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.     

Modulation of Mouse Anti-Trinitrophenyl Plaque-Forming Cell Affinity by Adjuvants or Lectins

The effects of several kinds of adjuvants or lectins, such as Corynebacterium parvum, dextran, poly AU, poly IC, dibutyryl cAMP, concanavalin A (Con A), phytohemagglutinin (PHA) and pokeweed mitogen (PWM) 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. The numbers of anti-TNP PFC in the spleens of mice which had been injected with C. parvum 7 days in advance were greater than those in controls after immunization with TNP-coupled heterologous erythrocytes, while the affinity of antibodies released by these PFC was not affected. On the contrary, simultaneous injection of dextran with TNP-erythrocytes did not increase the numbers of splenic anti-TNP PFC, but heightened the affinity of antibodies released by these PFC. Copolymers of nucleotides, poly AU and poly IC, were capable of enhancing splenic anti-TNP PFC responses, but showed almost no altering of PFC affinity. Dibutyryl cAMP did not have any effect on this system. Con A had potencies to both augment the number of anti-TNP PFC and heighten the PFC affinity, while PHA seemed to lack these potencies. Injection of PWM in the presence of antigen increased the number of anti-TNP PFC and heightened slightly the PFC affinity. These results indicate that the heightening of the affinity at the cellular level is regulated in ways different from the augmenting effects on the number of anti-TNP PFC by adjuvants or lectins. These results are discussed in the light of the mode of action of the substances used.     

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.     

Immune response of guinea pig lymphoid cells in vitro: III. Induction by Concanavalin A of a secondary anti-TNP response in primed guinea pig cells

Spleen and lymph node cells of 2,4,6-trinitrophenyl-ovalbumin (TNP-OVA)-primed guinea pigs, show a secondary anti-TNP plaque-forming cell (PFC) response on culture with Concanavalin A which does not require the addition of TNP-OVA but this response may be modestly stimulated by soluble TNP-OVA. If TNP sheep red blood cells (SRBC) are added instead as antigen, the spontaneous anti-TNP response is suppressed but an anti-SRBC response is induced.     

Immunosuppressive Effect of Bacterial Lipopolysaccharide on Antibody Response

Injection of endotoxins (bacterial lipopolysaccharide: LPS) several days prior to immunization causes the suppression of antibody response. The suppressive effects of several kinds of LPS preparations on the plaque-forming cell (PFC) antibody response in the spleen of mice were examined after immunization with sheep red blood cells (SRBC). Glycolipids obtained from heptoseless mutants (Re form) of salmonella or its lipid A preparation coupled artificially with bovine serum albumin (BSA) are capable, like LPS obtained from a wild type (S form) strain, of inducing suppression of the PFC response, while alkaline-detoxified LPS can not. The refractory periods of the PFC response induced by LPS injection last only a few days. However, the use of cyclophosphamide (CY) together with LPS can extend the refractory periods of antigenic stimulation for several weeks. Injections of LPS and CY can also induce unresponsive states of OH agglutinin antibody response to antigenic stimulation with formalin-killed organisms of Escherichia coli or Salmonella enteritidis (presumably both thymus-independent antigens). These unresponsive states induced by LPS and CY are easily terminated by a transfer of syngeneic bone marrow cells but not by thymocyte transfer.     

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.     

Nonspecific Elicitation of Antibody-Forming Cells in the Mouse Spleen by Bacterial Lipopolysaccharide

Mechanisms of nonspecific elicitation of anti-sheep erythrocyte (SRBC) hemolytic antibody plaque-forming cells (PFC) in mouse spleens with an injection of bacterial endotoxin (lipopolysaccharide (LPS)) were studied in comparison with the genesis of naturally occurring ‘background’ PFC in normal mouse spleens and of rapidly arising PFC in mouse spleens after immunization with SRBC. The cytokinetic pattern of anti-SRBC PFC response after an injection of LPS was quite different from that of the response elicited after immunization with SRBC. In addition, even though LPS nonspecifically elicited anti-SRBC PFC response in mice, LPS could not confer any immunological memory on mouse immunocytes for a ‘secondary-type’ anti-SRBC PFC response to restimulation with LPS or SRBC. The administration of rabbit anti-mouse thymocyte immunoglobulin or anti-SRBC antiserum in mice markedly suppressed the PFC response after immunization with SRBC, but did not do so after stimulation with LPS. Neonatally thymectomized mice could still respond to stimulation with LPS, producing anti-SRBC PFC in their spleens. Injections of actinomycin D or cyclophosphamide into mice resulted in obvious reductions of the PFC responses elicited by either LPS or SRBC. However, injections of these immunosuppressive antisera or drugs did not affect the number of anti-SRBC PFC in normal mouse spleens. These results suggest that the geneses of anti-SRBC PFC developed under different conditions, i.e., background PFC, LPS-stimulated PFC, and antigen-stimulated PFC, are quite different from each other, and that the nonspecific elicitation of anti-SRBC PFC by LPS does not require the helper function of T lymphocytes. No obvious difference, however, was observed in the time of ontogenic maturation among these three different anti-SRBC PFC in the mouse spleens judging from when they were first manifested after birth.     

Modulation of mouse anti-trinitrophenyl plaque-forming cell affinity by adjuvants or lectins.

The efffects of several kinds of adjuvants or lectins, such as Corynebacterium parvum, dextran, poly AU, poly IC, dibutyryl cAMP, concanavalin A (Con A), phytohemagglutinin (PHA) and pokeweed mitogen (PWM) 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. The numbers of anti-TNP PFC in the spleens of mice which had been injected with C. parvum 7 days in advance were greater than those in controls after immunization with TNP-coupled heterologous erythrocytes, while the affinity of antibodies released by these PFC. Copolymers of nucleotides, poly AU and poly IC, were capable of enhancing splenic anti-TNP PFC responses, but showed almost no altering of PFC affinity. Dibutyryl cAMP did not have any effect on this system. Con A had potencies to both augment the number of anti-TNP PFC and heighten the PFC affinity, while PHA seemed to lack these potencies. Injection of PWM in the presence of antigen increased the number of anti-TNP PFC and heightened slightly the PFC affinity. These results indicate that the heightening of the affinity at the cellular level is regulated in ways different from the augmenting effects on the number of anti-TNP PFC by adjuvants or lectins. These results are discussed in the light of the mode of action of the substances used.     

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.     

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.     

Differential immunosuppressive effects of ascites fluid from mastocytoma-bearing mice on primary vs secondary immunocyte responses.

The effects of immunosuppressive ascites fluids from mastocytoma-bearing mice on the primary vs secondary immune response to sheep red blood cells (SRBC) was examined. Injection of mice with ascites fluid from tumor-bearing mice markedly depressed the primary immune response of normal syngeneic mice challenged with SRBC. However, there was a preferential depression of the 19S IgM antibody response as compared with the 7S IgG response. Injection of ascites fluid shortly before secondary immunization of mice with SRBC also resulted in depressed IgM PFC responses but only a slight to moderate depression of IgG PFC. Treatment of mice with the ascites fluid before primary immunization had little if any effect on the secondary IgG PFC response, although the IgM response was moderately depressed. These results indicate that the immunosuppressive factor(s) present in the ascites fluid of mastocytoma-bearing mice has a differential effect on distinct classes of immunocytes. Those immunocytes or their precursors involved in formation of low efficiency 7S IgG antibody are more resistant to immunodepression. Such differences appear due to different sensitivities of cells involved in the immune response system.     

Antibody formation in mouse bone marrow. VIII. Dependence on potentially circulating memory cells: a study with parabiotic mice.

Mouse bone marrow barely contains antibody-producing plaque-forming cells (PFC) during the primary response to sheep red blood cells (SRBC). However, during the secondary response, the number of IgM, IgG, and IgA PFC in the bone marrow can rise to a level which surpasses the number of PFC in all the other lymphoid organs together. In the present paper we investigated whether the capacity of immune mice to react upon a booster injection of SRBC with a bone marrow PFC response can be transferred from immune to nonimmune mice. Therefore, mice primed with SRBC 6 months previously and nonprimed syngeneic mice were joined for parabiosis and were separated from each other at various intervals after joining. These separated mice were subsequently immunized with SRBC. It was found that, after 3 weeks of parabiosis, the nonprimed members reacted upon an injection of SRBC with a bone marrow IgM, IgG, and IgA PFC response as high as did the previously primed members. Furthermore it could be demonstrated by means of cell transfer experiments that, after a period of parabiosis of 3 weeks, the bone marrow and spleen of the normal mice contained about as many memory cells as the bone marrow and spleen of the immune mice. These results suggest that antibody formation in mouse bone marrow is dependent on a population of potentially circulating memory cells.     

Antibody formation in mouse bone marrow. IX. Peripheral lymphoid organs are involved in the initiation of bone marrow antibody formation.

Mouse bone marrow is barely capable of plaque-forming cell (PFC) activity during the primary response to sheep red blood cells (SRBC). However, during secondary-type responses, it becomes the major organ, containing IgM, IgG, and IgA PFC. In the present paper, the influence of splenectomy (Sx) upon the secondary bone marrow PFC response to SRBC was investigated. When previously primed mice were splenectomized just before the second intravenous (iv) injection of SRBC, the effect of Sx upon the height of the bone marrow PFC response was dependent on the booster dose. Sx just before a booster of 10⁶ SRBC iv almost completely prevented bone marrow PFC activity, whereas an iv booster dose of 4 × 10⁸ SRBC evoked a normal IgM, IgG, and IgA PFC response in Sx mice. Apparently low doses of iv administered antigen require the spleen in order to evoke antibody formation in the bone marrow. Experiments with parabiotic mice, consisting of Sx and sham-Sx mice, showed that this facilitating influence of the spleen upon bone marrow antibody formation occurs via the blood stream. In a subsequent study, it was investigated whether the spleen is required throughout the bone marrow PFC response or only during the few days of the initiation phase. Therefore, mice were splenectomized at different intervals after a booster injection of 10⁶ SRBC iv. It appeared that Sx 2 days after the booster injection could still prevent the normal bone marrow PFC activity, whereas Sx at Day 4 could no longer do so. Apparently, after an iv booster injection, the spleen is only required for initiation of the bone marrow PFC response and not for the maintenance of this PFC activity thereafter.     

Immunological properties of Propionibacterium acnes. I. Potentiation and Suppression on antibody response to sheep and hamster erythrocytes in mice.

Adjuvant activity of phenol-treated cells of Propionibacterium acnes C-7 in antibody response was investigated in ICR mice. Simultaneous administration (day 0) of P. acnes (i.p.) and sheep red blood cells (SRBC) (i.v.) enhanced the formation of direct plaque-forming cells (PFC) on days 2, and the formation of indirect PFC response on day 7 and thereafter. Conversely, pretreatment from 11 to 14 days before antigen injection suppressed markedly the antibody response. The potentiation and the suppression of immune response depended on doses of antigen and of P. acnes, the timing of adjuvant injection and the time of assay. The two opposite phenomena caused by P. acnes were also confirmed in antibody response against hamster red blood cells (HRBC). Pretreatment with P. acnes 1 to 14 days before antigen injection suppressed markedly anti-HRBC antibody response, whereas P. acnes injected simultaneously with HRBC or one day after injection of the antigen induced prolongation of antibody response and the production of 2-mercaptoethanol-resistant antibody.     

Effects of a novel perfluorochemical emulsion on lymphoid tissues and immunocompetence in rats: time course effects relative to immunological challenge

1. The effects of intravenous (i.v.) injection of low doses of a novel perfluorochemical (PFC) emulsion on lymphoid tissues and antibody production against i.v.-injected sheep red blood cells (SRBC) have been studied relative to the timing of immunization in rats. 2. Spleen and, to a lesser extent, liver weights were significantly increased in response to injection of emulsion but no consistent pattern was observed. 3. Thymus weight was consistently decreased following injection of emulsion; in contrast, mesenteric lymph node (MLN) weights were unchanged throughout except for a significant increase in animals injected with emulsion 24 hr prior to immunization. 4. The mean plasma antibody titre to SRBC showed a variable response in animals receiving the emulsion: titres were significantly increased in rats injected with emulsion 1 hr prior to immunization whereas they were significantly reduced in animals injected with emulsion 7 days before SRBC. 5. These results show that lymphoid tissue weights and plasma antibody titres to SRBC vary according to the time of a previous or subsequent injection of PFC emulsion.     

Effects of microwave exposure on the hamster immune system. IV. Spleen cell IgM hemolytic plaque formation

Microwave exposure has been reported to affect various components of the immune system. In this study, we examined the effect of a single whole-body exposure of hamsters to microwave (mw) energy (2.45 GHz; 5-25 mW/cm2; 1 h) on the IgM antibody (Ab) response of spleen cells to sheep red blood cells (SRBC). MW-exposed, sham-exposed, and cage-control hamsters were immunized with SRBC and plaque-forming cells (PFC) in spleens assayed using the direct hemolytic plaque assay. In cage-control hamsters the Ab response was highest between days 4 and 5, returning to baseline by day 9. MW exposure (25 mW/cm2 for 1 h) significantly augmented PFC response only on days 4 and 5 postimmunization, causing approximately a 4.3- and 3.5-fold increase over controls, respectively. Exposure to 15 mW/cm2 caused a lesser, but significant increase in PFC. Exposure to intensities below 15 mW/cm2 for 1 h did not produce any increase in Ab response. Immunization with different concentrations of SRBC following 1 h of 25 mW/cm2 MW exposure revealed a stimulation in PFC at all concentrations ranging from 5 X 10(7) to 5 X 10(8) SRBC. Pretreatment of hamsters with MW radiation prior to immunization showed that the animals retained an increased sensitivity to SRBC for as long as 4 days after MW exposure. In contrast, exposure of hamsters to MW energy on different days after immunization showed an effect of the PFC response only if given between 0 and 1 day after immunization. These results suggest that MW exposure augments the primary IgM response to SRBC by affecting some early event in the immune response process. The various possible explanations for this phenomenon are discussed.     

Variations in the responses of C57BL and a mice to sheep red blood cells: II. Analysis of plaque-forming cells

The number of direct and indirect plaque-forming cells (PFC) and the serum hemolytic activity was determined for A/He, C57BL/6J, and B6AF1 mice responding to multiple injections of sheep red blood cells (SRBC). Although the kinetics of the primary response differed, all mice had high numbers of both direct and indirect PFC and low-titered 2-mercaptoethanol (2-ME) sensitive serum antibody. Following multiple SRBC injections, the A/He spleens contained predominantly IgG producing PFC. Their serum antibody activity was resistant to 2-ME signifying the presence of IgG. The serum activity of both the C57BL/6J and B6AF1 mice was sensitive to 2-ME (IgM antibody) over the course of immunization, and although there was a definite IgM PFC memory response, the presence of 7S memory PFC was questionable. The results are discussed in terms of the maturation of the antibody response to SRBC and of the question of the postulated IgM and IgG switch.     

Immune response of the Mongolian gerbil (Meriones unguiculatus) to sheep red blood cells.

The immune responses of Mongolian gerbils, Meriones unguiculatus, to sheep red blood cells (SRBC) were studied as compared to those of mice. After a single injection of SRBC, hemagglutinin titers in gerbils were significantly lower and hemolytic plaque-forming cells (PFC) in the spleen were less in number as compared to the response of mice. In gerbils the PFC response to a higher dose of bacterial lipopolysaccharide (LPS) was rather higher than in mice. The delayed-type hypersensitivity (DTH) assay on the foot-pad revealed that the responsiveness was considerably lower in gerbils than in mice.     

Antibody formation in mouse bone marrow. II. Evidence for a memory-dependent phenomenon

Mouse bone marrow is barely capable of plaque-forming cell (PFC) activity in a primary response to sheep red blood cells (SRBC), while PFC activity in the secondary response to SRBC can be clearly demonstrated. This phenomenon was studied by means of cell transfer experiments.T cells, which are involved in an anti-SRBC PFC response, were shown to be very scarce in normal mouse bone marrow. This is considered to be the cause of the low PFC activity in the marrow during the primary response to SRBC.In normal mouse bone marrow precursors of IgM-PFC but not of IgG- and IgA-PFC could be found. Priming with SRBC induced the appearance of IgM-, IgG-, IgA- and T-memory cells in the marrow. These B- and T-memory cells were shown to be specific for the antigen which induced their appearance. It is thought that after a second injection of SRBC the IgM-, IgG- and IgA-memory cells can differentiate with the help of the T-memory cells within the bone marrow into IgM-, IgG- and IgA-PFC respectively.The sequence of appearance of the B-memory cells in the bone marrow was shown to be IgM-IgG-IgA.Six months after the intravenous injection of SRBC, the presence of B-memory cells could be demonstrated not only in spleen and bone marrow, but also in peripheral lymph nodes, mesenteric lymph node, Peyer's patches, thymus and blood. The increase in amount of B-memory cells was most prominent in the spleen.     

Characterization of Background Anti-Trinitrophenyl Plaque-Forming Cells Observed in Several Strains of Mice

Normal mice have a large number of background anti-trinitrophenyl (TNP) antibody-forming cells (AFC) in their spleens (about 40–50 anti-TNP PFC/10⁶ cells). We investigated this among several mouse strains, i.e., C57BL/6, C3H/He, Balb/c, ddd, and ICR mice, and found that all strains had a similar number of anti-TNP PFC (plaque-forming cells). Developmental aspects of background anti-TNP PFC in the ontogenic process were also investigated. The number of anti-TNP PFC increased logari thmically during the first few days of age, reached a peak on the 13th day and attained a constant value within 30 days. Neonatal thymectomy did not decrease the number of background anti-TNP PFC but such treatment decreased the anti-TNP PFC response to TNP-HRBC (horse red blood cells) immunization. Germ-free ICR mice had a number of background anti-TNP PFC similar to that of conventional ICR mice. Avidity of background anti-TNP PFC was compared among mice of several ages and it was shown that there were no differences among them. These results suggest that the occurrence of these background anti-TNP PFC is not elicited by the immune response but by the natural maturation of precursors of AFC without antigenic stimulation.