cells by the bindhlg of EAG complexes hz the PWM model (18).
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Figure 4. Aggregated lgG-htdaced suppression mediated by TG cells: restriction to the lgG class.
(Fig. 4). Such isotype-restricted suppression is demonstrated in cultures of cell suspensions obtained from defibrinated blood and, therefore, containing only a few monocytes. Under these experimental conditions, aggregated lgG do not induce B-cell activation in the absence of mitogen. It may be concluded that in the presence of monocytes, aggregated IgG induce either polyclonal activation or suppression. These effects are not isotype-specific. Conversely, when appropriate conditions for the binding of IgG to T cells are achieved, only suppression can be demonstrated and the maturation into IgG-producing cells appears selectively inhibited.
binding factor, IBF). This factor suppresses the antibody response in vitro as well as polyclonal B-cell activation induced by lipopolysaccharides. IBF quite likely represents the soluble form of Fc~/R or a part of it. It has been partially purified as several glycoproteins containing a major component of 60 kd. Interestingly, this fraction was found to bind to Ia antigens by a possible interaction through the osidic residues (5). Human peripheral blood lymphocytes or polymorphonuclear neutrophils spontaneously release a soluble material that can be purified by affinity chromatography on IgG immunosorbents. This material binds to the Fc part of IgG since it is not absorbed on F(ab')2 fragments. It is recovered only from supernatants of Fc~,Rbearing cells (T- or B-enriched suspensions, neutrophils) but not from that of Fc~/R-negative cell or cell lines (human erythrocytes, T cell line Molt 4, fibrobtasts MRC5) (I6). The material prepared by elution at pH 2.8 is
Suppression by IgG Fc Binding Factor Released from Fc'yR-Bearing Cells Activated rnurine T cells or T-cell hybridoma were shown by Fridman et al. (5) to release a factor that binds to the Fc part of IgG (immunoglobulin-
Figure 5. Selective suppression of lgG-contahlhzg cells and presecretory block produeed by soluble Fc-lgG binding material.
Isotype - restricted suppression
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heterogenous and contains several glycoproteins. Among them, two fractions of 30 kd and 17 kd have the highest binding affinity for Fc IgG (25). When added to lymphocyte cultures stimulated by PWM or NDCM, the Fc'/-binding material suppresses the late stages of B-cell maturation (Fig. 5). Two distinct effects can be demonstrated: a selective decrease of the numbers of IgG-containing cells (15) and a presecretory block (14,17). With either PWM or NDCM, IgGcontaining cell counts are reduced to 50% of control values, whereas those of IgA are not changed. With respect to IgM-containing cells, they are not modified in PWM-stimulated cultures, whereas they are markedly increased in NDCM-stimulated cultures, suggesting a possible block of the switch from IgM- to IgG-containing cells with the latter PBA. Although the molar concentration of the suppressor factor cannot be estimated due to the absence of precise biochemical characterization, it should be noted that suppression is still achieved at a 10 - 4 dilution of the eluate without any measurable absorbance at 280 nm (14). The second effect of Fc IgG-binding material is characterized by a decrease in the number of IgM, IgG, and IgA secreting cells. However, the same material does not impair Ig secretion by fully differentiated B cells. Indeed, this material was not suppressive when added after 6 days of culture or during the first 3 days of culture (17). Finally, the suppression could be induced on allogenic as well as autologous cells and even on murine B cells (16). Because of the heterogeneity of the IgG-Fc binding material, it cannot be excluded that the IgG-restricted suppression and the presecretory block are mediated by two distinct factors sharing the property to bind to Fc IgG but not to F(ab')2 fragments. Whatever the mechanism involved and the nature of the suppressor factor, these experiments clearly indicate that two distinct steps controlled by different regulatory mechanisms can already be defined during the late stages of polyclonal Bcell activation: the first leads to Igcontaining cells and the second to Igsecreting cells.
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This concept may be extended to other isotypes since, in both human and murine myelomas, an expansion of T-cell subsets bearing FcR that match the heavy chain isotype specificity of the myeloma proteins has been reported (9). Recently, Hoover and Lynch demonstrated that the T a cells obtained from the mice bearing the lgA myeloma MOPC 315 tumor specifically inhibited the generation of IgA response in immunized mice (I0). It is tempting to speculate that data collected from a variety of different models will soon be organized into a coherent concept of isotype-restricted regulation. It is already predictable that two classes of regulatory factors will be identified: those acting on the switch of heavy chain gene expression and those modulating the Ig synthesis by B cells already committed to the expression of one isotype.
lg Binding-Factors and Heavy Chain Specific Regulation of B-Cell Maturation Among the above-described models on the regulation of human B cell polyclonal activation, two were characterized by the restriction of the suppressive effect of the IgG class. The first involves the interaction between aggregated IgG and T C cells and the second, the effect of IgG-binding material released from Fc'yR-bearing cells. Therefore, a possible relationship may be hypothesized between Ig heavy chain regulation and FcR of the matching isotype (15). Other examples of a similar relationship may be mentioned. It was shown that T cells bearing receptors for IgA may help preferentially the differentiation of IgA-producing cells in PWM-stimulated cultures (3). So far, the most extensively investigated model of classspecific regulation of antibody production concerns IgE. Ishizaka et al. have shown that T cells bearing the W3/25 antigen released IgE binding-factors (IgE IBF) that control the differentiation of precursors into IgE-synthesizing cells (7,8). Furthermore, these investigators demonstrated that a glycosylation inhibiting factor acts on W3/25 + T cells and prevents them from glycosylating the IgE BF they were synthesizing. Such an IgE BF, therefore, is provided with suppressive activities on the IgE production. The glycosylation-inhibiting factor was shown to have a molecular weight of approximately 16 kd, to come from the Ox8 + T cells in rats, and to be a fragment of lipomodulin (27). Conversely, a glycosylation enhancing factor was shown to enhance the glycosylation of the IgE BF during their biosynthesis by W3/25 + T cells and the resulting IgE BF is an IgE production potentiating factor. The glycosylation-enhancing factor is produced by W3/25 + Fc~/R + T cells, has a molecular weight of approximately 25 kd, and is different from IL-2 (11). Since T-cell receptors for Fc IgE may be induced by in vitro incubation with IgE (29), it is conceivable that such receptors may represent one component of an IgE specific regulatory network. •
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-
-
© 1983by ElsevierSciencePublishingCo.. Inc.
Freund's adjuvant. J. lmmunol. 127: 1810-1816. 9. Hoover, G. R. et al. (1981). Occurrence and potential significance of increased numbers of T cells with Fc receptors in myeloma. Immunol. Rev. 56:115-139. 10. Hoover, R. G. and R. G. Lynch. (1983). Isotype-specific suppression of IgA: Suppression of IgA responses in Balb/c mice by Ta cells. J. Immunol. 130:521-523. I 1. Iwata, M. et al. (1983). Modulation of the biologic activities of lgE BF. 11. Physico-chemical properties and cell sources of glycosylation-enhancing factor. J. Immunol. 130:1802-1808. 12. L. Thi Bich-Thuy, R. Ciorbaru, and J. Brochier. (1978). Human B cell differentiation. I. Immunoglobulin synthesis induced by Nocardia mitogen. Eur. J. lmmunol. 8:119-123. 13. L. Thi Bich-Thuy and d. Brochier. (1979). Human B cell differentiation. It. Suppression by T cells of T-dependent and T-independent plasma cell maturation. J. Immunol. I22:i8421848. 14. L. Thi Bich-Thuy el al. (1981). Suppression of the late stages of mitogen induced human B cell differentiation by Fc~/receptors released from polymorphonuclear neutrophils. J. Immunol. 127:1229-1303. 15. L. Thi Bich-Thuy and J. P. Revil. lard. (1982). Selective suppression of human B cell differentiation into IgG producing cells by soluble Fc"/receptors. J. Immunol. 129:150-152. 16. L. Thi Bich-Thuy et al. (1982). The suppressive activity of Fc"/receptors is not related to their T cell origin. Cell. lmmunol. 68:252-260. 17. L. Thi Bich-Thuy and J. P. Revil. lard. (1982). Polyclonal activation of human B iymphocytes: Characterization of the maturation stages susceptible to Fc~/receptors. In Serrou B. et al. (eds.), Current concepts in human immunology and cancer immunomodulation. Elsevier Biomedical Press, Amsterdam, 17:134-142. 18. Moretta, L., M. C. Mingari, and A. Moretta. (1979). Human T cell subpopulations in normal and pathological conditions. Immunol. Rev. 45:163-193. 19. Morgan, E. L. and W. O. Weigle. (1980). Regulation of Fc fragment induced murine spleen cell proliferation. J. Exp. Med. 151:1-11. 20. Morgan, E. L. and W. O. Weigle. (1980). Aggregated human gammaglobulin-induced proliferation and polyclonal activation of murine B iymphocytes. J. Immunol. 125:226-231. 21. Morgan, E. L. and W. O. Welgle. (1981). Polyclonal activation of human B lymphocytes by Fc fragments. I. Characterization of the cellular require-
References 1. Bona, C. et al. (1979). Polyclonal activation of human B lymphocytes by Nocardia water soluble mitogen. Immunot. Rev. 45:69-92. 2. Durandy, A., A. Fischer, and C. Griscelli. (1981). Dysfunctions of pokeweed mitogen stimulated T and B lymphocyte responses induced by gammaglobulin therapy. J. Clin. Invest. 67:867-877. 3. Endoh, M. et al. (1981). IgA specific helper activity of T cells in human peripheral blood. J. Immunol. 127: 2612-2613. 4. Fauci, A. S. (1979). Human B cell function in a polyclonally induced plaque forming cell system. Cell triggering and immunoregulation. Immunol. Rev. 45:93-116. 5. Fridman, W. H. et al. (1981). Characterization and function of T cell Fc receptors. Immunol. Rev. 56:51-88.
6. Gronowicz, E., A. Coutinho, and F. Melchers. (1976). A plaque assay for all cells secreting Ig of a given type or class. Eur. J. Immunol. 6:588-590. 7. Hirashima, M., J. Yodoi, and K. Ishizaka. (1981). Formation of IgEbinding factors by rat T lymphocytes. I[. Mechanisms of selective formation of IgE potentiating factors by treatment with Bordetella pertussis vaccine. J. Immunol. 127:1804-1810. 8. llirashima, M. et ai. (1981). Formation of IgE-binding factors by rat T lymphocytes. III. Mechanisms of selective formation of IgE suppressive factors by treatment with complete .
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ments for Fc fragment-mediated polyclonal antibody secretion by human peripheral blood B lymphocytes. J. Exp. Med. 154:778-790. 22. Morgan, E. L. and W. O. Weigle. (1983). Polyclonal activation of murine B lymphocytes by immune complexes. J. Immunol. 130:1066-1070. 23. Reviilard, J. P. and C. Samarut. (198 I). Interaction between immune complexes and lymphocyte Fc~ receptors. In A. S. Fauci (ed.), Clinics in immunology and allergy. W. B. Saunders Co. Ltd., London, 1:361-381.
24. Samarut, C. and J. P. Revillard. (1980). Active and passive re-expression of Fc"/receptors on human lymphocytes. Eur. J. Immunol. 10:352-358. 25. Samarut, C., L. Thi Bich-Thuy, and J. P. Revillard. (1982). Isolation of Fc~ receptors released by human lymphocytes. In H. Peeters (ed.), Protides of the biological fluids. Pergamon Press Ltd, Oxford, 29:409-414. 26. Thomann, M. L. and W. O. Weigle. (1982). Preliminary chemical and biological characterization of (Fc)TRF: An Fc fragment-inducedT ceil replacing factor. J. Immunol. 128:590-594.
27. Uede, T. et ai. (1983). Modulation of the biologic activities of IgE BF. I. Identification of glycosylation-inhibiting factor as a fragment of Lipomodutin. J. Immunol. 130:878-884. 28. Waidman, T. A. et ai. (1974). Role of suppressor T cells in the pathogenesis of common variable hypogammaglobulinemia. Lancet 11:609-613. 29. Yodoi, J. and K. Ishizaka. (1979). Lymphocytes bearing receptors for lgE. III. Transition of Fc'), R ( - ) cells to Fc"/R(+) cells by IgE. J. Immunol. 123:2004-2010.
Parasite Interaction with Host Immunoregulatory Circuits
which parasites activate suppressor cells. This study deals with the activation of suppressor cells capable of inhibiting cell-mediated immunity (CMI) in the course of infections with facultative or obligate intracellular microorganisms whose control is dependent on CMI. Some of these parasites are Mycobacterium, Brttcella, Sahnonella, Yershlia spp, Francisella tularensis, Treponema pallidum, Listeria monocytogenes, and certain fungi. According to several investigators, the suppressor cells that can be found in infections with these parasites belong to the monocyte-macrophage lineage whereas, according to others, they are T or B lymphocytes (4). It should be emphasized that several factors, such as the strain of the infecting microorganism, the route of infection, or the strain of the animal species, may influence the development, the nature and, thus, the properties of suppressor cells. For instance, no suppressor cells arise in the spleen of mice infected with M. bovis strain BCG in the footpad, in the lung, or subcutaneously, whereas intravenous or intraperitoneal infection may induce suppressor cells, lntraperitoneal injection of heat-killed BCG (as opposed to viable BCG) does not induce suppression. Furthermore, the type of suppressor cell induced may depend on the dose of the infecting microorganism. For instance, in mice, low doses of BCG favor the appearance of suppressor macrophages, whereas larger doses induce suppressor T cells as well (4). Also, the host may play a
crucial role in the expression of suppressor cell activity. It has been reported recently that patients with lepromatous leprosy have a weakened capacity to generate or respond to circulating suppressor cells (4).
Mario Campa, M.D. htstitute of Microbiology University of Pisa, Pisa, Italy Parasites have undergone a slow evolutionary process along with their hosts. This close and prolonged association appears to be the result of the parasite's ability to adapt to the everchanging conditions in the host's tissues, as well as the consequence of its capacity to influence the host's physiologic systems, including the immune system (22). A great deal of literature has shown that microorganisms or microbial constituents may influence the phenotypic and functional maturation of the cells of the immune system (10,15) and that, under appropriate conditions, parasites are able to potentiate the host's immune responses (22). On the other hand, in order to escape the host's antimicrobial activity, parasites have contrived sophisticated mechanisms capable of interfering either with natural immunity factors, or with the specific immune response, or with its effector mechanisms (18). There is increasing evidence that the outcome of an infectious disease is the result of a delicate balance between effector cells and suppressor cells. Although in the last few years there has been an explosion of information relating to these cells, relatively little attention has been focused on elucidating the mechanisms by
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Suppressor Macrophages and Suppressor T Lymphocytes Macrophages capable of preventing T lymphocytes from mounting a normal CMI in infections with M. tuberculosis, M. leprae, BCG, fungi, and other intracellular parasites are "activated" macrophages, even if it is still controversial whether the cells that display a suppressive activity are a subpopulation of activated macrophages (4). Suppressor macrophages may act via the release of soluble factors, such as arginase, cold thymidine, or prostaglandins (25). Among these factors, however, a suppressive role has been firmly established only for prostaglandins, which are capable of inhibiting lymphocyte proliferation as weII as secretion of mediators by T cells (14,25). Very recently, prostaglandins have been reported to act as an important regulator of lymphocyte traffic by retaining circulating lymphocytes at the site of antigen deposition (16). Thus, prostaglandin-mediated disturbance of lymphocyte circulation might represent an additional mechanism by which suppressor macrophages interfere with the host's CMI, thereby, limiting the expression of the specific subset of T cells at the periphery. It still remains to be proved, however, that the altered lymphocyte circulation found in sev-
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responses to either PBA are regulated by signals generated by other cell types, among which the most extensively investigated have been suppressor T cells and monocytes. By culturing relatively few peripheral blood mononuclear cells, it is now possible to assess subsequent steps of human B-cell maturation. The incorporation of 3H-thymidine into cell nuclei reflects the magnitude of the proliferative response to PBA. Also, with the help of fluorescent antibodies, it is possible to visualize and enumerate B cells synthesizing Ig (clg cells) of various isotypes. Furthermore, the quantitation of lg-secreting cells can be performed by a reverse hemolytic plaque forming cell (PFC) assay. The method initially described by Gronowicz, Coutinho, and Melchers (6) utilizes the capacity of staphylococcus protein A to bind to the Fc fragment of IgG molecules. Sheep erythrocytes are coated with protein A and mixed with Ig-secreting cells in an agarose gel. IgG fraction from a rabbit antihuman polyvalent or isotype-specific serum is added as a developing reagent and PFCs are formed after the addition of complement. This simple and reproducible method is now widely used. Finally, the total amount of Ig secreted in the culture supernatant, after appropriate culture periods, can be measured by RIA or by ELISA. Among the. many regulatory pathways that can be studied by employing human B cells cultured with PBA, those involving immune complexes
Figure 1. Polyclonal activation of B cells by Fc fragments (19.26).
Fc ( IgG1 ) Agg -IgG
(~---~l-~-~-~i
s8-6o Ka
and F c receptors deserve special attention. Indeed, Fc receptors (FcR), which are expressed on a variety of effector or regulatory cells (23), as well as immune complexes, have been implicated as factors regulating the immune response. The effect of antigenIgG antibody complexes, especially their capacity to activate the complement system and to bind to Fc',/R on lymphocytes or monocytes, can be mimicked to some extent by chemically crosslinked or heat-aggregated IgG. It is shown below that IgG preparations, which are extensively used as immunomodulators in several immune disorders, often contain IgG aggregates that are responsible for major alterations of in vitro human B cell differentiation. Although in vitro models may not reflect the complex interactions that occur in vivo, such models may shed some light on the still poorly understood mode of action of gammaglobulin therapy.
Polyclonal Activation of B L y m p h o c y t e s
by Fc Fragments Fc fragments from human IgGl myeloma were shown by Weigle, Morgan, and Thomann to activate murine and human B cells by inducing their proliferation and maturation into Ig-secreting cells (Fig. I). Comparable activation can be achieved using aggregated Ig (20) or antigen-antibody complexes (21). Such activation requires the cooperation of macrophages and T cells. Fc fragment bound to adherent cell FcR are enzymatically degradated into a Fc peptide of 17 kd. This peptide triggers the proliferation of murine or human B cells in the absence of helper T cells. In addition, it stimulates inducer T ceils bearing the Ly 1 + 2 - 3 phenotype to secrete an interleukin named (Fc)TRF. This factor allows the differentiation of activated B lymphocytes int o Ig-secreting cells. (Fc)TRF has been partially characterized as two moieties of 3 5 - 4 0 kd and 5 8 - 6 0 kd, respectively, and it is distinct from IL-2 (26). The activation requires the Fc part of the antibody since antigen F(ab') 2 complexes are in-
1
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efficient. The mitogenic peptide bears Fc (19) determinants and acts directly on B cells (19). It is, thus, quite different from IL-I. The possibility of its binding to T-cell Fe receptors or to other structures has not been determined as yet, and its preferential triggering of one Ig isotype, although unlikely, remains to be investigated.
/
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Suppression of B-Cell Polyclonal A c t i v a t i o n by Aggregated IgG
Figure 2. Aggregated lgG-hlduced suppression mediated by monocytes and PGE,_ (2).
Monocytes and PGE2The addition of commercial preparations of human IgG (used for intramuscular injections) to peripheral blood lymphocyte cultures stimulated with PWM results in an altered proliferative response and in a marked decrease of the number of cells containing IgM, IgG, or IgA (2). Suppression requires the presence of IgG aggregates with intact Fc fragments and this is attributed to the release of prostaglandin PGE 2 from activated monocytes (Fig. 2). Indeed, the addition of indomethacin or antiserum to PGE2 was shown to inhibit this suppression. PGE 2 induces suppressor T cells, each having different membrane markers but all sharing the sensitivity to 2000 rad irradiation and to 24-hr incubation at 37°C. Such suppressor T cells could be detected among peripheral blood lymphocytes in children treated by intramuscular injections of gammaglobulins, up to 4 mo after the last injection (2). It was assumed, therefore, that PGE2 induces suppressor T cells that remain active long after the cessation of PGE2 secretion. Prevention of the induction of suppressor T cells by concomitant administration of indomethacin together with gammaglobulin in vivo might be attempted to further document the mechanisms of this suppression.
T Cells Bearing Fc',/Receptors (Tc) The presence of Fc~/R on a subset of T cells (TG) can be demonstrated by several techniques of which the most extensively used has been the formation of erythrocyte-IgG antibody (EA G) rosettes. Optimum binding of the Fc
© 1983by ElsevierSciencePublishingCo., inc.
part of aggregates or complexed IgG to T cell Fc3'R requires a short incubation at 37°C followed by centrifugation at 4°C (23). Conversely, the binding of IgG to monocytes is readily achieved at 37°C without centrifugation. Using the PWM, Moretta, Mingari, and Moretta (18) showed that positively selected TG cells suppressed B cell differentiation into Ig-containing cells without altering the proliferative response to the mitogen (Fig. 3). The suppression involves the release of soluble suppressor factor, which impairs the helper activity of T cells bearing receptors for IgM (TM) (18). Since only fluorescent antibodies to whole human Ig were used in this study, it is not known whether the three major Ig classes were equally suppressed.
We have investigated the suppressive effect of aggregated IgG on B cell stimulated by PWM or by NDCM. When assessed by 3H-thymidine incorporation, these two PBA act on distinct B cell subsets (1). The binding of aggregated IgG to rig cells was found to suppress the maturation of B cells and this suppression required intact Fc fragments since aggregated F(ab')2 fragments were inefficient. It was also abrogated by in vitro irradiation at 3000 rads or by T-cell depletion (13). The most striking feature of T G cellmediated suppression induced by lgG aggregates is its restriction to the IgG isotype: the number of IgG-containing cells and IgG-secreting cells averages 50% of control levels, whereas that of IgM and IgA cells are not modified
Figure 3. Stimulation of suppressor T6 cells by the bindhlg of EAG complexes hz the PWM model (18).
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Figure 4. Aggregated lgG-htdaced suppression mediated by TG cells: restriction to the lgG class.
(Fig. 4). Such isotype-restricted suppression is demonstrated in cultures of cell suspensions obtained from defibrinated blood and, therefore, containing only a few monocytes. Under these experimental conditions, aggregated lgG do not induce B-cell activation in the absence of mitogen. It may be concluded that in the presence of monocytes, aggregated IgG induce either polyclonal activation or suppression. These effects are not isotype-specific. Conversely, when appropriate conditions for the binding of IgG to T cells are achieved, only suppression can be demonstrated and the maturation into IgG-producing cells appears selectively inhibited.
binding factor, IBF). This factor suppresses the antibody response in vitro as well as polyclonal B-cell activation induced by lipopolysaccharides. IBF quite likely represents the soluble form of Fc~/R or a part of it. It has been partially purified as several glycoproteins containing a major component of 60 kd. Interestingly, this fraction was found to bind to Ia antigens by a possible interaction through the osidic residues (5). Human peripheral blood lymphocytes or polymorphonuclear neutrophils spontaneously release a soluble material that can be purified by affinity chromatography on IgG immunosorbents. This material binds to the Fc part of IgG since it is not absorbed on F(ab')2 fragments. It is recovered only from supernatants of Fc~,Rbearing cells (T- or B-enriched suspensions, neutrophils) but not from that of Fc~/R-negative cell or cell lines (human erythrocytes, T cell line Molt 4, fibrobtasts MRC5) (I6). The material prepared by elution at pH 2.8 is
Suppression by IgG Fc Binding Factor Released from Fc'yR-Bearing Cells Activated rnurine T cells or T-cell hybridoma were shown by Fridman et al. (5) to release a factor that binds to the Fc part of IgG (immunoglobulin-
Figure 5. Selective suppression of lgG-contahlhzg cells and presecretory block produeed by soluble Fc-lgG binding material.
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heterogenous and contains several glycoproteins. Among them, two fractions of 30 kd and 17 kd have the highest binding affinity for Fc IgG (25). When added to lymphocyte cultures stimulated by PWM or NDCM, the Fc'/-binding material suppresses the late stages of B-cell maturation (Fig. 5). Two distinct effects can be demonstrated: a selective decrease of the numbers of IgG-containing cells (15) and a presecretory block (14,17). With either PWM or NDCM, IgGcontaining cell counts are reduced to 50% of control values, whereas those of IgA are not changed. With respect to IgM-containing cells, they are not modified in PWM-stimulated cultures, whereas they are markedly increased in NDCM-stimulated cultures, suggesting a possible block of the switch from IgM- to IgG-containing cells with the latter PBA. Although the molar concentration of the suppressor factor cannot be estimated due to the absence of precise biochemical characterization, it should be noted that suppression is still achieved at a 10 - 4 dilution of the eluate without any measurable absorbance at 280 nm (14). The second effect of Fc IgG-binding material is characterized by a decrease in the number of IgM, IgG, and IgA secreting cells. However, the same material does not impair Ig secretion by fully differentiated B cells. Indeed, this material was not suppressive when added after 6 days of culture or during the first 3 days of culture (17). Finally, the suppression could be induced on allogenic as well as autologous cells and even on murine B cells (16). Because of the heterogeneity of the IgG-Fc binding material, it cannot be excluded that the IgG-restricted suppression and the presecretory block are mediated by two distinct factors sharing the property to bind to Fc IgG but not to F(ab')2 fragments. Whatever the mechanism involved and the nature of the suppressor factor, these experiments clearly indicate that two distinct steps controlled by different regulatory mechanisms can already be defined during the late stages of polyclonal Bcell activation: the first leads to Igcontaining cells and the second to Igsecreting cells.
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This concept may be extended to other isotypes since, in both human and murine myelomas, an expansion of T-cell subsets bearing FcR that match the heavy chain isotype specificity of the myeloma proteins has been reported (9). Recently, Hoover and Lynch demonstrated that the T a cells obtained from the mice bearing the lgA myeloma MOPC 315 tumor specifically inhibited the generation of IgA response in immunized mice (I0). It is tempting to speculate that data collected from a variety of different models will soon be organized into a coherent concept of isotype-restricted regulation. It is already predictable that two classes of regulatory factors will be identified: those acting on the switch of heavy chain gene expression and those modulating the Ig synthesis by B cells already committed to the expression of one isotype.
lg Binding-Factors and Heavy Chain Specific Regulation of B-Cell Maturation Among the above-described models on the regulation of human B cell polyclonal activation, two were characterized by the restriction of the suppressive effect of the IgG class. The first involves the interaction between aggregated IgG and T C cells and the second, the effect of IgG-binding material released from Fc'yR-bearing cells. Therefore, a possible relationship may be hypothesized between Ig heavy chain regulation and FcR of the matching isotype (15). Other examples of a similar relationship may be mentioned. It was shown that T cells bearing receptors for IgA may help preferentially the differentiation of IgA-producing cells in PWM-stimulated cultures (3). So far, the most extensively investigated model of classspecific regulation of antibody production concerns IgE. Ishizaka et al. have shown that T cells bearing the W3/25 antigen released IgE binding-factors (IgE IBF) that control the differentiation of precursors into IgE-synthesizing cells (7,8). Furthermore, these investigators demonstrated that a glycosylation inhibiting factor acts on W3/25 + T cells and prevents them from glycosylating the IgE BF they were synthesizing. Such an IgE BF, therefore, is provided with suppressive activities on the IgE production. The glycosylation-inhibiting factor was shown to have a molecular weight of approximately 16 kd, to come from the Ox8 + T cells in rats, and to be a fragment of lipomodulin (27). Conversely, a glycosylation enhancing factor was shown to enhance the glycosylation of the IgE BF during their biosynthesis by W3/25 + T cells and the resulting IgE BF is an IgE production potentiating factor. The glycosylation-enhancing factor is produced by W3/25 + Fc~/R + T cells, has a molecular weight of approximately 25 kd, and is different from IL-2 (11). Since T-cell receptors for Fc IgE may be induced by in vitro incubation with IgE (29), it is conceivable that such receptors may represent one component of an IgE specific regulatory network. •
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Freund's adjuvant. J. lmmunol. 127: 1810-1816. 9. Hoover, G. R. et al. (1981). Occurrence and potential significance of increased numbers of T cells with Fc receptors in myeloma. Immunol. Rev. 56:115-139. 10. Hoover, R. G. and R. G. Lynch. (1983). Isotype-specific suppression of IgA: Suppression of IgA responses in Balb/c mice by Ta cells. J. Immunol. 130:521-523. I 1. Iwata, M. et al. (1983). Modulation of the biologic activities of lgE BF. 11. Physico-chemical properties and cell sources of glycosylation-enhancing factor. J. Immunol. 130:1802-1808. 12. L. Thi Bich-Thuy, R. Ciorbaru, and J. Brochier. (1978). Human B cell differentiation. I. Immunoglobulin synthesis induced by Nocardia mitogen. Eur. J. lmmunol. 8:119-123. 13. L. Thi Bich-Thuy and d. Brochier. (1979). Human B cell differentiation. It. Suppression by T cells of T-dependent and T-independent plasma cell maturation. J. Immunol. I22:i8421848. 14. L. Thi Bich-Thuy el al. (1981). Suppression of the late stages of mitogen induced human B cell differentiation by Fc~/receptors released from polymorphonuclear neutrophils. J. Immunol. 127:1229-1303. 15. L. Thi Bich-Thuy and J. P. Revil. lard. (1982). Selective suppression of human B cell differentiation into IgG producing cells by soluble Fc"/receptors. J. Immunol. 129:150-152. 16. L. Thi Bich-Thuy et al. (1982). The suppressive activity of Fc"/receptors is not related to their T cell origin. Cell. lmmunol. 68:252-260. 17. L. Thi Bich-Thuy and J. P. Revil. lard. (1982). Polyclonal activation of human B iymphocytes: Characterization of the maturation stages susceptible to Fc~/receptors. In Serrou B. et al. (eds.), Current concepts in human immunology and cancer immunomodulation. Elsevier Biomedical Press, Amsterdam, 17:134-142. 18. Moretta, L., M. C. Mingari, and A. Moretta. (1979). Human T cell subpopulations in normal and pathological conditions. Immunol. Rev. 45:163-193. 19. Morgan, E. L. and W. O. Weigle. (1980). Regulation of Fc fragment induced murine spleen cell proliferation. J. Exp. Med. 151:1-11. 20. Morgan, E. L. and W. O. Weigle. (1980). Aggregated human gammaglobulin-induced proliferation and polyclonal activation of murine B iymphocytes. J. Immunol. 125:226-231. 21. Morgan, E. L. and W. O. Welgle. (1981). Polyclonal activation of human B lymphocytes by Fc fragments. I. Characterization of the cellular require-
References 1. Bona, C. et al. (1979). Polyclonal activation of human B lymphocytes by Nocardia water soluble mitogen. Immunot. Rev. 45:69-92. 2. Durandy, A., A. Fischer, and C. Griscelli. (1981). Dysfunctions of pokeweed mitogen stimulated T and B lymphocyte responses induced by gammaglobulin therapy. J. Clin. Invest. 67:867-877. 3. Endoh, M. et al. (1981). IgA specific helper activity of T cells in human peripheral blood. J. Immunol. 127: 2612-2613. 4. Fauci, A. S. (1979). Human B cell function in a polyclonally induced plaque forming cell system. Cell triggering and immunoregulation. Immunol. Rev. 45:93-116. 5. Fridman, W. H. et al. (1981). Characterization and function of T cell Fc receptors. Immunol. Rev. 56:51-88.
6. Gronowicz, E., A. Coutinho, and F. Melchers. (1976). A plaque assay for all cells secreting Ig of a given type or class. Eur. J. Immunol. 6:588-590. 7. Hirashima, M., J. Yodoi, and K. Ishizaka. (1981). Formation of IgEbinding factors by rat T lymphocytes. I[. Mechanisms of selective formation of IgE potentiating factors by treatment with Bordetella pertussis vaccine. J. Immunol. 127:1804-1810. 8. llirashima, M. et ai. (1981). Formation of IgE-binding factors by rat T lymphocytes. III. Mechanisms of selective formation of IgE suppressive factors by treatment with complete .
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ments for Fc fragment-mediated polyclonal antibody secretion by human peripheral blood B lymphocytes. J. Exp. Med. 154:778-790. 22. Morgan, E. L. and W. O. Weigle. (1983). Polyclonal activation of murine B lymphocytes by immune complexes. J. Immunol. 130:1066-1070. 23. Reviilard, J. P. and C. Samarut. (198 I). Interaction between immune complexes and lymphocyte Fc~ receptors. In A. S. Fauci (ed.), Clinics in immunology and allergy. W. B. Saunders Co. Ltd., London, 1:361-381.
24. Samarut, C. and J. P. Revillard. (1980). Active and passive re-expression of Fc"/receptors on human lymphocytes. Eur. J. Immunol. 10:352-358. 25. Samarut, C., L. Thi Bich-Thuy, and J. P. Revillard. (1982). Isolation of Fc~ receptors released by human lymphocytes. In H. Peeters (ed.), Protides of the biological fluids. Pergamon Press Ltd, Oxford, 29:409-414. 26. Thomann, M. L. and W. O. Weigle. (1982). Preliminary chemical and biological characterization of (Fc)TRF: An Fc fragment-inducedT ceil replacing factor. J. Immunol. 128:590-594.
27. Uede, T. et ai. (1983). Modulation of the biologic activities of IgE BF. I. Identification of glycosylation-inhibiting factor as a fragment of Lipomodutin. J. Immunol. 130:878-884. 28. Waidman, T. A. et ai. (1974). Role of suppressor T cells in the pathogenesis of common variable hypogammaglobulinemia. Lancet 11:609-613. 29. Yodoi, J. and K. Ishizaka. (1979). Lymphocytes bearing receptors for lgE. III. Transition of Fc'), R ( - ) cells to Fc"/R(+) cells by IgE. J. Immunol. 123:2004-2010.
Parasite Interaction with Host Immunoregulatory Circuits
which parasites activate suppressor cells. This study deals with the activation of suppressor cells capable of inhibiting cell-mediated immunity (CMI) in the course of infections with facultative or obligate intracellular microorganisms whose control is dependent on CMI. Some of these parasites are Mycobacterium, Brttcella, Sahnonella, Yershlia spp, Francisella tularensis, Treponema pallidum, Listeria monocytogenes, and certain fungi. According to several investigators, the suppressor cells that can be found in infections with these parasites belong to the monocyte-macrophage lineage whereas, according to others, they are T or B lymphocytes (4). It should be emphasized that several factors, such as the strain of the infecting microorganism, the route of infection, or the strain of the animal species, may influence the development, the nature and, thus, the properties of suppressor cells. For instance, no suppressor cells arise in the spleen of mice infected with M. bovis strain BCG in the footpad, in the lung, or subcutaneously, whereas intravenous or intraperitoneal infection may induce suppressor cells, lntraperitoneal injection of heat-killed BCG (as opposed to viable BCG) does not induce suppression. Furthermore, the type of suppressor cell induced may depend on the dose of the infecting microorganism. For instance, in mice, low doses of BCG favor the appearance of suppressor macrophages, whereas larger doses induce suppressor T cells as well (4). Also, the host may play a
crucial role in the expression of suppressor cell activity. It has been reported recently that patients with lepromatous leprosy have a weakened capacity to generate or respond to circulating suppressor cells (4).
Mario Campa, M.D. htstitute of Microbiology University of Pisa, Pisa, Italy Parasites have undergone a slow evolutionary process along with their hosts. This close and prolonged association appears to be the result of the parasite's ability to adapt to the everchanging conditions in the host's tissues, as well as the consequence of its capacity to influence the host's physiologic systems, including the immune system (22). A great deal of literature has shown that microorganisms or microbial constituents may influence the phenotypic and functional maturation of the cells of the immune system (10,15) and that, under appropriate conditions, parasites are able to potentiate the host's immune responses (22). On the other hand, in order to escape the host's antimicrobial activity, parasites have contrived sophisticated mechanisms capable of interfering either with natural immunity factors, or with the specific immune response, or with its effector mechanisms (18). There is increasing evidence that the outcome of an infectious disease is the result of a delicate balance between effector cells and suppressor cells. Although in the last few years there has been an explosion of information relating to these cells, relatively little attention has been focused on elucidating the mechanisms by
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Suppressor Macrophages and Suppressor T Lymphocytes Macrophages capable of preventing T lymphocytes from mounting a normal CMI in infections with M. tuberculosis, M. leprae, BCG, fungi, and other intracellular parasites are "activated" macrophages, even if it is still controversial whether the cells that display a suppressive activity are a subpopulation of activated macrophages (4). Suppressor macrophages may act via the release of soluble factors, such as arginase, cold thymidine, or prostaglandins (25). Among these factors, however, a suppressive role has been firmly established only for prostaglandins, which are capable of inhibiting lymphocyte proliferation as weII as secretion of mediators by T cells (14,25). Very recently, prostaglandins have been reported to act as an important regulator of lymphocyte traffic by retaining circulating lymphocytes at the site of antigen deposition (16). Thus, prostaglandin-mediated disturbance of lymphocyte circulation might represent an additional mechanism by which suppressor macrophages interfere with the host's CMI, thereby, limiting the expression of the specific subset of T cells at the periphery. It still remains to be proved, however, that the altered lymphocyte circulation found in sev-
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