Effect of mouse antiserum against isologous aggregated immunoglobulins on the accumulation of rosette- and antibody-forming cells in mice immunized with ram erythrocytes

The paper describes the effect of mouse antiserum against isologous aggregated immunoglobulins (termed MAAS) on the kinetics of rosette-forming and antibody-forming cells (RFC and AFC, respectively) in mice immunized with SRBC. MAAS effect was assessed in vivo by injecting this serum for 5 days to mice CBA, combining the first injection with the injection of 5.10(7) SRBC. MAAS administration to mice immunized with SRBC induced a marked reduction of RFC in the spleen on the 5th and 9th days after the immunization. At the same periods MAAS produced no significant effect on the proliferation of AFC producing IgM-hemagglutinins. At the same time MAAS intensified the IgG-AFC proliferation in the period of the maximal content of these cells in the spleen of the immunized mice. After the MAAS absorption with the immune complexes formed by the mouse IgG-antibodies this serum largely lost its capacity to block RFC in vivo. On the basis of the data obtained it is suggested that the property of MAAS to influence the accumulation of RFC and AFC producing IgG-hemagglutinins is caused by the factor reacting with the immune complex formed by mouse IgG-antibodies. Possibly this factor represented antibodies against the aggregated immunoglobulins of this class.     

Structural organization of the antigen-binding receptors of immunocompetent cells]

It was demonstrated in this work that rabbit antimouse serum against the aggregated immunoglobulins (RAAS) and mouse serum against the aggregated mouse immunoglobulins (MAAS) inhibited the rosette-forming B-cells (RFC) on the 5th day after the immunization of mice CBA with SRBC in a dose of 5 X 10(-8) cells in vitro in 1:20--1:80, and 1:10--1:40 dilutions in 83--55 and 72--39%, respectively. In difference from RAAS, MAAS in a dilution of 1:20 induced a statistically significant suppression of the antigen-binding receptors of RFC of-B type in the intact animals, and on the 8th--9th day after their immunization with SRBC. In vivo MAAS induced inactivation of the antigen-binding receptors of B-lymphocytes only. Results of the work carried out served as a confirmation of the fact that immunoglobulins in the form of an antigen-antibody complex (functioning in the capacity of the antigen-binding receptors) were sorbed on B-lymphocytes of the spleen.     

Requirement for a bacterial flora before mice generate cells capable of mediating the delayed hypersensitivity reaction to sheep red blood cells.

A delayed-type hypersensitivity (DTH) reaction can be elicited by an injection of 10(8) sheep red blood cells (SRBC) into a rear footpad of conventional (CV) mice previously immunized with small doses of SRBC. In contrast, immunization of germ-free (GF) mice with the same doses of SRBC produced no DTH when immunization was by the intravenous (i.v.) route, and only weak reactions when immunization was by the subcutaneous (footpad) route. Varying the immunizing dose of SRBC, or the time at which DTH was elicited, did not produce a state of DTH responsiveness in i.v. immunized GF mice. However, the transfer of lymphocytes from CV mice, immunized 4 to 5 days previously with SRBC, into GF mice, conferred on GF mice the capacity to express DTH. Although DTH was not readily demonstrable in GF mice immunized with SRBC, they nevertheless produced normal levels of hemagglutinating antibody to SRBC. Finally, it was shown that GF mice could generate a normal DTH response to SRBC if they were first monoassociated with a Gram-negative bacterial flora.     

Delayed-type hypersensitivity (DTH) in BCG-sensitized mice I. Lack of suppressor T cell activity on DTH to sheep red blood cells.

Mice pretreated with an intravenous (i.v.) injection of BCG (BCG-sensitized mice) and then immunized intravenously with a high dose (10(8)--10(9)) of sheep red blood cells (SRBC) 2 weeks later developed strong delayed-type hypersensitivity (DTH) to SRBC, as in mice pretreated with cyclophosphamide (CY) (CY-treated mice) and then immunized with SRBC 2 days later; normal mice given the same dose of SRBC did not show such DTH. The mechanism of this strong DTH to SRBC which developed in BCG-sensitized mice was studied, by comparing it with that in CY-treated mice. The transfer of either whole spleen cells or thymus cells, but not serum, obtained from mice immunized with i.v. injections of 10(9) SRBC 4 days previously (hyperimmune mice) did not suppress either the induction or the expression of DTH to SRBC in BCG-sensitized mice, but suppressed those in CY-treated mice. The suppressor cells were SRBC-specific T cells. Adoptive transfer of DTH to SRBC by spleen cells from either BCG-sensitized mice of CY-treated mice to hyperimmune recipients failed. The adoptive transfer of DTH from BCG-sensitized mice to normal recipients also failed if the spleen cells from hyperimmune mice were cotransferred. Whole body irradiation (600 rad) of mice 2 hr before or after the time of immunization with SRBC reduced significantly DTH to SRBC in both BCG-sensitized and CY-treated mice. It was noticed that the total number of spleen cells in BCG-sensitized mice was 3--4 times larger than that in CY-treated mice. From these results, we conclude that the entity of effector T cells of DTH to SRBC induced in BCG-sensitized mice and in CY-treated mice was not different in terms of susceptibility to suppressor T cells and irradiation, but that the total numbers of effector T cells generated in these mice differed remarkably, resulting in the above-described different responsiveness to suppressor T cells transferred passively.     

Difference in thymus dependency between effector and suppressor T cells for delayed footpad reaction to sheep erythrocytes in mice

Effects of thymectomy at various times after birth on effector and suppressor T cells for a delayed footpad reaction were determined in 6-week-old mice immunized intraperitoneally (ip) with sheep erythrocytes (SRBC). Mice thymectomized 1 day after birth (Tx-1 mice) gave delayed footpad reactions weaker than those of mice thymectomized 7 days after birth (Tx-7 mice) or sham operated (SH mice) after immunization with a low dose of SRBC. After immunization with a high dose of SRBC, on the other hand, Tx-1 mice showed reactions stronger than those of Tx-7 or SH mice. Pretreatment with cyclophosphamide (CY) augmented the delayed footpad reaction in Tx-7 or SH mice, but not in Tx-1 mice, immunized with a high dose of SRBC. The presence of T cells suppressive for the delayed footpad reaction in the spleen of Tx-7 or SH mice was confirmed by cell transfer experiments. These results suggest that effector T cells responsible for a delayed footpad reaction to SRBC are less thymus dependent and require the presence of the thymus for a shorter period in their development compared to suppressor T cells.     

Different time course patterns of local expression of delayed-type hypersensitivity to sheep red blood cells in mice.

The delayed-type hypersensitivity (DTH) reaction, a peripheral expression of cell-mediated immunity is still a crucial in vivo immunological test. Nevertheless, the biological significance of its time course remains unclear. Thus, an exhaustive study of DTH was undertaken in mice immunized with increasing doses of sheep red blood cells (SRBC) inoculated intravenously (iv) or subcutaneously. The results showed that overall DTH reactions peaked at 18 hr except in mice iv immunized with the lowest doses (10(5) and 10(6)) and elicited at Day 4. The protracted DTH reaction was shown to be associated with an histological picture of tuberculin-type reaction. A part of the 18-hr DTH reaction is mediated by serum in mice inoculated with large doses of SRBC; nevertheless, numeration by limiting dilution analysis of circulating DTH cells showed that the frequency of these cells correlates with the 18-hr DTH level. The protracted DTH shown at 42 and 48 hr, 4 days after immunization with 10(5) and 10(6) SRBC, could not be transferred in naive recipients with immune spleen cells; it was independent of the antigen life span and did not result from immunization modulation at the bone marrow level on recruitable cells.     

The effect of Bordetella pertussis lymphocytosis-promoting factor (LPF) on antibody response in mice: its enhancing and suppressive effects

The effect of lymphocytosis-promoting factor (LPF) on antibody response in mice was estimated under different sets of experimental conditions. Four- and 6-week-old mice were intravenously inoculated with LPF. Three days later these mice were inoculated either intraperitoneally or intravenously with sheep red blood cell (SRBC) or human serum albumin (HSA) as an antigen. The adjuvant effect of LPF was demonstrated on antibody response in 6-week old mice to intraperitoneally inoculated SRBC but not to intravenously-inoculated one. When 4-week-old mice were immunized, hemagglutinin production in response to intraperitoneally inoculated SRBC was not enhanced by LPF. In addition, a rather suppressive effect of LPF at a comparatively high dose was demonstrated on hemagglutinin production in response to intravenously inoculated SRBC. Anti-HSA production was enhanced by inoculation of LPF in any combination of the mouse age and the route of antigen administration. These findings indicate that the adjuvant effect of LPF on antibody response in mice depends upon experimental conditions: the age of mice, the quality of antigen and the route of antigen administration used for immunization.     

In Vivo Production of Various Cytokines in Splenocytes of Sheep Erythrocyte-Immunized Mice after Intravenous Administration of Bacterial Lipid A

The effects of an intravenous administration of lipid A from Salmonella minnesota R595 lipopolysaccharide on the in vivo production of interleukin-2 (IL-2), IL-4, IL-5 and IL-6 in the spleens of mice intravenously immunized with sheep red blood cell (SRBC) antigen were investigated. The increased number of antigen-specific IgM antibody-producing cells and the titer of the IgM serum antibody were measured using the plaque-forming cell (PFC) assay and an enzyme-linked immunosorbent assay (ELISA). Simultaneous injections of SRBC antigen and lipid A adjuvant enhanced IgM-PFC number on days 3 and 4 and the serum IgM titer on days 4 and 5 after the immunization. We found that the enhanced IL-4 and IL-5 levels correlated with the PFC number and IgM titer. When lipid A was injected intravenously 2 days after immunization with SRBC, the PFC number in lipid A-treated groups were similar to those in controls 3 and 4 days after the immunization. However, it was found that a twofold increase in the IgM titer in serum was induced by lipid A 5 days after immunization. In relation to this increase, lipid A stimulated the production of only IL-5 among the cytokines tested.     

Efficacy of intracerebral immunization against pseudorabies virus in mice

To evaluate the efficacy of intracerebral (IC) immunization, mice were immunized with formalin-inactivated pseudorabies virus (PRV) by either subcutaneous (SC) or IC injection, and then 10(6) plaque-forming units of PRV were introduced into the hindleg of the immunized or non-immunized mice by intramuscular injection. The antibody titer in serum was elevated and boosted by additional immunization via both the SC and IC routes, but was higher after IC immunization. Intracerebrally immunized mice were completely protected from mortality and neurological signs, whereas all the non-immunized and 80% of the subcutaneously immunized mice died after developing neurological signs. In mouse models, IC immunization is more effective at inducing a protective immune response against the transneural spread of PRV than SC immunization.     

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.     

Differential effects of concanavalin A on T helper dependent and independent antibody responses

The in vivo immunosuppressive effects of Concanavalin A (Con A) on the thymus (T) helper dependent response to sheep erythrocytes (SRBC) and the T helper independent response to E. coli lipopolysaccharide 055: B5 have been investigated. Maximum suppression was observed in BALB/c mice treated with 3 successive ip injections of 100 μg each of Con A administered on Days ?1, 0, and +1 relative to the day of immunization (Day 0) with SRBC (splenic PFC on Day 4 reduced from 74,000 down to 1400). As little as 10 μg × 3 of Con A was capable of depressing both the PFC and serologic response while 2.5 μg × 3 was ineffective. A single ip injection of 300 μg of Con A administered simultaneously at the time of immunization with SRBC reduced splenic PFC from 74,000 down to 9990 and serum antibody titers by 3–4 log₂ units. Significant depression was noted if mice were treated 1, 2, or 3 days prior to but not following immunization. Immunosuppression was noted in mice which had been treated and immunized ip or iv or treated iv and immunized ip. Heat inactivation reduced if not abolished the immunosuppressive properties of Con A.Mice immunized with varying doses of a bacterial vaccine of E. coli 055: B5 (15–1500 × 10⁶ killed organisms) and treated with Con A on days ?1, 0, and +1 had no significant depression of splenic PFC when compared to nontreated controls. Mice treated with Con A and simultaneously immunized with both SRBC and E. coli had a 37-fold reduction in the PFC response to SRBC but only a 2-fold reduction in the response to E. coli. This differential immunosuppressive effect on T helper dependent and independent responses is consistent with the recently reported in vitro specificity which Con A has for theta antigen bearing lymphocytes.     

Preparation of mouse antiserum against isologous aggregated immunoglobulins and its effect on rosette forming cells]

A method of obtaining antisera against isologous aggregated mouse immunoglobulins is described. This serum designated MAAS blocked in vitro the antigen-binding receptors of the immune rosette-forming cells. MAAS was injected to mice immunized with SRBC. In comparision with the immunized mice given normal isologous serum rosette-forming B-cells were absent in the spleen of mice given MAAS at the peak of isologous response. But the antibody-forming cell count was not decreased under the influence of MAAS.     

Effect of antiserum against isologous aggregated immunoglobulins on the delayed-type hypersensitivity in mice immunized with sheep red blood cells

The mouse antiserum against isologous aggregated immunoglobulins (MAAS) injected to mice sensitized with 10(5) sheep red blood cells (SRBC) did not influence the delayed-type hypersensitivity (DTH) tested on the peak of sensitization (the 4th day) but enhanced significantly DTH tested on the 6th day. MAAS completely abolished the DTH suppression observed after sensitization with 5 x 10(7) SRBC. In transfer experiments the number of the DTH suppressor cells decreased in the spleen of sensitized mice under the MAAS action. MAAS did not affect the proliferation of antibody-forming cells (AFC) and hemagglutinin production but reduced by 70% the number of rosette-forming cells (RFC) in the spleen on the peak of the initial immune response. The data obtained may indicate that RFC participate in DTH suppression.     

Primary and secondary immune response in animals following spontaneous loss of tolerance

Mice subjected to tolerogenic treatment by sheep red blood cells (SRBC) and cyclophosphamide were immunized at various intervals (from 1 to 8 weeks after treatment) either by a single injection of 5 X 10(8) SRBC or by a double-injection of 1 X 10(6) SRBC. In control mice both immunization methods proved to be equally successful. In the experimental animals the immunological memory formation and/or its realization was destroyed to a greater extent and was restored more slowly than the capacity to the primary response.     

Types of cells involved in antigen-stimulated and spontaneous rosette formation with Toxoplasma gondii.

Antigen-binding cells were identified by using rosette formation of Toxoplasma gondii and defined lymphoid populations under different experimental conditions. Treatment of immunized spleen cell suspensions with anti-Thy 1 serum plus complement inhibited 5 to 29% of the rosette-forming cells (RFC). Higher numbers of thymus-derived lymphocyte-RFC were obtained after incubation at 4 degrees C and by the centrifugation method than by simple incubation at 20 degrees C. RFC were also observed with thymocytes. Combined treatment with anti-Thy 1 serum plus complement and depletion of adherent cells indicated that the major proportion, 46 to 70%, of RFC were B cells. Spleenocytes of nu/nu mice formed similarly high numbers of rosettes. Spontaneous RFC were observed in nonimmunized mice with both spleen and thymus populations. Numbers of rosettes varied considerably depending on the method and the source of cell population used. Removal of adherent cells from spleen suspensions resulted in RFC reduction of 14 to 25% in immunized and 14 to 33% in nonimmunized animals. Pretreatment with anti-mouse immunoglobulin inhibited completely the spleen and spontaneous thymus RFC and partially the thymus RFC in immunized animals.     

Analysis of certain mechanisms of antigen-specific suppression of the immune response

CBA mice were immunized with sheep red blood cells (SRBC) to obtain immune spleen cells (ISc) which were used to suppressor cells. Administration of ISC to intact syngeneic recipients on the immunization day led to a more powerful suppression of the immune response as compared to that seen one day after antigen injection. Four days after immunization the animals' immune response was not liable to be suppressed. ISC extract possessed similar effects with respect to the immune response of normal spleen cells which were transplanted to the cyclophosphamide-treated recipients. The immune response of spleen cells from mice immunized with SRBC in a dose of 10(6) was less liable to be suppressed. Hyperimmune spleen cells from donors immunized with SRC in a dose of 10(9) were insensitive to ISC or to the extract. Experiments with the use of adoptive transfer of a mixture of immune and intact T- and B-cells have disclosed that B-cells from hyperimmune donors were resistant to suppression. Therefore, B-lymphocytes are the most probable target cells exposed to T-suppressors in the given system. The mechanism is discussed of the selective effect of T-suppressors on B-cells in the course of the immune response development during immunization with high doses of antigen.     

Oral vaccination against Helicobacter pylori with recombinant cholera toxin B-subunit

BACKGROUND: The innocuous pure recombinant cholera toxin B-subunit (rCTB) is very attractive as a strong adjuvant for host immunization, but little is known about rCTB's gastric mucosal immunoadjuvanticity against Helicobacter pylori. The immunoadjuvanticity of rCTB against H. pylori was tested. MATERIAL AND METHODS: Mice were immunized with sonicated H. pylori and rCTB orally or intranasally and sacrificed on day 42 after immunization. Passive cutaneous anaphylaxis (PCA) test was performed to evaluate IgE-mediated anaphylaxis with serum from mice to which H. pylori-antigen with rCTB had been administered. Immunoglobulin titer specific to H. pylori in serum, lavation of the gastrointestinal tracts and feces were examined. Gastritis in vaccinated mice after a challenge was assessed with the scoring defined from grading of gastric inflammation. H. pylori proliferation after immunization was investigated by counting colony forming units (CFU) per gram of stomach tissue. RESULTS: PCA test exhibited no reactions against the serum from mice immunized with H. pylori-antigen with rCTB administered orally and intranasally. Oral and nasal coadministrations of rCTB significantly raised systemic and mucosal immunities against H. pylori and suppressed proliferation of H. pylori in gastric mucosa. The score of gastritis in mice immunized orally was significantly higher than that of mice immunized nasally due to postimmunization gastritis. Only oral administration of rCTB suppressed H. pylori proliferation as compared with intranasal administration and without rCTB. CONCLUSIONS: The present study indicated that rCTB has systemic and mucosal immunoadjuvanticities against H. pylori and that oral vaccination with rCTB might additively support antibiotic eradication.     

Activation of T cells by glutaraldehyde-fixed erythrocyte antigens: radiosensitivity of cells mediating delayed-type hypersensitivity reactions in the mouse.

Mice immunized with glutaraldehyde-fixed sheep red blood cells (G-SRBC) show delayed-type hypersensitivity (DTH) reactions to G-SRBC or SRBC. The specificity of the DTH reaction of mice sensitized with glutaraldehyde-fixed antigens is similar to that found after sensitization with unfixed antigens. The dose-response curve for sensitization by glutaraldehyde-fixed SRBC was very different from the curve for normal SRBC. At low doses, both antigens were effective in sensitizing to show DTH but neither induced an antibody response. However, at high antigen doses, only the glutaraldehyde-fixed antigen was efficient in sensitizing to show DTH and it failed to raise an antibody titer. Spleen cells of mice sensitized with fixed RBC can transfer DTH locally but if the donor cells are irradiated (500 R), the transfer is abrogated. In contrast, the transfer of DTH by spleen cells of mice immunized with unfixed antigen is not affected by 500 R. The transfer of DTH by spleen cells of mice immunized with fixed antigen can be blocked by “in vitro desensitization” while the transfer of DTH by spleen cells from mice primed with normal antigen is resistant to “in vitro desensitization.” These results suggest that immunization of mice with different physical states of the same antigen can result in the activation of antigen-specific T cells which exhibit markedly different properties.     

Higher ADCC of murine peritoneal cells after immunization with allogenic tumor cells as compared with stimulation by adriamycin, BCG, and thioglycolate

Normal peritoneal cells or spleen cells from C57BL mice could not lyse SRBC in an ADCC assay. After intraperitoneal injection of Adriamycin, BCG or thioglycolate the ADCC of peritoneal cells toward antibody-coated SRBC was elevated to 30% in contrast to the ADCC of spleen cells. However, peritoneal cells but not spleen cells of mice immunized with allogenic tumor cells (DBA SL2) showed ADCC levels at least two times higher than the levels observed after stimulation by other agents. Maximal ADCC levels (55.8%) were observed 10 to 15 days after immunization. Direct cytotoxicity towards SRBC increased to a maximum of 17.7% at 9 days after immunization. The effector cells in this system are thought to be macrophages, for ADCC activity was only present in the plastic-adherent cell fraction. Cell to cell contact was necessary for ADCC to occur; nonsensitized erythrocytes were not lysed when added to a mixture of effector cells and sensitized erythrocytes. Concentrations of antibody of 1 pg/ml were sufficient to induce ADCC, and effector cell to target cell ratios could be as low as 0.05. The finding that macrophages of mice immunized with allogenic tumor cells exhibit higher ADCC levels than macrophages elicited in other ways can contribute to the investigation of combined cancer therapy with antibodies and biological response modifiers.     

Fumonisin B1 alters sphingolipid metabolism and immune function in BALB/c mice: Immunological responses to fumonisin B1

Fumonisins have been reported to have diverse effects on animals, including immunosuppression in chickens and feeder calves; therefore, the effects of fumonisin B₁ (FB₁) on immune function in BALB/c mice was investigated. When administered i.p. with sheep red blood cells (SRBC), 5 to 100 µg of FB₁ reduced the number of plaque-forming cells (PFC) produced against SRBC; however, when administered daily, 1 to 50 µg of FB₁ caused a 4 to 12-fold increase in the number of PFC after SRBC injection. Therefore, FB₁ is not only immunosuppressive; but also, immunostimulatory. To test the possibility that there may have been an immune response to FB₁ as an antigen, FB₁ was injected into mice and the number of splenic cells forming rosettes on FB₁-treated SRBC was determined. There were dose-dependent increases in the antigen-binding cells, with up to 4.9- and 4.6-fold increases, respectively, upon primary and secondary immunization. FB₁-binding immunoglobulins could be detected in sera from some treated mice, but this response was not obtained in every experiment. In summary, these results show that FB₁ has diverse effects on the immune system, causing both stimulation and suppression of the response to foreign antigens, and apparently inducing an antigenic response to FB₁.Abbreviations DMEM Dulbecco's Modified Eagle's Medium - FB₁ fumonisin B₁ - FCS fetal calf serum - PBS phosphate buffered saline - PFC plaque forming cells - RFC rosette forming cells - SRBC sheep red blood cells