Antibody formation in mouse bone marrow. VII. Evidence against the migration of plaque-forming cells as the underlying cause for bone marrow plaque-forming cell activity: a study with parabiotic mice

After intravenous immunization of mice with Escherichia coli lipopolysaccharide (LPS) or sheep red blood cells (SRBC), the bone marrow can contain large numbers of plaque-forming cells (PFC). By means of parabiosis, it was studied whether or not this appearance of PFC in the bone marrow might be due to a migration of such cells from peripheral lymphoid organs into the marrow, as has been suggested in the literature. Using parabionts consisting of nonimmunized mice and mice immunized with LPS, only background numbers of PFC could be demonstrated in the bone marrow of the nonimmunized mice. In similar experiments, with SRBC as antigen, mice showing high anti-SRBC PFC activity in the bone marrow could only provide for minor numbers of anti-SRBC PFC in the bone marrow of affixed normal mice. These results suggest that migration of PFC can not be the main cause for bone marrow PFC activity in the mouse. This provides additional evidence for our view presented in previous papers of this series that the appearance of PFC activity in the bone marrow is dependent on local maturation of B cells into PFC rather than on immigration of PFC.     

Effect of Bacterial Lipopolysaccharides on Anti-Trinitrophenyl Antibody-Producing Cells

The effect of lipopolysaccharide (LPS) on anti-trinitrophenyl (TNP) direct plaque-forming cells (PFC) in the spleen of mice and the affinity of antibodies produced by these PFC were examined. Simultaneous injection of bacterial LPS and TNP-coupled sheep red blood cells(SRBC) induced an obvious increase in anti-TNP PFC numbers and heightened the antibody affinity at cellular levels. The higher the doses of LPS, the greater the effects. Concomitant injection of LPS in TNP-coupled homologous mouse red blood cells (MRBC) also elicited good anti-TNP PFC response and slightly heightened the affinity. Priming with LPS and SRBC together 7 days prior to immunization did not enhance the anti-TNP PFC response and it was difficult to alter the affinity. Preinjection with small amounts of TNP-MRBC or -rabbit red blood cells and LPS simultaneously did not induce any significant increase in anti-TNP PFC secondary response after reimmunization with TNP-SRBC, but obviously heightened the antibody affinity. Injection of LPS simultaneously with the secondary immunization was effective for both the anti-TNP PFC response and the alteration of antibody affinity. These results suggest that LPS affects the control mechanisms of anti-TNP antibody affinity via the non-thymus-derived helper cell function, and the adjuvant action and alteration of antibody affinity induced by LPS are regulated by different mechanisms.     

Effect of bacterial lipopolysaccharides on anti-trinitrophenyl antibody-producing cells. Nonspecific modification of the affinity at the cellular level.

The effect of lipopolysaccharide (LPS) on anti-trinitrophenyl (TNP) direct plaque-forming cells (PFC) in the spleen of mice and the affinity of antibodies produced by these PFC were examined. Simultaneous injection of bacterial LPS and TNP-coupled sheep red blood cells(SRBC) induced an obvious increase in anti-TNP PFC numbers and heightened the antibody affinity at cellular levels. The higher the doses of LPS, the greater the effects. Concomitant injection of LPS in TNP-coupled homologous mouse red blood cells (MRBC) also elicited good anti-TNP PFC response and slightly heightened the affinity. Priming with LPS and SRBC together 7 days prior to immunization did not enhance the anti-TNP PFC response and it was difficult to alter the affinity. Preinjection with small amounts of TNP-MRBC or -rabbit red blood cells and LPS simultaneously did not induce any significant increase in anti-TNP PFC secondary response after reimmunization with TNP-SRBC, but obviously heightened the antibody affinity. Injection of LPS simultaneously with the secondary immunization was effective for both the anti-TNP PFC response and the alteration of antibody affinity. These results suggest that LPS affects the control mechanisms of anti-TNP antibody affinity via the non-thymus-derived helper cell function, and the adjuvant action and alteration of antibody affinity induced by LPS are regulated by different mechanisms.     

Structural and serological studies of lipopolysaccharides of Citrobacter O35 and O38 antigenically related to Salmonella

Abstract Lipopolysaccharide (LPS) was administered into sheep red blood cells (SRBC)-primed mice, and the effect of LPS on SRBC-specific memory cells was investigated. Spleen cells from SRBC-primed mice which were injected with LPS exhibited much lower in vitro secondary plaque-forming cells (PFC) responses to SRBC than those from untreated SRBC-primed mice. The in vitro anti-SRBC response of the spleen cells to LPS was also reduced. The combination experiments of B cells and T cells from SRBC-primed mice which were injected with or without LPS demonstrated that the reduction of immune responses to SRBC after administration of LPS was caused by the defect of SRBC-specific B memory cells, but not T memory cells. B cell type rosette-forming cells (RFC) for SRBC markedly decreased after injection of LPS, while PFC as antibody-forming cells did not increase subsequently. Therefore, the reduction of RFC was not due to their differentiation into PFC. The lymphoid follicles in the spleens from mice injected with LPS were stained positively by in situ nick end labeling specific for fragmented DNA. A large percentage of Ig⁺ spleen cells from SRBC-primed mice which were injected with LPS was also stained positively. The injection of glucocorticoids into SRBC-primed mice induced similar reduction of B memory cells. It was suggested that LPS might induce apoptosis of B memory cells and regulate B cell memory in antigen-nonspecific manner.     

A study of the mechanism of Con A-induced immunosuppression in vivo

The plaque-forming cell (PFC) response to sheep erythrocytes (SRBC) is suppressed in a dose-related manner when concanavalin A (Con A) is administered intravenously to mice prior to or after immunization with antigen. The magnitude of suppression as well as the duration of the Con A effect greatly depends on the concentration of antigen used for immunization. Although profound suppression of the anti-SRBC PFC response is observed in intact mice pretreated with Con A for 4-24 hr, spleen cells from these mice do not exhibit suppressive activity when transferred into normal recipients or when cotransferred with normal spleen cells into irradiated recipients. Moreover, the cells from Con A-treated mice respond as normal spleen cells to SRBC when transferred alone into irradiated hosts. Suppression of the anti-SRBC PFC is only observed when adoptive hosts of cells from Con A-treated mice are also injected with Con A within 48 hr (but not 72 hr) of cell transfer and immunization. This time course of responsiveness to the suppressive effects of Con A is similar to that observed in normal mice and in irradiated recipients of normal spleen cells. The immune response to SRBC is also suppressed in adoptive hosts of normal spleen cells that are pretreated with Con A 4-24 hr prior to irradiation and cell transfer. Although functionally inactive when transferred into adoptive hosts, spleen cells from mice pretreated with Con A for 4-24 hr can suppress a primary antibody response to SRBC in vitro. The suppressive activity, which cannot be detected in the spleens of mice when the interval between pretreatment and assay is longer than 24 hr, is present in a subpopulation that bears the Thy 1.2 and Lyt 2 phenotype. Taken together the results obtained in in vivo and in vitro functional assays suggest that a suppressor cell population is activated following in vivo treatment with Con A, but that the cells rapidly lose their state of activation when removed from a Con A environment. This phenomenon is in all probability responsible for the failure to demonstrate suppressive activity in the spleens of Con A-treated mice using in vivo functional assays.     

Ineffectiveness of Lipopolysaccharide for Preventing the Tolerance Induction in Bone Marrow-Derived Lymphoid Cells with Dinitrophenyl-Poly-l-(Glutamic Acid,Lysine)

The effect of endotoxin or lipopolysaccharide (LPS) on tolerance induction in bone marrow-derived lymphoid cells (B cells) was investigated. Dinitrophenylated amino acid copolymer-l-(glutamic acid, lysine) (DNP-GL) acts as a potent tolerogen on normal and DNP-primed B cells. LPS significantly enhanced the anti-sheep red blood cell plaque-forming cell (anti-SRBC PFC) response that occurred after the immunization with a low dose of SRBC. LPS did not induce the primary anti-DNP PFC response after the injection of DNP-GL, nor did it prevent the tolerance induction in normal and DNP-primed B cells that occurred after the administration of DNP-GL.     

Suppressive action of Candida albicans on the immune response in mice.

This study was carried out to determine whether Candida albicans infection has a suppressive effect on the immune response in mice and, if so, whether the suppressive effect influences the response towards T-dependent or T-independent antigens. ICR mice were injected with SRBC with or without C. albicans, or with bacterial LPS with or without C. albicans. The immune response of the mice towards SRBC or towards the LPS was compared by the assay for PFC, hemagglutination and hemolysis tests. The results showed a decrease in the number of PFC in spleens of mice inoculated with SRBC and C. albicans as compared to mice inoculated with SRBC alone, but no decrease in animals injected with LPS and C. albicans as compared to those immunized with LPS alone. No significant differences in the titers of hemagglutinins and hemolysins in sera of mice inoculated with SRBC or with SRBC and C. albicans were observed. C. albicans infection had no effect at all on the hemagglutinins and hemolysins titers in sera of mice inoculated with LPS. These data indicate that C. albicans affects the early phase of the immune response primarily towards T dependent antigens.     

Murine bone marrow IgA responses to orally administered sheep erythrocytes

Specific immunization protocols have been established for the induction of murine bone marrow IgA responses to the T cell-dependent (TD) antigen sheep red blood cells (SRBC). Systemic immunization, either i.p. or i.v., followed by a second injection, induced splenic IgM and IgG responses and a bone marrow IgM response. No significant IgA responses were observed in either lymphoid tissue compartment. Oral immunization with SRBC by gastric intubation for 2 days, followed 1 wk later by an i.p. injection of SRBC resulted in a splenic IgA plaque-forming cell (PFC) response, but did not elicit a bone marrow IgA response. Repeated daily gastric intubation of SRBC to C3H/HeN and C3H/HeJ mice led to the previously reported pattern of systemic unresponsiveness in C3H/HeN mice and good anamnestic type IgM, IgG, and IgA splenic anti-SRBC PFC responses in the C3H/HeJ strain upon parenteral challenge. Oral administration of SRBC for 14 days to C3H/HeN mice, followed by systemic SRBC challenge, resulted in diminished splenic PFC responses of all isotypes, whereas gastric intubation of SRBC for 28 days led to complete systemic unresponsiveness to antigen in C3H/HeN mice. Interestingly, the repeated oral administration of SRBC resulted in significant bone marrow IgA PFC responses upon i.p. challenge in both C3H/HeN and C3H/HeJ mouse strains. The bone marrow IgA responses were clearly dependent upon chronic oral exposure to SRBC, because gastric intubation with SRBC for 2 consecutive days/wk for 10 wk also induced bone marrow and splenic IgA anti-SRBC PFC responses in C3H/HeN mice. These results suggest that memory B cells reside in the bone marrow of orally immunized mice and can yield anamnestic-type responses to challenge with the inducing antigen. The memory cells may arise in the Peyer's patches of the gut and migrate to the bone marrow. The possibility that the bone marrow is a component of the common mucosal immune system in mammals is suggested by this study.     

Relation between Enhanced Antibody Responses Elicited by Adjuvant Injection and Those Elicited by Secondary Antigenic Stimulation

An attempt was made to determine if there is any common mechanism in the enhanced antibody response caused either by injection of adjuvant, such as bacterial endotoxin (LPS) and complexed polynucleotides, or by secondary antigenic stimulation. LPS inoculated in mice 4 days before injection of sheep red blood cells (SRBC) and polyA:U invalidated the adjuvant effect of polyA:U injected together with SRBC, and the hemolysin plaque-forming cell (PFC) response of such mice was similar to that of the mice which received SRBC alone. When mice primed with SRBC 24 days in advance were injected with LPS and 4 days later re-stimulated with SRBC, their PFC response to the secondary stimulation was suppressed to less than one tenth of the normal secondary PFC response. The suppressive effect of LPS on the secondary antibody response was abolished if the serum collected from mice injected with LPS was given to the primed and LPS-injected mice at the time of the secondary antigenic stimulation. From these results we discussed the possibility that some common mediator might play a role in the enhanced antibody response elicited by either adjuvant injection or secondary injection of antigen.     

Immunocycte stimulation in vitro by nontoxic bacterial lipopolysaccharide derivatives.

Intact lipopolysaccharides (LPS), considered nonspecific enhancers of B cell responses, as well as nontoxic derivatives from Serratia marcescens LPS, were studied with regard to their ability to stimulate in vitro immune responses to a T-dependent antigen, sheep erythrocytes. Intact LPS, at a dose of 10 to 50 microgram, consistently enhanced the in vitro anti-SRBC immune response by normal splenocytes. The LPS also increased the background PFC response to SRBC in nonimmunized cultures. A chemically detoxified preparation derived from LPS (Mex B) had no stimulatory activity in vitro. A completely nontoxic, relatively small m.w., polysaccharide-rich preparation (PS), free of detectable lipid and protein, was stimulatory in vitro and at a dose of 10 microgram resulted in a 40 to 70% enhancement of the anti-SRBC response. The PS also stimulated an enhanced background response to SRBC as well as several other RBC species in nonimmunized cultures. PS had no mitogenic effect in vitro since addition of this bacterial derivative failed to stimulate thymidine incorporation into mouse splenocytes, as occurred with the intact LPS. The use of nontoxic preparations from gram-negative bacterial LPS for dissecting the stimulatory vs antigenic properties of bacterial products provides a model system for determining the role of a mitogenic stimulus in B cell activation.     

Combinations of two synthetic adjuvants: synergistic effects of a surfactant and a polyanion on the humoral immune response

Synergistic effects of two synthetic adjuvants, dimethyldioctadecylammonium bromide (DDA) and dextran sulfate (DXS) on the humoral response to sheep red blood cells (SRBC) were investigated. Mice received intraperitoneal (ip) injections of adjuvant and antigen simultaneously. The number of plaque-forming cells (PFC) in the spleen were determined 5 days later and circulating anti-SRBC antibodies were measured till 16 weeks after immunization. Although combinations of DDA and DXS were very effective in enhancing the PFC response to both moderate (2 X 10(7] and low (2 X 10(6] doses of SRBC, synergy between the adjuvants was only observed at the low dose of SRBC. Optimal augmentation of the primary response to the low antigen dose was evoked by the combination of the highest dose tested of either adjuvant (1 mumol DDA and 1 nmol DXS) resulting in a 560-fold increase of the number of PFC in the spleen as compared to controls. Even combinations of relatively small amounts of both adjuvants were very effective in augmenting the response to SRBC. Mice receiving half the amounts of both adjuvants with 2 X 10(6) SRBC displayed increased numbers of PFC in the spleen at Day 5 as well as increased titers of total anti-SRBC antibodies at Week 1 and Week 2 and 2-mercaptoethanol-resistant antibodies from Week 4 till Week 16 as compared to the calculated sum of responses in mice which received either DDA (0.05 mumol per mouse) or DXS (0.05 nmol per mouse). The mechanism behind the synergy between these adjuvants is discussed and the possibility of discerning adjuvants on their modes of action is suggested.     

Difference in genesis between antigen-induced IgM antibody-forming cells and B-cell mitogen-induced IgM antibody-forming cells in mouse spleen cell cultures.

To determine the mechanisms in the triggering of thymus-independent lymphocytes (B cells) for development into antibody-forming cells (AFC), genesis of IgM AFC elicited polyclonally by nonspecific stimulation with B-cell mitogen, such as nystatin and bacterial lipopolysaccharide, was compared with that of IgM AFC specifically elicited by antigenic stimulation, using mouse spleen cell cultures as an experimental system and sheep erythrocytes (SRBC) as a test antigen. Considering that differentiation and proliferation are necessary cellular events for precursor B cells to develop into AFC, the effect of different antimetabolic agents on the generation of each type of AFC in spleen cell cultures was examined. The generation of anti-SRBC IgM hemolysin plaque-forming cells (PFC) in B-cell mitogen-stimulated spleen cell cultures was found to be less susceptible to X-irradiation or mitomycin C than that in the SRBC-stimulated cultures. These apparently paradoxical results were affiirmed using colcemid as an inhibitor of cell mitosis and hydroxyurea (HU) as an inhibitor of cellular DNA synthesis. Thus, when spleen cell cultures responding to either SRBC or B-cell mitogen were exposed to colcemid or HU during a period from 2 days to 3 days after the stimulation, the exponential generation of anti-SRBC IgM PFC in the cultures responding to SRBC was completely halted, whereas that in the cultures responding to B-cell mitogen was not. Furthermore, N⁶, O^2′ -dibutyryl adenosine 3′, 5′ -cyclic monophosphoric acid was found to halt the exponential generation of antigen-induced anti-SRBC IgM PFC but not that of the B-cell mitogen-induced anti-SRBC IgM PFC. From these results it was suggested that B-cell mitogen might stimulate precursor Bμ cells at a late stage in the differentiative pathway to develop into AFC without cell division, and that antigenic stimulation might stimulate relatively primitive precursor Bμ cells to proliferate and then differentiate into AFC. Based on this idea, mechanisms in the triggering of B-cell activation are discussed.     

Studies on the Maturation of Immune Responsiveness in the Mouse

The ability of early post-natal mice to respond to sheep erythrocytes (SRBC) by formation of plaque-forming cells (PFC) was studied. When 1–2 day old BDF, mice were injected with SRBC, no PFC could be detected in their spleens five days after immunization. However, if animals were given a second injection of antigen two days after the initial immunization, PFC could be detected within five days of the initial injection. Experiments with other heterologous erythrocytes attest to the specificity of the two-injection schedule, and examination of a variety of strains of mice indicate that our findings may be generally applicable to the emerging immune system of the mouse.     

The enhancement of the immune response by pain stimulation in mice. I. The enhancement effect on PFC production via sympathetic nervous system in vivo and in vitro

Effects of catecholamines and osmotical and physical stimuli on the induction of anti-sheep red blood cells (SRBC) plaque-forming cells (PFC) were investigated in (C57BL/6 X BALB/c)F1 mice in vivo and in vitro. The anti-SRBC PFC from mice immunized with 5 X 10(7) SRBC was markedly increased by daily s.c. injections of epinephrine. The enhancement of PFC by epinephrine was completely blocked by preadministration with propranolol and hexamethonium, but not with phentolamine. The PFC was increased by osmotic and physical stimuli given once a day for 4 days after immunization with SRBC. The enhancement of PFC by these stimuli was completely blocked by preadministration with propranolol and hexamethonium. The enhancement of PFC by physical stimuli was observed in nonimmunized mice when spleen cells from stimulated mice were cultured with SRBC in vitro. In normal mice, the enhancement of PFC was observed 2 hr after one physical stimulation. However, spleen cells from mice given two physical stimuli did not show the enhancement of PFC after treatment with anti-Thy-1.2 antibody and complement, nor after removal of nonadherent cells. Next, the serum obtained from mice 30 to 60 min after a physical stimulation enhanced PFC of normal mice spleen cells in vitro, but the enhancement was abolished by the addition of propranolol. The enhancement of anti-SRBC PFC by s.c. injection of epinephrine suggested that the autonomic nervous system, especially the sympathetic nervous system, was activated by a local stimulus effect of the injection. This enhancement of anti-SRBC PFC appear to be due to the activation of antigen non-specific helper T lymphocytes by the beta-actin of endogenous catecholamines from the adrenal gland.     

Role of T Cells in the Mitogen-Induced Proliferation and Polyclonal Antibody Response of Murine B Cells

The effect of thymus-derived lymphocytes (T cells) on the responsiveness of bone marrow-derived lymphocytes (B cells) to bacterial lipopolysaccharide (LPS) was determined in in vitro experiments. Radiation resistant splenic T cells obtained from euthymic nu/+ mice increased the number of proliferating cells in the cultures of splenic B cells from athymic nu/nu mice even in a nonstimulated state. The radiation resistant T cells augmented significantly the responsiveness of B cells to LPS, as determined by an increase in proliferating cells and polyclonally induced anti-sheep erythrocyte (SRBC) IgM hemolysin plaque-forming cells (PFC). Addition of the T cells to B cell cultures not only augmented the responsiveness of B cells to suboptimal doses of LPS but also enabled B cells to respond to supraoptimal doses of LPS. As is well documented, the radiation resistant T cells were unable to induce the generation of anti-SRBC PFC in B cell cultures, unless the cultures were simultaneously stimulated with SRBC. Colcemid, a specific inhibitor of cell mitosis, blocked almost completely the exponential generation of anti-SRBC PFC in B cell cultures responding to SRBC with the aid of radiation resistant T cells. In contrast, colcemid did not affect the exponential generation of anti-SRBC PFC of a polyclonal nature in B cell cultures responding to LPS, either in the presence or absence of radiation resistant T cells.     

IgM-mediated enhancement of in vivo anti-sheep erythrocyte antibody responses: isotype analysis of the enhanced responses

The direct splenic anti-sheep erythrocyte (anti-SRBC) responses as well as the serum IgG1, IgG2a, IgG2b, and IgG3 anti-SRBC responses of CBA/CaJ mice were monitored 4-35 days after immunization with: (1) a suboptimal dose of SRBC, (2) a suboptimal dose of SRBC plus monoclonal IgM anti-SRBC, or (3) a high dose of SRBC. The direct plaque-forming cell (PFC) responses of mice in treatment group 2 were significantly higher than those in group 1 but similar to the responses in group 3. The serum anti-SRBC antibody responses of all IgG subclasses were significantly enhanced by IgM anti-SRBC and were generally even higher than the responses obtained with high doses of SRBC. The relative proportions of each serum IgG subclass were similar in all three groups. These data suggest that the enhancement of suboptimal anti-SRBC antibody responses by IgM anti-SRBC extends through IgM and all of the IgG subclasses and, further, that the isotype profile in antibody-enhanced responses is similar to that obtained with high doses of SRBC.     

Antigen-reactive cell opsonization: a mechanism of antibody-mediated immune suppression.

Antisera against sheep red blood cells (SRBC) specifically suppressed the direct anti-SRBC plaque-forming cell (PFC) response in mice when passively administered with the antigen. The suppressive activity of mouse and rabbit anti-SRBC sera was found to correlate with anti-SRBC opsonic activity but not with hemagglutination or hemolysin titers. Macrophage depletion of mice, using carrageenan treatment, inhibited antibody-mediated immune suppression. When mice immunized with SRBC were given ¹²⁵I-labeled Udr, radiolabeled spleen lymphocytes were obtained which specifically formed rosettes with SRBC. These radiolabeled antigen-reactive cells (1ARC) were specifically opsonized in mice treated with antigen-antibody complexes but not in mice treated with antigen or antibody alone. These results suggest that antibody-mediated immune suppression may be due to specific opsonization (and subsequent destruction) of ARC in the presence of antigen-antibody complexes.     

IgA responses in xid mice: oral antigen primes Peyer's patch cells for in vitro immune responses and secretory antibody production

Because the gut-associated lymphoreticular tissue (GALT), e.g., Peyer's patches (PP), of X-linked immunodeficient (xid) mice possesses a subpopulation of mature B cells, we have characterized the ability of xid mice to respond to the thymic-dependent antigen sheep erythrocytes (SRBC) given by the oral route. Gastric intubation of SRBC to xid (CBA/N X DBA/2) F1 male or CBA/N mice, followed by the in vitro culture of dissociated PP cells with SRBC, resulted in IgM, IgG1, IgG2, and high IgA anti-SRBC plaque-forming cell (PFC) responses. The addition of unprimed PP but not splenic T cells to splenic xid B cell cultures resulted in IgM anti-SRBC PFC responses, suggesting the importance of GALT T cells for support of the immune responses to SRBC by splenic B cells from xid mice. Furthermore, purified PP T cells from SRBC orally primed xid mice supported in vitro IgA anti-SRBC PFC responses in B cell cultures from either the PP or the spleens of nonprimed xid mice. Higher IgA responses, however, occurred in PP, when compared with splenic B cell cultures. Additional evidence that the GALT of xid mice contains functional IgA precursor cells was provided by the finding that cloned H-2k PP T helper cells (PP Th A) supported IgA responses in PP B cell cultures derived from (CBA/N X C3H/HeN) F1 male (xid) mice. On the other hand, splenic B cells from these xid mice, in the presence of PP Th A cells, did not support in vitro responses. These results suggest that unique subpopulations of T cells occur in the GALT of xid and normal mice; one T cell subpopulation may induce immature B cells to become precursor IgA cells in the PP. A separate GALT T cell subpopulation, e.g., isotype-specific helper T cells, effectively collaborates with mature IgA B cells for the induction of IgA responses to orally administered antigen. When xid mice were gastric intubated with SRBC, followed by i.p. injection of SRBC, good splenic IgA anti-SRBC PFC responses were seen. Salivary and serum IgA antibodies were also detected in these xid mice. Nevertheless, the magnitude of the anti-SRBC response in xid mice was lower than that seen in similarly treated normal mice. These studies indicate that the GALT of both xid and normal mice possess unique populations of T cells that support in vitro responses in xid B cell cultures from either the spleen or the PP, which direct the mature B cell populations present toward IgA isotype-specific responses.(ABSTRACT TRUNCATED AT 400 WORDS)     

Measurement and comparison of the proliferative and antibody response of neonatal, immature and adult murine spleen cells to T-dependent and T-independent antigens.

Mouse spleen cell antigenic responses to the thymic-dependent antigen sheep red blood cells (SRBC), and the thymic-independent antigens, E. Coli lipopolysaccharide (LPS) and pneumococcal polysaccharides Type I and II (SI, SII) were studied as as a function of age, employing both in vitro spleen cell stimulation and plaque-forming cell (PFC) assay systems. Primary spleen cell proliferative and PFC responses to SRBC, were either absent or meager in comparison to adult (8–12 weeks) values for the first 3 weeks of life. Thereafter responses rose achieving adult values between 4 and 8 weeks of age. The inability of young mice to respond to SRBC was not because of a different immunizing dose requirement for SRBC, since immunization with SRBC over a 200-fold range did not enhance their capability to respond. Also, addition of adherent cells or macrophages from adult mice did not enhance the immune responses of young mice. Furthermore, immunization of 2–4 week old mice with SRBC inhibited the secondary response to SRBC. In contrast, young murine spleen cell proliferative and PFC responses to SI, SII, and LPS were approximately the same as the adult by 7–14 days of life. These data suggest that B-cell immunologic activity, as measured by immunologic assays utilized in this study, develops much earlier than does T-cell responsiveness.     

Different effects of the capsular polysaccharide of Klebsiella pneumoniae on the functions of various subpopulations of B cells in the immune response of mice to T-dependent antigen.

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, we studied the effects of PBA on the functions of various subpopulations of B cells in the immune response of mice to T-dependent antigen. Antibody-forming cells (AFC) of IgM and IgG types were estimated as anti-SRBC direct and indirect plaque-forming cells (PFC), and the B cells with precursor activities involving generation of AFC and supplementing new B cells as rosette-forming cells (RFC) of the B-cell type. Stimulation of normal mice by CPS-K caused a definite increase in the number of direct PFC but not in that of indirect PFC and RFC in the spleens. The responsiveness of spleen cells of CPS-K-treated mice to generate PFC and RFC responses to a subsequent injection of SRBC was lower than that of CPS-K-untreated normal mice. In this case, the responsiveness to generate RFC and indirect PFC was inhibited more strongly by CPS-K than that to generate direct PFC. When CPS-K was injected into normal mice simultaneously with SRBC, CPS-K never decreased but increased the levels of PFC and RFC responses to SRBC. In the spleens of SRBC-primed mice, the number of RFC was markedly decreased following injection of CPS-K, the number of direct PFC was increased only slightly and the number of indirect PFC was increased very slightly. The responsiveness of spleen cells of these CPS-K-treated SRBC-primed mice to generate secondary PFC and RFC responses to a subsequent injection of SRBC was much lower than that of CPS-K-untreated SRBC-primed mice. In this case, the responsiveness to generate the secondary RFC and indirect PFC responses was more strongly inhibited by CPS-K than that to generate the secondary direct PFC response. When CPS-K was injected into SRBC-primed mice simultaneously with the secondary injection of SRBC, there were marked decreases in the level of the secondary RFC response and slight decreases in that of the secondary indirect PFC response, but little change in that of the secondary direct PFC response. From these results it has been concluded that CPS-K provides the positive signal (the minor action) and the negative signal (the major action) to various subpopulations of B cells functioning at various stages of the immune response to T-dependent antigen in different ways, and acts to regulate the levels of B-cell responses to the antigen-mediated positive signal.