Enhancement of the in vivo antibody response by an 8-derivatized guanine nucleoside

The capacity of the C8-substituted guanine ribonucleosides to enhance the in vivo humoral immune response to the protein antigen, human gamma globulin (HGG), in A/J mice was evaluated. It has been shown recently that the C8-substituted guanine ribonucleosides are a new class of potent adjuvant for humoral immune responses to the sheep erythrocyte antigen. The current studies extend these findings to HGG with 8-bromoguanosine (8BrGuo), a representative of this group of nucleosides. The adjuvant activity of 8BrGuo in this system is highly dose and time dependent. Although 8BrGuo enhanced responses when injected either early (Day 0) or late (Day 4 or 5) after immunization, its administration on Day 1 or 2 most often led to no enhancement, suggesting that 8BrGuo may act on two events separated by a resistant stage in an ongoing immune response. The plaque-forming cell (PFC) response to HGG was enhanced optimally at doses as low as 1 mg 8BrGuo/mouse administered either on the day of immunization or 4 days thereafter. In contrast, however, serum anti-HGG antibody concentration assayed by enzyme-linked immunosorbent assay (ELISA) was enhanced only at doses of 10 mg or more, injected on the day of immunization, but doses as low as 1 mg were effective on Day 4. 8BrGuo was also an effective adjuvant when injected after antigen administration in incomplete Freund's adjuvant or when administered by several different routes (intraperitoneal, subcutaneous, oral).     

Modulation of Immune Response by Bacterial Lipopolysaccharide (LPS): Roles of Macrophages and T Cells in In Vitro Adjuvant Effect of LPS on Antibody Response to T Cell-Dependent and T Cell-Independent Antigens

The roles of macrophages and T cells in the adjuvant effect of lipopolysaccharide (LPS) were studied. In vitro anti-trinitrophenyl (anti-TNP) antibody responses to TNP-Ficoll and TNP-keyhole limpet hemocyanine (TNP-KLH) in spleen cells of C57BL/6 mice showed the most enhancement, when LPS was added to cultures at 1 μg/ml 48 hr after culture was started. The responses to these antigens were enhanced markedly by LPS in whole and macrophage-depleted spleen cells. The enhancement was greater in the latter group than in the former. The adjuvant effect among whole, T cell-depleted, macrophage-depleted and both macrophage- and T cell-depleted spleen cells was compared. The response to TNP-Ficoll was enhanced markedly by LPS in all groups. The enhancement was greater in the latter two groups than in the first two groups. The response to TNP-KLH was enhanced by LPS strongly in macrophage-depleted spleen cells, moderately in whole and both macrophage- and T cell-depleted spleen cells, and only slightly in T cell-depleted spleen cells. Enhancement was restored to T cell-depleted spleen cells by adding T cells. The response to TNP-KLH of macrophage-depleted spleen cells of LPS-responsive C3H/HeN mice which was enhanced by LPS was suppressed by adding splenic macrophages of C3H/HeN mice, but not of LPS-nonresponsive C3H/HeJ mice. The response to TNP-KLH of macrophage-depleted spleen cells of C3H/HeJ mice was not enhanced by LPS, irrespective of the addition of macrophages of C3H/HeN mice. The results indicate that B cells are activated directly by LPS, and T cells enhance and macrophages suppress the adjuvant effect of LPS.     

Strain Differences in the Immunogenicity of Aggregated Human IgG and the Adjuvant Action of Lipopolysaccharide on the Low-Responder Strain of Mice

Strain differences in the antibody response to human IgG (HGG) were observed when aggregated HGG was injected intravenously. Lipopolysaccharide (LPS) administered subsequently markedly enhanced the antibody response to HGG in low responder C57BL/6 mice as compared with that in high responder DDD, C3H/He or (C57BL/6 × DDD)F₁ mice. Aggregate-free preparation of HGG at a dose of 0.5 mg induced immunological tolerance in all strains of mice tested. LPS injected subsequently converted tolerogenic, aggregate-free HGG into immunogen in DDD mice but not in C57BL/6 mice. To determine the correlation between adjuvanticity and mitogenicity of LPS, spleen cells from normal mice were cultured in the presence of LPS and ³H-thymidine uptake was measured. Spleen cells of DDD mice incorporated three times as much ³H-thymidine as those of C57BL/6 mice. There seems no strong correlation between both activities of LPS. The data obtained are discussed in terms of strain differences in the macrophage function for processing the antigen.     

Genetic control of the immune response of mice to highly dinitrophenylated human gamma-globulin (DNP56HGG)

The immune response to highly dinitrophenylated human gamma-globulin (DNP56HGG) was tested in inbred strains of mice. Significant differences in the anti-DNP response among inbred strains were found, including the magnitude of serum antibody and the location of plaque-forming cells (spleen or lymph nodes). The strain differences persisted when the dose and adjuvant were changed. The genetic control of the anti-DNP response to DNP56HGG was investigated. The analysis of the response of congenic and F1 hybrid mice to DNP56HGG suggests that at least two genes are involved in the control of the anti-DNP response. The two genes are demonstrated by complementation in the F1 generation, and show no correlation with H-2 haplotype or IgG2a allotype. A third gene may be implicated by differences in response observed between male and female mice.     

Specific, transient suppression of the immune response by HGG tolerant spleen cells. II. Effector cells and target cells.

In a previous report, it was shown that spleen cells from mice made tolerant to human gamma-globulin (HGG)5 could specifically inhibit the immune response of normal spleen cells after adoptive transfer to lethally irradiated recipients. However, that report also showed that the suppressive activity was only transiently associated with tolerant spleen cell populations. It was concluded from those experiments that while suppressive activity could be demonstrated in tolerant spleen cells under certain conditions, such activity was not obligatory for the maintainance of the tolerant state. The experiments presented here were performed to determine the nature of the effector cell(s) and the target cell(s) involved in this system of suppression of the immune response. Treatment of cells from tolerant animals with anti-thymocyte serum and complement to remove thymus-derived (T) cells completely abrogated suppresive activity. Removal of adherent cells from tolerant spleen cells by passage over glass wool columns resulted in partial loss of the suppression. The inhibitory activity of the suppressor cells was resistant to 900 R irradiation regardless of whether the tolerant spleen cells were irradiated before or after adoptive transfer. The cellular target(s) for the supprssor cells was examined by using lipopolysaccharide (LPS) as an alternative source of helper activity for the response to HGG. LPS, injected at the time of the initial antigenic challenge of mice that had been reconstituted with tolerant and normal spleen cells, prevented the expression of suppression against bone marrow-derived (B) cells. However, when LPS was presented only at the time of secondary antigenic challenge, it was unable to overcome suppression of the immune response of reconstituted recipients. Thus, LPS could produce a state where the B cells were resistant to suppression, but LPS could not rescue the responsiveness of B cells once the cells in the reconstituted recipient had been suppressed. In addition, the immune response to both the hapten dinitrophenol (DNP) and the carrier (HGG) were suppressed when recipients of tolerant and normal spleen cells were challenged with DNP6HGG. This indicates that T helper cells are also a target for suppression. The results presented in this paper are discussed in relation to a possible mechanism of suppression which proposes that suppressive activity represents the induction of tolerance in immunologically competent cells by HCG which is closely associated with the tolerant spleen cells.     

A synthetic analogue of Escherichia coli lipoprotein, tripalmitoyl pentapeptide, constitutes a potent immune adjuvant

Lipoprotein from the outer membrane of Escherichia coli and other Enterobacteriaceae constitutes a potent B lymphocyte mitogen and polyclonal activator in various species. Tripalmitoyl pentapeptide (S-(2,3-bis-(palmitoyloxy)-(2RS)-propyl)-N-palmitoyl-(R)-cysteinyl -(S)-seryl-(S)-seryl-(S)-asparaginyl-(S)-alanine) is a synthetic analogue of the N-terminal part of lipoprotein and has, in all assays tested, a biologic activity similar to native lipoprotein. It also exhibits a strong adjuvant activity in vitro: In the presence of 3.3 to 33.3 micrograms/ml of tripalmitoyl pentapeptide, the stimulation of the primary antibody response toward underivatized sheep red blood cells (SRBC) and toward trinitrophenylated (TNP-) SRBC was markedly enhanced, as measured by a direct hemolytic plaque assay. At optimal mitogen- and antigen-doses, plaque formation was increased up to 100-fold, and at suboptimal doses (0.03 to 0.3 microgram/ml) a 10- to 60-fold increase of plaque numbers was achieved. In the presence of tripalmitoyl pentapeptide, the antigen-specific IgM response was increased about sevenfold and the IgG response was augmented about 10-fold, as measured by ELISA. Similarly, in the secondary in vitro response to TNP-SRBC, a 7 to 10-fold enhancement of the antibody titer was obtained in the presence of the adjuvant. The application of tripalmitoyl pentapeptide and antigen had to occur concurrently in order to achieve a strong adjuvant effect. Addition of tripalmitoyl pentapeptide to the cell cultures 1 day after or 1 day before antigen application had no significant positive effect, and in several instances a decrease in antibody production was found. Thus, tripalmitoyl pentapeptide, a well-characterized synthetic product available in major amounts, constitutes a potent immune adjuvant for potential animal and clinical use.     

Altered Innate Immune and Glial Cell Responses to Inflammatory Stimuli in Amyloid Precursor Protein Knockout Mice

Amyloid precursor protein (APP) and its cleaved products have been reported to have important functions in CNS health, including in memory and synapse formation, cell survival and neuroprotection. Furthermore APP and its cleaved products have been shown to be transiently increased in response to various CNS stressors, suggesting a role in response to acute cellular injury. In an attempt to further understand the function of APP in response to CNS injury, we have used intracranial LPS injection as an inflammatory injury model in APP knock out mice (APPKO). Our data show that innate immune responses to LPS injection is significantly blunted in APPKO mice compared to APP sufficient wild type (BL6) mice. Morphologically, glial cells in APPKO mice appear less reactive, with shorter ramified processes and smaller cell bodies in response to LPS. Additionally, quantitative RT-PCR analysis for several glia markers and innate immune cytokine levels (e.g. TNFα, IL-6, IL-1β and IL-10) showed significantly reduced expression levels in LPS injected APPKO mice. In vitro cell culture assays confirmed this attenuated response to LPS stimulation by primary microglial cells isolated from APPKO mice. Our data suggests that APP full length protein and/or its cleaved products are necessary to mount a complete and effective innate immune cell response to inflammatory injury.     

In vivo and in vitro regulation of type I IFN synthesis by synergistic effects of CD40 and type II IFN

During cognate interaction with CD40 ligand (CD154)-expressing T cells, Ag-presenting accessory cells are activated for increased cytokine synthetic and costimulatory function. We examined whether CD40 modulates in vivo innate immune function over time, hypothesizing that distinct cytokine responses evolve to delayed microbial exposure. C3H/HeN mice pretreated with activating anti-CD40 Ab (FGK45) produced 10-fold more serum IFN-gamma and IL-12 p70 to delayed, but not synchronous, challenge with LPS. A novel finding was that LPS-induced IFN-alpha increased by 20-fold in mice pretreated for 24 h, but not 6 h or less, with anti-CD40. Anti-CD40-pretreated C57BL/6 RAG-2(-/-) mice similarly increased IFN-alpha responses to delayed LPS challenge, confirming mediation by innate immunity. Type I IFNR- and IFN-gamma-deficient mice treated with anti-CD40 failed to expand serum IFN-alpha responses to LPS challenge. Combined pretreatment with anti-CD40 and anti-IFN-gamma mAb showed that IFN-gamma produced after anti-CD40 pretreatment, but before LPS challenge, was necessary for IFN-alpha synthetic enhancement. Anti-CD40 also increased polyinosinic-polycytidylic acid (poly(I:C))-inducible IFN-alpha by 5-fold in an IFN-gamma-dependent fashion, but did not significantly increase IFN-alpha production to CpG or Pam(3)Cys challenges. Poly(IC)-stimulated splenocytes from anti-CD40-pretreated mice produced 4-fold more IFN-alpha than controls and production associated with CD11c(+) cells. Finally, rIFN-gamma and anti-CD40 combined synergistically to increase poly(IC)-inducible IFN-alpha synthetic capacity in bone marrow dendritic cells. We conclude that innate immune production of IFN-alpha is cooperatively regulated by CD40 and IFN-gamma acting on dendritic cells, suggesting a unique mechanism by which innate immune function evolves in response to specific adaptive immune signals.     

Immune enhancement by tumor-therapeutic drugs and endotoxins

Summary Five tumor chemotherapeutic drugs: adriamycin (ADM), formyl leurosine (FLE), 1,2–5,6-dianhydrogalactitol (DAD), 5-fluorouracyl (5-FU), and vincristine (VCR) were tested for their effect on the immune response to sheep red blood cells (SRBC) in mice in the Jerne plaque assay. It was found that four of these can act as both immunostimulators and immunosuppressants, depending upon the time elapsing between their application and immunization. VCR was found to be slightly immunopotentiating.Proper combination and timing of injections of DAD and FLE enhanced the immune response over 23fold. Administration of the same drugs in reversed sequence led to an almost complete immune suppression. Repeated injections of some drugs alone or in combination also led to marked or complete immunosuppression.The combined use of drugs and endotoxic lipopolysaccharide (LPS) or its nontoxic, polysaccharide-rich hydrolytic breakdown product (PS) could compensate for some of the immunosuppressive effects. The most striking adjuvant effect was elicited by the combined use of VCR and LPS or VCR and the nontoxic PS preparation.     

Suppressor cells in tolerance to HGG: kinetics and cross-suppression in high dose tolerance--absence in low dose tolerance.

Spleen cells from mice made tolerant with high doses of human gamma-globulin (HGG) specifically suppress the immune response of normal, syngeneic, spleen cells. These suppressor cells were found to be cross-reactive in that they would suppress the immune response of normal spleen cells to bovine gamma-globulin (BGG) as well as to HGG. In contrast, suppressor cells could not be demonstrated in spleens of mice made tolerant with low doses of HGG (i.e., T-cell tolerance), nor could they be found in high dose tolerant mice following a second injection of DHGG at a time when the initial suppressor activity had waned. The role of suppressor cells in the induction, maintenance, and loss of tolerance is discussed.     

Popliteal lymph node (PLN) assay to study adjuvant effects on respiratory allergy

Different variants of the popliteal lymph node (PLN) assay have been published. Here we describe the adjuvant popliteal lymph node assay, an immune response assay to study the adjuvant activity of soluble substances as well as particulate matter. The substance to be studied for adjuvant activity is injected into the hind footpad of mice or rats together with an antigen. Adjuvant activity is determined as the increase in PLN weight and cell numbers in animals receiving antigen together with the substance under study, compared with PLN weight and cell numbers in animals given the antigen without the substance in question, and animals given the putative adjuvant alone. Because lymph node weight and cell numbers are immunologically non-specific parameters, specific immune response assays like serum antibody responses or antibody-forming cell numbers should additionally be performed. Different antigens and immune response assays may be used, depending on the research question asked. In relation to respiratory (or food) allergy, the assays should as a minimum include determination of specific IgE in serum, and preferably also IgG1 (mouse). Serum specific IgG2a antibody determination may be added to get an indication of the Th1-Th2-balance of the response. The adjuvant PLN assay, with cellular response assays performed in the draining popliteal lymph node and antibody determinations in serum, requires small amounts of test material. The assay offers a practical, sensitive and reproducible method to determine the adjuvant activity of soluble substances as well as particulate material, with the possibility to also perform mechanistic studies.     

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.     

Corticosteroids and the humoral immune response of mice. II. Enhancement of bone marrow antibody formation to lipopolysaccharide by high doses of corticosteroids.

The effect of injection of the synthetic corticosteroid dexamethasone sodium phosphate upon the primary response to Escherichia coli lipopolysaccharide (LPS) was studied in mouse spleen and bone marrow. Daily corticosteroid injections, starting 1 day before immunization with LPS, could suppress the anti-LPS plaque-forming cell (PFC) response in the spleen. The higher the dose of corticosteroids, the more the splenic PFC response was suppressed. On the other hand, the bone marrow PFC response showed a dose-dependent enhancement after corticosteroid injections. This effect was maximal when tested 7 days after antigen injection, and constituted a 3- to 15-fold increase after daily injection of 16 mg dexamethasone/kg body wt. The same effect was found in genetically athymic nude mice, showing that the corticosteroid-mediated enhancement of the anti-LPS PFC response in the bone marrow is not due to elimination of T suppressor cells. Probably the differential effect of corticosteroids upon antibody formation in spleen and bone marrow is due to a redistribution of B-lineage cells, with a resulting accumulation in the bone marrow.     

Maturation of the lymphoid system. II. Characterization of the cellular levels of unresponsiveness induced in neonates by a T-dependent antigen that is an obligate immunogen in adults.

It has previously shown that AHGG, a form of HGG that is highly immunogenic in euthymic adult mice, is capable of inducing specific unresponsiveness when injected into neonatal animals. This report extends this finding and indicates that such a neonatal treatment results in the induction of tolerance in T as well as B cells. Furthermore, a similar conclusion was reached regarding specific T lymphocyte function in animals treated as neonates with OVA. The ability of LPS to modulate responses of neonatal animals to AHGG or DHGG was also examined. It appeared that such mice were not susceptible to the adjuvant effects of LPS until the 4th week of life. Furthermore, LPS was incapable of inhibiting the unresponsiveness induced in mice by either DHGG or AHGG until the 3rd or 4th week of life.     

The effect of aging on the induction of humoral and cellular immunity and tolerance in two long-lived mouse strains.

Aging is a complex process that adversely affects most if not all components of the immune system. In this report, two long-lived mouse strains have been compared in ability to generate both antigen-specific immunity and tolerance. Although CBA/CaJ mice produced high levels of antibody following injection of aqueous preparations of aggregated human gamma-globulin (AHGG), C57BL/6 mice made only meager antibody responses to such preparations. Age dramatically affects the humoral anti-HGG response to aqueous AHGG in both strains, but the meager response of young C57BL/6 mice was at insignificant levels in aged C57BL/6 mice. Conversely, both mouse strains generated good responses following injection of HGG in complete Freund's adjuvant at both the T and B cell level as evidenced by in vitro antigen-specific T cell proliferation and anti-HGG antibody production. Aged mice of both strains showed a marked decrease in the production of serum anti-HGG antibody in comparison to young mice. Although the antigen-specific T cell proliferative response was significantly decreased in aged CBA/CaJ mice, such proliferation was not affected in aged mice of the C57BL/6 strain. Removal of CD8+ cells from lymph node T cells of either young or aged C57BL/6 mice did not increase the antigen-specific proliferative response, suggesting that loss of CD8+ suppressors during the aging process is not responsible for the high level of antigen-specific T cell proliferation in aged C57BL/6 mice. Tolerance to HGG was readily induced in both young and aged C57BL/6 and CBA/CaJ mice although aged mice demonstrate a modest resistance to tolerance induction when compared to their young counterparts. This resistance was observed in both antibody production and antigen-specific T cell proliferation.     

Effects of the adjuvants SGP and Quil A on the induction of experimental autoimmune thyroiditis in mice

In the present study, two adjuvants, SGP and Quil A, were assessed for their ability to induce experimental autoimmune thyroiditis (EAT) in mice. SGP (a synthetic copolymer of starch, acrylamide, and sodium acrylate) and Quil A (a plant saponin) were compared with lipopolysaccharide (LPS) and complete Freund's adjuvant (CFA) given together with mouse thyroglobulin (MTg) for their ability to induce EAT in CBA/J mice. Immunization with MTg and LPS, MTg and CFA, or MTg with SGP was effective in inducing anti-MTg antibodies and histologic EAT, while MTg with Quil A was ineffective in inducing either anti-MTg antibodies or EAT. MTg with LPS was able to prime mice for the development of an in vitro spleen cell proliferative response to MTg while MTg with SGP or with Quil A was unable to prime spleen cells to proliferate detectably in response to MTg. MTg with LPS given in vivo primes CBA/J spleen cells for further activation by in vitro culture with MTg to transfer EAT to naive CBA/J recipients. MTg with SGP was also effective in priming CBA/J spleen cells for in vitro activation and transfer of EAT while MTg with Quil A was ineffective. The effective adjuvant activity of SGP and its lack of toxicity relative to LPS should make it a useful agent for further studies in murine models of EAT.     

Adjuvant effect on maintenance and escape from tolerence.

When mice are injected with deaggregated human gamma globulin (HGG), and HGG-tolerant state ordinarily is produced and persists despite subsequent challenges with an immunizing dose of HGG in saline or with an immunizing dose of HGG in Mycobacterium adjuvant. Subsequent administration of an immune elimination dose of radiolabeled HGG, at 27 days and 47 days does not break the tolerant state. Of special interest is the observation that when complete adjuvant containing increasing amounts of mycobacterial components was administered in conjunction with antigen very early in the tolerance induction phase 5 days after TID, it appears to prevent tolerance production. Mice challenged 5 and 17 days after the tolerance-inducing inoculation exhibit a statistically significant increase in circumvention of tolerance when compared with individuals challenged on the 7 and 17 day schedule. This increased circumvention of tolerance, as evidenced by 5 day challenge mice, seems to be related both to the mycobacterial content of the adjuvant and murine strain.     

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.     

Leishmania tropica: differences in the antigenicity of promastigotes and amastigotes

In vitro bioassays were used to compare T-cell responses induced by the intramacrophage amastigote stage and the sandfly-borne promastigote stage of Leishmania tropica. Lymph node cells from mice immunized with sonicated promastigotes or amastigotes incorporated in Freund's complete adjuvant were assayed for their ability to proliferate and to release interleukin 2 following in vitro challenge with promastigote or amastigote antigen. The levels of the proliferative and interleukin 2 synthetic responses of cells from promastigote and amastigote immunized mice were quite distinct. Cells from mice immunized with promastigotes demonstrated a vigorous in vitro response to the homologous antigen, but a reduced response to the potentially cross-reactive amastigotes. In contrast, cells from mice immunized with amastigotes mounted a weak response to the homologous antigen, but a consistently greater response to promastigote antigen. This unusual response was similar when 8 M urea extracts were used as immunogens and test antigens. In general, interleukin 2 production by immune mice paralleled the results from the T-cell proliferation assays. These results are discussed in relation to evasion of host immunologic detection by the intramacrophage amastigote stage.