Immune response to chemically modified flagellin. IV. Further studies on the relationship between humoral and cell-mediated immunity

Acetoacetylation converts flagellin from an antigen which preferentially induces humoral antibodies to an antigen which exclusively provokes cell-mediated immunity and, under certain circumstances, induces antibody tolerance. Studies reported in this paper revealed that the acetoacetylated flagellins expressed similar immunological properties in flagellin primed rats as in normal rats. Thus, on the one hand, acetoacetylation destroyed the capacity of flagellin to trigger a secondary antibody response, but on the other hand, the acetoacetyl-flagellins very effectively induced delayed-type hypersensitivity reactions in flagellin primed animals. It was concluded from these results that humoral and cell-mediated immunity may be opposing immunological processes in both unprimed and primed animals.Acetoacetylated flagellin induced antibody tolerance in both strain W (low responder) and J (high responder) Wistar rats. Maximum tolerance was induced 12 hr after injection of antigen, but in strain J animals the tolerance had disappeared by 48 hr, whereas in strain W rats tolerance persisted for >28 days. The potential to recover from tolerance in strain J rats appeared to coincide with the level of delayed hypersensitivity at the time of challenge. However, this delayed hypersensitivity disappeared when breaking of tolerance occurred. These results suggest that the T cells which participate in delayed hypersensitivity reactions may also act as “helper” cells in antibody responses. On the other hand, it was found that priming for a secondary antibody response by flagellin appeared to coincide with development of primary antibodies rather than with induction of delayed-type hypersensitivity. The relative importance of specific T and B cells in these phenomena is discussed.     

A reverse effect of in vitro and in vivo concanavalin A-induced spleen cells on anti-sheep red blood cells response detected with a new method based on flow cytometry

A simple flow cytometric method for detecting humoral immunity against sheep red blood cells (SRBC) is described. The SRBC were incubated with the serum from SRBC-immunized mice, monoclonal anti-SRBC, or the supernatant which was obtained from the in vitro primary culture of spleen cells with SRBC. The antibodies which bound to SRBC were estimated by means of an immunofluorescence and a flow cytometry. When the channel number of the peak in the histogram of flow cytometry was measured as an index of fluorescence intensity of SRBC, the number significantly correlated with the concentration of IgM and IgG classes of anti-SRBC. The flow cytometry method and hemagglutination (HA) test, as a classic method, were compared in SRBC-immune sera and monoclonal anti-SRBC antibody. The sensitivity determined with flow cytometry was much higher than that with HA. The minimum detectable concentration of anti-SRBC antibody was found to be 3.4 ng/ml by the flow cytometry. The dose response of SRBC in in vitro primary culture was detected by the flow cytometry, not by HA, and the response increased with the dose of SRBC. Using this method, the effect of in vitro and in vivo concanavalin A (Con A)-induced spleen cells on humoral response against SRBC was examined in an in vitro culture system. Anti-SRBC response (IgM and IgG) was found to be suppressed by in vitro Con A-induced lymphocytes, but enhanced by in vivo Con A-induced lymphocytes. Thus, this new approach is found to be a good method for detecting the in vitro primary humoral antibody response, which is known to have a low reactivity.     

Immunomodulation activity of new vaccines for pertussis prophylaxis--acellular pertussis vaccine and adsorbed DPT vaccine with acellular component

The immunomodulating activity of acellular pertussis vaccine (APV) and adsorbed DPT vaccine with acellular pertussis component (DPTA vaccine) was studied. The study revealed that only large doses of APV, 10 immunizing doses (ID), suppressed humoral and cell-mediated response to sheep red blood cells (SRBC). 1 ID produced no influence on the formation of antibody producing cells, but increased the development of delayed hypersensitivity (DH) to SRBC. The modulation of cell-mediated immune response, induced by APV, returned to normal after the injection of purified staphylococcal toxoid, used as immunomodulator, in doses of 0.15 BU per mouse and 1.5 BU per mouse. DPTA vaccine containing 1 ID, as well as 10 ID, produced no immunomodulating effect. This was established by the evaluation of humoral response to SRBC in CBA mice and the study of the formation of DH to SRBC in BALB/c mice. As indicated by the total of the presented data, the inclusion of APV into DPTA vaccine enhanced the immunological safety of its pertussis component.     

The response of foetal sheep to the somatic and flagellar antigens of Salmonella typhimurium.

Following the injection of polymeric flagellin (POL), foetal sheep older than 70 days gestation produced haemagglutinating antibody and synthesized IgM. The maximum titre of antibody in the blood increased with the age at which the foetus was injected. All foetuses synthesized 2-mercapto-ethanol-sensitive antibodies, while older foetuses (approximately 120 days gestation) also produced 2-mercaptoethanol-resistant antibodies and synthesized IgG1. During the primary immune response, there was a poor correlation between the antibody titre and the amount of immunoglobulin synthesized. The majority of IgM synthesized and almost all IgG1 had no demonstrable specificity for POL. During the secondary response to POL, the majority of IgG1 synthesized was specific and in one case appeared to be monoclona. There was no detectable primary antibody response in foetal sheep to the somatic antigens of Salmonella typhimurium, although all foetuses synthesized IgM. Only one of six foetuses receiving a second injection of antigen produced antibody. There was an increase in the numbers of blood lymphocytes following the injection of both POL and S. typhimurium, but only POL induced a rapid increase in the numbers of neutrophils in the blood and produced histological changes in the draining lymph nodes and spleen.     

The IgM and IgG antibody responses in iron-deficient and iron-loaded mice

The humoral immune response was evaluated in male CD-1 mice fed the iron deficient (7 ppm Fe), iron sufficient (120 ppm Fe), and high-iron diets (3000 or 5000 ppm Fe) for 54 d. The IgM and IgG antibody responses against sheep erythrocytes (SRBC) determined by hemolytic plaque assay were suppressed by 65.4 and 51.2%, respectively, in the iron deficient mice. Subclinical iron deficiency was manifested by a marked reduction in hepatic iron concentration without any changes in hematocrit or body weight gain. In contrast, consumption of high-iron diets caused a marked accumulation of iron in the liver and a twofold reduction in the IgM antibody response without alteration in the IgG response. The suppression of the IgG antibody response in the iron deficient mice, however, did not result in a compensatory increase in delayed type hypersensitivity response.     

Anti-IgD enhancement of primary antibody responses in rats

The role of membrane IgD in immune responses was examined by treating adult rats with anti-IgD. Anti-IgD when administered to rats in conjunction with optimal or suboptimal doses of either SRBC, a T-dependent antigen, or DNP-Ficoll, a T-independent antigen, enhanced the antibody responses. The greatest enhancement was obtained when anti-IgD was administered before the antigen. The effects of anti-IgD on antibody responses to SRBC were: (i) significant antibody responses to suboptimal antigen concentrations; (ii) greater antibody responses to optimal antigen concentrations; (iii) accelerated antibody responses; (iv) an early shift from IgM to IgG antibodies; (v) prolonged antibody responses. Similar effects on the immune response to DNP-Ficoll were observed with the exception that all antibodies were 2ME sensitive (IgM). These results suggest that an anamnestic type of immune response can be induced in anti-IgD-treated rats when given a primary antigen exposure. Injection of anti-IgD without SRBC or DNP-Ficoll induces B-cell proliferation without detectable antibody production to these antigens, indicating at least two signals are required for the enhanced antibody responses.     

Immunoregulative effects of homocarnosine and gamma-aminobuthyric acid

The effects of homocarnosine and GABA on antibody production (PFC reaction) and cellular immunity (delayed hypersensitivity reaction, DHR) were examined in vivo. In mice treated with these agents, PFC reaction to 2 X 10(7) SRBC was enhanced but that to 1 X 10(9) SRBC was suppressed; moreover, immunoreaction was reduced in immature mice (2-2.5 weeks old) but was increased in aged mice (30 weeks old or above). These agents had optimal doses on the PFC reaction in mice given 1 X 10(8) SRBC and DHR, and induced recovery of immunofunction suppressed by the administration of MMC.     

Mycoplasma pulmonis depresses humoral and cell-mediated responses in mice

Humoral and cell-mediated immune responses to sheep red blood cells (SRBC) were studied in mice infected experimentally with Mycoplasma pulmonis. The hemagglutinating (HA) antibody against SRBC was evaluated at 0, 3, 5, 7, 14, 21 and 28 days postinfection (PI). Antibody tiers during all days PI were depressed significantly (p less than 0.05) in infected mice as compared to noninfected controls. The HA antibody, which is of the IgM class, peaks at day 5 PI. There is no shift in the kinetics of the humoral response in M. pulmonis infected mice. Cellular immune responses were evaluated by a delayed-type hypersensitivity (DTH) reaction and the lymphocyte transformation technique. Mice were sensitized at 0,3,5,7,14, 21 and 28 days PI with SRBC and challenged by footpad injection of SRBC 7 days later. The DTH reaction measured at 24 hours after challenge was depressed significantly (p less than 0.05) in all infected animals. After a transient enhancement on day 3 PI, the DTH responses remained depressed through day 28 PI. The lymphocyte transformation test showed a significantly (p less than 0.05) depressed response except on days 5 and 7 PI. These results indicate that M. pulmonis infection in mice suppresses the humoral antibody and cell-mediated immune responses.     

The effect of anti-adhesion molecule antibody on the development of collagen-induced arthritis.

In order to study how inflammatory cells including autoimmune lymphocytes interact with each other to develop collagen-induced arthritis (CIA), we injected monoclonal antibodies against mouse LFA-1 and ICAM-1 into DBA/1 mice immunized with type II collagen (CII). Both antibodies suppressed the development of CIA. These antibodies showed no effect on anti-CII antibody response, although they both significantly suppressed DTH response. It was suggested that anti-adhesion molecule antibodies suppress CIA mainly through their effect on cell-mediated immunity, without affecting humoral immunity under the conditions used.     

Antibody-mediated regulation of T cell responses. I. Characterization of a monoclonal antibody which specifically regulates contact hypersensitivity to DNFB in BALB/c mice

Contact hypersensitivity (CS) to 2,4-dinitrofluorobenzene (DNFB) in BALB/c mice is regulated by autoanti-idiotypic antibody. This report describes the preparation and characterization of a monoclonal antibody, 2-16.1, which has characteristics previously described for the serum anti-idiotypic antibodies. Monoclonal 2-16.1 was prepared by fusing lymph node (LN) cells from optimally sensitized BALB/c mice to the P3X myeloma. The monoclonal product of the cloned hybridoma is an IgM (K) immunoglobulin which does not bind to DNP-protein but which does bind to other immunoglobulins with anti-DNP specificity, primarily of the IgM class. Functionally, 2-16.1 inhibits the efferent limb of the CS reaction as measured by passive transfer of immunity. This inhibition is antigen-specific and appears to require the presence of a subset of Ia+ T cells in the DNFB-immune LN cell population. Suppression of transfer of immunity is strain-specific. Finally, suppression occurs only in the absence of complement, indicating that a lytic mechanism is not involved and that 2-16.1 does not recognize determinants expressed on the effector T cells of the CS reaction. Collectively, these results indicate that 2-16.1 is a monoclonal anti-idiotypic antibody, and that the hybridoma CSDNP 2-16.1 represents a clone of B cells which is stimulated during the primary CS response to DNFB and whose antibody product is involved in the endogenous, active regulation of this T cell-mediated response.     

IgM-mediated, T cell-independent suppression of humoral immunity.

The immunological unreactive state occurring in (T,G)-A-L nonresponder mice after secondary antigen challenge was investigated. Syngeneic IgM anti-(T,G)-A-L antibody-containing plasma, transferred at the time of the time of primary challenge, induced persistent suppression of autologous specific antibody production. Removal of plasma IgM with goat anti-mu antisera removed the ability of the plasma to supress. The induction and maintenance of the suppressed state were not different in thymectomized or sham-thymectomized animals. Primed animals subjected to graft-vs-host reaction (GVHR) at the time of secondary challenge switched over to IgG production. Animals suppressed by passive antibody transfer reacted to GVHR, at the time of secondary challenge, with specific IgM but not IgG antibody production. Transfused normal spleen cells partially abrogated suppression only when (suppressed) hosts had been lethally irradiated. Spleen cells from antigen-plus-antibody suppressed donors, upon transfer to previously normal, syngeneic hosts, were less immunocompetent than spleen cells from untreated donors. These data are consistent with a model of IgM mediated, T cell-independent persistent suppression of humoral immunity.     

Class-specific antibody in human dermatophytosis reactive withTrichophyton rubrum derived antigen

Dermatophyte infections induce a humoral immune response and an enzyme linked immunosorbent assay was used to detect specific antibody classes against antigen derived fromTrichophyton rubrum. Sera from 19 acute patients, 18 chronic patients, and 27 normal controls were evaluated. Mean IgG titers against dermatophyte antigen were significantly higher in all patients than in controls. Mean IgM levels were significantly higher in acute patients than in controls. No significant difference was detected in IgE titers between the patients and controls. These results do not reveal whether the humoral immune response has a role in the progression of the infection.Abbreviations CMI cell-mediated immunity - PBS phosphate buffered saline - Tr antigen Trichophyton rubrum antigen     

Antibody-mediated modulation of the immune response

We have studied the ability of monoclonal IgM and IgG antibodies to enhance or suppress immune responses and attempted to dissect the underlying mechanisms. Both IgM and IgG1 antibodies increased the rate of clearance of antigen from the circulation. Monoclonal IgM antibody to SRBC was found to specifically increase antibody responses, enhancement being insensitive to low doses of irradiation (150 R). IgM antibody specifically depressed the delayed hypersensitivity response to SRBC in vivo. Following administration of IgM in vivo, in vitro responses to SRBC were also enhanced. This in vitro enhancement appeared to depend on both T cells and B cells. In contrast, monoclonal IgG1 antibody to SRBC specifically depressed antibody responses in vivo. Such depressed antibody responses were also seen in vitro following IgG1 in vivo and did not appear to be due to the induction of suppressor T cells.     

Type II collagen-induced murine arthritis. I. Induction and perpetuation of arthritis require synergy between humoral and cell-mediated immunity

Immunization of DBA/1 mice with type II collagen resulted in typical and progressive arthritis, which is associated with the production of high titer of anti-collagen antibody and the induction of cell-mediated immunity as exemplified by delayed type hypersensitivity response as well as lymphokine production. In contrast, administration of heat-denatured collagen into DBA/1 mice failed to induce the arthritis. These mice produced only marginal antibody, whereas they developed comparable cell-mediated immunity to that induced by immunization with native collagen, and therefore the inoculation of heat-denatured collagen provided the regimen capable of inducing preferentially cell-mediated immunity without the generation of high level of antibody. Inasmuch as administration of antibody induced only marginal and transient joint swelling not associated with typical histologic lesion, the synergistic effect of humoral and cell-mediated immunities was investigated using antibody preparation and the regimen to induce selectively cell-mediated immunity. The results demonstrate that administration of antibody into DBA/1 mice pre-sensitized with heat-denatured collagen resulted in potent and progressive arthritis. Such synergy was further confirmed by the induction of arthritis in T cell-depleted DBA/1 mice that had been adoptively transferred with antibody and lymphoid cells from heat-denatured collagen-sensitized mice. Moreover, it was revealed that the nature of cells capable of transferring cell-mediated immunity was of Thy-1+ and L3T4+ Lyt-2-. These results indicate that anti-collagen antibody and L3T4+ T cell-mediated cellular immunity are crucially required for the perpetuated development of type II collagen-induced arthritis.     

Altered humoral immune response to sheep red blood cells by sheep erythrocyte-soluble hemolysate.

Adult C3H/He mice were rendered unresponsive to a primary injection of sheep red blood cells (SRBC) by pretreatment with sheep hemolysate supernatant (SHS) or subfractions of SHS isolated by column chromatography. The following effects of SHS on the immune response were observed: SHS did not kill antigen-reactive cells, it did not prevent the release of antibody by cells actively synthesizing and secreting antibody, and SHS-induced tolerance was not inhibited or abrogated by methods which terminate or abolish tolerance. In addition, cell-mediated responses were not affected in animals whose humoral responses were suppressed; however, the secondary plaque-forming cell (PFC) response was enhanced by SHS treatment. SDS gel electrophoresis revealed SHS to contain several proteins ranging from 12,000 to approximately 500,000 daltons.     

Effects of antigen-feeding on intestinal and systemic immune responses. III. Antigen-specific serum-mediated suppression of humoral antibody responses after antigen feeding.

Feeding mice sheep erythrocytes (SRBC) caused a significant decrease in splenic IgM antibody responses to SRBC given ip. Reduced IgM responses were due to a suppressor factor in the serum of fed mice rather than due to a lack of IgM antibody-forming cell precursors or to the presence of suppressor T cells. Although feeding initially primed mice to produce greater IgA and IgG anti-SRBC responses after SRBC challenge, the initial primed state was transitory. Mice fed SRBC for longer than 8 weeks had significantly reduced splenic IgG and IgA responses after SRBC challenge.Suppression of IgM responses by serum from fed mice was antigen-specific and not H-2 restricted. Serum from fed mice inhibited the induction of IgM anti-SRBC responses but did not block the expression of already established responses. The size of the suppressor factor and the ability to remove suppressor activity from serum by anti-mouse immunoglobulin suggested that suppression was mediated by antibody. However, the determinants against which the antibody was directed appeared to differ among batches of suppressor sera. Suppressor activity did not appear to be mediated by immune complexes, or soluble antigen. Oral feeding of antigen can have a marked influence on host systemic immune responses when the antigen used for feeding is subsequently administered parenterally. Thus, oral antigen administration may provide a way for specifically manipulating systemic immune responses in vivo. In addition, antigen-feeding may provide a means for producing transferable factors that suppress humoral antibody responses.     

Enhancement of murine in vitro antibody formation by hyperthermia

The influence of hyperthermia on primary in vitro antibody formation by C57BL/6 splenocytes to a T-dependent antigen, sheep red blood cells (SRBC), and a T-independent antigen, trinitrophenyl-lipopolysaccharide (TNP-LPS), was evaluated. Following heat treatment (39 or 40 °C), spleen cells demonstrated two- to fivefold increases in antibody production to SRBC. This enhancement of humoral immunity is critically dependent on the timing of hyperthermia administration relative to antigenic exposure. Examination of the kinetics for the SRBC response revealed that heat significantly lengthens the time period of antibody production. Although a number of parameters were examined, antibody production to TNP-LPS from hyperthermically treated cultures were comparable to control (37 °C treated) cultures. Finally, we have determined that the heat-induced increases in antibody formation to SRBC are mediated through T lymphocytes.     

Ascaris suum: Immunosuppression in mice during acute infection

Mice inoculated by stomach intubation with 10,000 embryonated Ascaris suum eggs, 4, 11, or 21 days before an intraperitoneal (ip) immunization with 2 × 10⁸ sheep erythrocytes (SRBC) had reduced numbers of direct (IgM) splenic hemolytic plaques measured at 4 days after immunization and only a marginal reduction in indirect plaques (IgG) measured at 9 days after immunization. Lower dosages of Ascaris eggs or simultaneous inoculation of Ascaris eggs and SRBC did not suppress antibody responses to SRBC. No reduction in a secondary antibody response to SRBC injected 4 days after Ascaris inoculation was observed. IgM and IgG hemagglutinin titers, as distinguished by 2-mercaptoethanol sensitivity, were suppressed in mice injected ip with 10⁸ SRBC 10 days following inoculation of 10,000 Ascaris eggs, but titers in both Ig classes were similar in infected and control mice injected with 2 × 10⁹ SRBC. At Day 20, antibody titers following ip injection of 1.0 or 100 μg of ovalbumin in alum were reduced in mice infected with 10,000 Ascaris eggs 4 days before antigen injection.Contact hypersensitivity to oxazalone was not altered in mice sensitized at 5 or 14 days after inoculation of 10,000 Ascaris eggs. The delayed hypersensitivity response, measured by footpad swelling, to an optimum intravenous sensitizing dosage of SRBC was inhibited in mice sensitized 10 days after Ascaris infection, but not inhibited in mice sensitized at 21 or 32 days after infection. In contrast, the delayed hypersensitivity response to subcutaneous sensitization with SRBC 10 days after Ascaris infection was not altered.     

Relationships between cell-mediated immunity and the IgE antibody response: II. Delayed hypersensitivity and antibody production to DNP-Ascaris conjugates

Rats of the W/F strain were immunized with DNP-Ascaris conjugates using complete Freund's adjuvant (CFA), Al(OH)₃ gel (alum), or B. pertussis vaccine as adjuvants. Cell-mediated immunity was assessed by lymphotoxin in vitro and by delayed hypersensitivity in vivo. IgE and IgG antibody determinations were made on serum pools obtained at various times during the primary and secondary responses. Although delayed hypersensitivity appeared earlier than lymphotoxin, these two parameters correlated during the primary but not during the secondary response. The discrepancies suggested that different cells may be responsible for these two phenomena. Antibody production was influenced by the adjuvant used. CFA led to IgG antibody responses to both hapten and carrier but not to IgE antibody production. The use of B. pertussis resulted in both IgE and IgG antibody production. In the case of alum, anti-hapten antibodies appeared during the primary response while anti-carrier antibodies of both IgE and IgG classes were detected after booster. The results indicated that cell-mediated immunity, IgE, and IgG antibodies appeared independently in an ordered, temporal sequence, and that these responses were not mutually exclusive but were under strong modulatory influences of the various adjuvants used.     

Failure of IL-1 receptor antagonist and monoclonal anti-IL-1 receptor antibody to inhibit antigen-specific immune responses in vivo.

rIL-1R antagonist (rIL-1ra) and 35F5, a neutralizing mAb, have been shown to inhibit the ability of IL-1 alpha and IL-1 beta to bind to type I but not type II murine receptors. Additionally, IL-1ra and 35F5 inhibit a variety of inflammatory responses in vitro and in vivo. In the present report we have evaluated the activity of human IL-1ra and 35F5 in murine Ag-specific cell-mediated and humoral immune response models. Administration of IL-1ra, either twice daily or as a continuous infusion, did not inhibit the cytolytic T lymphocyte response to allogeneic splenocytes. The CTL response was also not inhibited by daily administration of 35F5. The delayed type hypersensitivity response to oxazolone was similarly refractory to administration of IL-1ra and 35F5. In the humoral immune response models, neither the splenic plaque response to SRBC nor the IgG or IgM response to TNP-keyhole limpet hemocyanin was inhibited by treatment with IL-1ra or 35F5. These data suggest that signaling through the type I IL-1R is not required for these Ag-specific immune responses.