Induction and protective role of antibodies in Legionella pneumophila infection

Legionella pneumophila (Lpn) is a ubiquitous Gram-negative bacterium found in aquatic environments and is the causative agent of Legionnaires' disease, a severe form of pneumonia. We have used Lpn-permissive A/J mice as a model to analyze the B cell response upon intravenous (i.v.) and intranasal (i.n.) infection with Lpn. A strong antibody (Ab) response was observed upon i.v. infection with wild-type (WT) Lpn and an icmT mutant strain, which is unable to replicate within permissive host cells. In contrast to i.v. infection, only WT but not icmT mutant Lpn was able to induce specific Ab responses upon i.n. infection. After primary i.n. infection with WT Lpn, a strict compartmentalization of Lpn-specific Ab isotypes was observed, as IgG was found exclusively systemically, while IgA was detectable only locally in the lung. Regardless of the infection route, isotype switching to IgG and to IgA was strictly dependent on CD4+ T cells, whereas IgM production was completely Th-independent. Finally, we analyzed the protective capacity of the Lpn-specific Ab response. Actively or passively immunized mice or mice that were infected with opsonized Lpn had 50-100-fold reduced bacterial titers compared to naive animals, clearly demonstrating the capacity of Ab to protect against infection with Lpn.     

Vitamin A metabolism and mucosal immune function are distinct between BALB/c and C57BL/6 mice

The vitamin A metabolite retinoic acid (RA) has been reported to suppress Th1 responses and enhance Th2 responses. Here, we investigated whether differences in vitamin A metabolism could underlie the differences between C57BL/6 and BALB/c mice, which are reportedly seen as Th1 and Th2 responders, respectively. BALB/c mice were shown to have higher intestinal epithelial expression of RALDH1 (where RALDH is retinaldehyde dehydrogenase), and, consequently, higher RALDH activity in MLN‐DCs, leading to an increased ability to induce IgA class switching in B cells. Furthermore, within BALB/c mice, induction of IgA secretion as well as increased accumulation of regulatory T cells (Treg) in the intestinal lamina propria was observed. Additionally, as BALB/c mice are more resistant to dextran sulphate sodium (DSS) induced colitis, mice that lacked vitamin A in their diet had a more severe form of DSS‐induced colitis compared to control mice. Therefore, the level of RA production and consequently the degree of RA‐mediated signaling is crucial for the efficiency of the mucosal immune system.     

T cell cytokines determine the severity of experimental IgA nephropathy by regulating IgA glycosylation.

Hyperfunction of Th2 cells and aberrant glycosylation of IgA have been proposed independently as factors in the pathogenesis of IgA nephropathy (IgAN), the most common form of glomerulonephritis. To investigate the relationship between Th2 cytokines and IgA glycosylation in the genesis of IgAN, we induced IgAN in C3HeB and BALB/c mice by oral immunization and intranasal challenge with Sendai virus. Although both strains of mice developed microhaematuria and glomerular IgA immune deposits to similar degrees, only BALB/c mice developed significant renal insufficiency. More profound reductions of terminal galactosylation and sialylation occurred in Sendai virus-specific IgA from BALB/c versus C3HeB mice, and splenocytes from immunized BALB/c mice produced more Th2 and less Th1 cytokines compared to C3HeB mice when stimulated with antigen in vitro. Furthermore, the decreased glycosylation of IgA elicited by Th2 cytokines in vitro was blunted by the addition of IFN-gamma. We conclude that increased production of Th2 cytokines can lead to abnormalities in IgA glycosylation, which in turn promote heightened phlogistic responses to IgA immune complexes lodging in the glomerulus. We suggest that a relative or absolute increase in Th2 cytokine production in response to mucosal infection is a significant pathogenic factor in human IgAN.     

Modulation of the Th1/Th2 bias by lipopeptide and saponin adjuvants in orally immunized mice

We compared the adjuvanticity of the synthetic lipopeptide P3CSK4 of bacterial origin and the plant-derived adjuvant saponin using the wheat storage protein gliadin as antigen. Gluten sensitive BALB/c mice were orally immunized with gliadin in a mixture with either lipopeptide or saponin. The gliadin-specific serum IgG response was markedly enhanced by the saponin adjuvant. The lipopeptide adjuvant enhanced the IgG2a response, but reduced IgG1 production. In contrast, the saponin adjuvant enhanced both IgG2a and IgG1, and the sera showed elevated specific IgE concentrations. Enhanced specific IgA levels were detected in sera and in faeces especially after immunizations with gliadin in combination with P3CSK4 Enhanced specific IgG and IgA levels could also be detected in supernatants of cell cultures prepared from mesenteric lymph nodes and Peyer's patches of immunized mice. Our data suggest that both adjuvants enhance the mucosal as well as the systemic immune response; P3CSK4 predominantly elicits the activation of the Th1 subset, whereas saponin activates both the Th1 and Th2 subser. Our findings are of importance for the improvement of mucosal immunizations, and might be a tool for the immunotheraphy of food allergies.     

Enhancement of mucosal immune responses by chimeric influenza HA/SHIV virus-like particles

To enhance mucosal immune responses using simian-human immunodeficiency virus-like particles (SHIV VLPs) as a mucosal HIV vaccine, we have produced phenotypically mixed, chimeric influenza HA/SHIV 89.6 VLPs and used them to immunize C57B/6J mice intranasally. Systemic and mucosal antibody responses, as well as cytotoxic T cell (CTL) responses, were compared in groups immunized with SHIV 89.6 VLPs or HA/SHIV 89.6 VLPs. Intranasal immunizations were given using VLPs either with or without the addition of the mucosal adjuvant cholera toxin. Total serum IgG, IgG1 and IgG2a, and IgA in saliva, vaginal lavage, lung wash, and fecal extracts were evaluated by enzyme-linked immunosorbent assay (ELISA). The level of serum IgG production to HIV Env was highest in the group immunized with chimeric HA/SHIV 89.6 VLPs. Similarly, mucosal IgA production was also enhanced in the mucosal HA/SHIV 89.6 VLP-immunized group. Analysis of the IgG1/IgG2a ratio indicated that a Th1-oriented immune response resulted from these VLP immunizations. High levels of serum IgG and mucosal IgA against influenza virus were also detected in mice immunized with HA/SHIV VLPs. HA/SHIV 89.6 VLP-immunized mice also showed significantly higher CTL responses than those observed in SHIV 89.6 VLP-immunized mice. Furthermore, a Major Histocompatibility Complex (MHC)-class-I-restricted T cell activation ELISPOT assay showed elevated interferon-gamma, interleukin-2, and interleukin-12 production in HA/SHIV 89.6 VLP-immunized mice, indicating that phenotypically mixed HA/SHIV 89.6 VLPs can enhance both humoral and cellular immune responses at multiple mucosal sites. Therefore, chimeric HA-containing VLPs represent a potential approach for mucosal immunization for prevention of HIV infection.     

Production of secretory immunoglobulin A against Shiga toxin-binding subunits in mice by mucosal immunization

The toxicity of Shiga toxins (Stx) depends on the binding of their B subunits to carbohydrate ligands on host cells. The production of antibodies against B subunits, especially immunoglobulin A (IgA) secreted on the mucosal surface, should contribute to host defense. One of the major problems in attempts to produce IgA against Stx was the poor immunogenicity of B subunits. We were able to produce serum IgA as well as IgG against Stx1B in mice of the H-2d haplotype by means of intranasal immunization with recombinant B subunits of Stx (Stx1B) together with cholera toxin as a mucosal adjuvant. Secretory IgA (S-IgA) was detected in nasal washes but not in feces. We prepared chemically cross-linked Stx1B for use as an immunogen, and the formation of stable oligomers was revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry. When the cross-linked Stx1B was used together with cholera toxin for the intranasal immunization of BALB/c mice, strong enhancement of the immune response was observed. The S-IgA titers in nasal washes were 16- to more than 64-fold higher than those in mice immunized with native Stx1B plus cholera toxin. Furthermore, fecal IgA was detectable when the cross-linked Stx1B was used. The use of cholera toxin was necessary for the induction of high titers of S-IgA in the nasal washes. However, the effect of cross-linking was dependent on the major histocompatibility complex haplotype; that is, no enhancement of IgA production was observed in C57BL/6 mice. The present results provide a practical means of producing IgA against Stx1B in BALB/c mice.     

Abnormal behavior of gamma-committed B lymphocytes probed by a lymphocyte blastogenesis inhibitory factor in autoimmune MRL mice

Recently, we have characterized a lymphocyte blastogenesis inhibitory factor (LBIF) which was purified from the culture supernatant of a human histiocytic lymphoma U-937 (Sugimura, K. et al., Eur. J. Immunol. 1989. 19: 1357). In this study, we investigated the effect of LBIF on the antibody production of autoimmune MRL mice in vitro. We demonstrated here that (a) LBIF inhibited the IgM, IgG and IgA antibody responses of lipopolysaccharide (LPS)-stimulated spleen cells of normal BALB/c mice, (b) in the case of old autoimmune MRL/Mp-lpr/lpr (MRL/l) and MRL/Mp-(+)/+ mice, however, LBIF inhibited IgM and IgA but not IgG responses of LPS-stimulated spleen cells, (c) the antibody production of all IgG subclasses, IgG3, IgG1, IgG2b and IgG2a, was not sensitive to LBIF inhibitory activity in these autoimmune mice, (d) in young MRL mice (3-5-week-old MRL/l), which were phenotypically normal, LPS-induced antibody production of all isotypes (IgM, IgG and IgA) was strongly inhibited by LBIF as shown in normal BALB/c mice and (e) in the case of 7-week-old MRL/l the insensitivity to LBIF was concomitant with the appearance of gamma + B lymphocytes. Thus, by employing LBIF as a probe, this study showed a correlation between the pathogenesis of MRL autoimmune disease and the lack of LBIF sensitivity of hyperactive B lymphocytes and suggested that the intrinsic abnormality of autoimmune MRL B lymphocytes might be confined to gamma- but not mu- or alpha-committed B cells.     

Mucosal immunization of CD4+ T cell-deficient mice with an inactivated virus induces IgG and IgA responses in serum and mucosal secretions

Immunoglobulin (Ig) class switching can occur in the absence of alphabeta+ or gammadelta+ T cells when mice are infected with certain live viruses, although CD4 T helper cells are believed to be essential for induction of a high-affinity antibody response and for efficient isotype switching from IgM to IgG and IgA production. However, little information is available about the immune responses after mucosal immunization of CD4+ T cell-deficient mice with inactivated virus. In this study, we show that intranasal immunization with formalin-inactivated influenza A/PR8/34 virus induces IgG and IgA responses in serum and IgA responses in mucosal secretions in CD4+ T cell-deficient mice. All four subclasses of IgG were produced. IgG1/IgG2a ratios were found to be from 1 to 1.75, indicating that both Th1 and Th2 immune responses are induced by the inactivated influenza virus. The sera and mucosal secretions were found to have neutralizing activity against influenza virus in vitro. In addition, the mucosally immunized CD4+ T cell-deficient mice were protected completely from challenge with a lethal dose of live, pathogenic influenza virus. To our knowledge, this is the first demonstration that mucosal immunization with an inactivated virus induces immune responses in serum and mucosal secretions in CD4+ T cell-deficient mice.     

Immunoglobulin A-deficient mice exhibit altered T helper 1-type immune responses but retain mucosal immunity to influenza virus

We have previously demonstrated that immunoglobulin A (IgA)(-/-) knockout (KO) mice exhibit levels of susceptibility to influenza virus infection that are similar to those of their normal IgA(+/+) littermates. To understand the mechanism of this apparent mucosal immunity without IgA, immunoglobulin isotype and T helper 1 (Th1)-type [interferon-gamma (IFN-gamma)] and Th2-type [interleukin (IL)-4, IL-5)] cytokine responses to influenza vaccine were evaluated. Intranasal immunization with influenza virus subunit vaccine plus cholera toxin/cholera toxin B subunit (CT/CTB) induced significant influenza virus-specific immunoglobulin G (IgG) antibody in the serum and nasal passages of both IgA(-/-) and IgA(+/+) mice, while IgA antibodies were induced only in IgA(+/+) mice. IgA KO mice exhibited an IgG1 subclass haemagglutinin (HA)-specific response but no detectable IgG2a and IgG2b responses. In contrast, IgA(+/+) mice exhibited significant IgG1 as well as IgG2a responses. This indicates a predominant Th2-type response in IgA KO mice compared to normal mice. Following stimulation with influenza virus in vitro, splenic lymphocytes from immunized IgA(-/-) mice produced significantly lower levels of IFN-gamma than IgA(+/+) mice (P < 0.001), but elaborated similar levels of IL-4 and IL-5. This was true at both protein and mRNA levels. Immunized mice were challenged intranasally with a small inoculum of influenza virus to allow deposition of virus in the nasal mucosal passages. Compared to non-immunized mice, immunized IgA(-/-) and IgA(+/+) mice exhibited significant, but similar levels of reduction in virus titres in the nose and lung. These results demonstrate that in addition to IgA deficiency, IgA gene deletion also resulted in down-regulated Th1-type immune responses and confirm our previous data that IgA antibody is not indispensable for the prevention of influenza virus infection.     

Immune responses induced by replication-defective adenovirus expressing the C-terminal portion of the Mycoplasma hyopneumoniae P97 adhesin

Mycoplasma hyopneumoniae, the causative agent of porcine enzootic pneumonia, colonizes the respiratory cilia of affected swine, causing significant economic losses to swine production worldwide. Vaccination is the most cost-effective strategy for the control and prevention of this disease. The goal of this study was to design and evaluate a replication-defective recombinant adenovirus, rAdP97c, expressing the C-terminal portion of P97 adhesin (P97c), an important pathogenesis-associated protein of M. hyopneumoniae, as a new vaccine candidate against M. hyopneumoniae infection. P97c-specific immune responses were evaluated in BALB/c mice following intranasal and intramuscular inoculation with rAdP97c. Mice inoculated by both routes of immunization produced significant levels of specific immunoglobulin G (IgG) antibodies in the serum and in bronchoalveolar lavage fluids (BALs). Animals immunized intranasally also produced a significant level of P97c-specific IgA in BALs. Intramuscular inoculation of rAdP97c induced a systemic and mucosal Th1-type biased response, evidenced by the predominance of IgG2a in the serum and BALs, whereas intranasal inoculation resulted in a mixed Th1/Th2-type response (balanced levels of IgG1 and IgG2a) in both sytemic and mucosal compartments. P97c-specific antibodies were able to inhibit the growth of M. hyopneumoniae cells in vitro. These data suggest that rAdP97c vaccine may represent a new strategy for controlling infection by M. hyopneumoniae.     

Effects of the Nature of Adjuvant and Site of Parenteral Immunization on the Serum and Mucosal Immune Responses Induced by a Nasal Boost with a Vaccine Alone

Outbred OF1 mice were immunized subcutaneously with flu vaccine, either in the neck or in the lumbar region (back), in combination with adjuvants inducing either a Th1- or a Th2-type response, referred to as adjuvants A1 and A2, respectively. After two parenteral immunizations, the mice were boosted intranasally with nonadjuvanted vaccine. The serum response was analyzed after each immunization by measuring specific immunoglobulin A (IgA), IgG1, and IgG2a antibody levels, while the local response (same isotypes) was measured in the salivary glands after the mucosal boost by ELISPOTs. We observed that systemic priming at any of the two sites with a Th2 rather than a Th1 adjuvant dramatically enhanced the mucosal IgG1 and IgA responses following a mucosal boost with unadjuvanted vaccine. In addition, as judged by the IgG2a/IgG1 ratios and serum IgA levels, immunization of mice in the back induced a rise in Th2 response compared to neck immunization with adjuvant A1. In contrast, such back immunization with adjuvant A2 reversed the Th1-Th2 balance in favor of the Th1 response compared to neck immunization. Similar differences were observed in mucosal antibody levels according to the site of priming with one given adjuvant; priming in the back with adjuvant A1 increased the mucosal IgA and IgG1 responses compared to neck priming, while the local IgG2a levels were decreased. The reverse was true for adjuvant A2. Back versus neck priming with this latter adjuvant decreased the mucosal IgG1 response, while local IgG2a levels were increased. The different lymphatic drainages of the two sites of parenteral immunization may explain these differences, due to the targeting of particular lymphoid inductive sites. Some of these sites may represent crossroads between systemic and mucosal immunity.     

Persistence of anti-donor allohelper T cells after neonatal induction of allotolerance in mice

BALB/c mice rendered tolerant to A/J alloantigens by neonatal injection of 10(8) (A/J X BALB/c)F1 spleen cells develop an autoimmune disease associated with a polyclonal activation of donor B cells. To study the mechanisms leading to donor B cell activation in tolerant mice, we prepared mixed lymphocyte cultures (MLC) between splenic T cells from neonatally injected mice and donor-type (A/J X BALB/c)F1 or third-party (C57BL/6 X BALB/c)F1 B cells. T cells from tolerized mice were unable to generate cytotoxic T lymphocytes, to proliferate or to secrete interleukin (IL)2 after stimulation with donor alloantigens in MLC. These T cell responses were present after MLC with third-party antigens, but were of lower intensity than those generated by control BALB/c T cells. In contrast, T cells from tolerized mice stimulated immunoglobulin production by donor-type (A/J X BALB/c)F1 B cells much more powerfully than T cells from control BALB/c mice. The stimulation of donor-type (A/J X BALB/c)F1 B cells was polyclonal, as attested by the levels of anti-hapten and anti-DNA antibodies in the MLC supernatants. IgM was the dominant isotype secreted in vitro, but IgG1 and IgG3 were also produced in significant amounts. Lysis experiments indicated that the T cells responsible for F1 B cell stimulation in MLC were CD4+ host T cells. These T helper cells were alloreactive since they did not stimulate syngeneic BALB/c B cells, and their effect on donor B cells was specifically blocked by anti-donor Ia monoclonal antibodies. Addition of anti-IL 4 monoclonal antibody to MLC between T cells from tolerant mice and (A/J X BALB/c)F1 B cells almost completely abolished the production of IgG1, but not that of IgM or IgG3. Taken together, these findings indicate that neonatal injection of alloantigens in BALB/c mice induces a state of dissociated tolerance, with unresponsiveness of anti-donor T cells secreting IL 2 on the one hand, and persistence of T cells responsible for B cell help and IL 4 secretion on the other hand.     

Short-term oral administration of ginseng extract induces type-1 cytokine production

Ginseng radix (Panax ginseng C.A. Meyer) is a popular herbal medicine used as a major ingredient in tonic recipes in eastern Asian countries. In our study, male BALB/c mice were treated orally with various doses of ginseng root extract for 5 consecutive days. The extract reduced the serum level of IgG but elevated the level of IgA. Under in vitro condition, the lipopolysaccharide-stimulated spleen cells from the ginseng-treated mice also showed a significant decrease in IgG production but an increase in IgA production. The serum level and production of IgM was unaffected. The interleukin-2, interferon-γ (Th1-type cytokines), and interleukin-10 (Tr1-type cytokine) production by Con A-stimulated spleen cells from the ginseng-treated mice showed an upregulation relative to the control group. However, the production of interleukin-4 (Th2-type cytokine) showed no significant change. The activity of natural killer cells was increased in the ginseng group, but the percentages of T-lymphocytes (CD3⁺) and CD4⁺8^-, CD4^-8⁺ subset were reduced. Thus, short-term oral administration of ginseng extract appears to enhance Th1-type cytokine production.     

Antigen dose defines T helper 1 and T helper 2 responses in the lungs of C57BL/6 and BALB/c mice independently of splenic responses

To investigate the effect of antigen dose on immune response, C57BL/6 and BALB/c mice were sensitized with aluminum hydroxide gel (alum)-precipitated ovalbumin (OVA) then challenged with aerosolized OVA. Low-dose sensitization (less than 8 microg of OVA) elicited T helper 2 (Th2)-type immunoglobulins (Igs) secretion from C57BL/6 mice, including high levels of serum IgE, IgG1 and low levels of IgG2a, while BALB/c mice secreted T helper 1 (Th1)-type Igs, including low levels of IgE, IgG1 and high levels of IgG2a. In contrast, high-dose sensitization (more than 50 microgram) elicited Th1-type Igs secretion in C57BL/6mice, while BALB/c mice exhibited Th2-type Igs secretion. Furthermore, the number of eosinophils infiltrating into the lungs of low-dose OVA-sensitized C57BL/6 mice was significantly greater than in BALB/c mice sensitized with the same amount of OVA. Only a very high dose of OVA (1 mg) could induce greater eosinophil infiltration into the lungs of BALB/c mice compared with C57BL/6 mice. Additionally, low-dose sensitization generated Th2-type cytokines, including high levels of interleukin (IL) -4, IL-5 and a low level of interferon-gamma (IFN-gamma) in the lungs of C57BL/6 mice, while BALB/c mice generated Th1-type cytokines in their lungs, including low levels of IL-4, IL-5 and a high level of IFN-gamma. In contrast, high-dose sensitization elicited Th1-type cytokines production in the lungs of C57BL/6 mice, while BALB/c mice generated Th2-type cytokines in their lungs. Interestingly, splenocyte cultures from C57BL/6 mice produced Th1-type cytokines, while cultures from BALB/c mice produced Th2-type cytokines regardless of OVA sensitization dose (100 ng-1 mg). These results indicate that C57BL/6 and BALB/c mice have different susceptibilities to OVA-sensitization and OVA-induced pulmonary eosinophilia regulated by Th1- and Th2-type cytokines, independent of splenic Th1- and Th2-type cytokines production.     

CD19-deficient mice exhibit poor responsiveness to oral immunization despite evidence of unaltered total IgA levels, germinal centers and IgA-isotype switching in Peyer's patches

CD19 exhibits a critical role as a response regulator in B cells, influencing activation, differentiation and survival. Accordingly, CD19-deficient mice largely lack B-1 cells, and their conventional B-2 cells are poor responders to thymus-dependent antigen. Since both B-1 and B-2 cells may contribute to the total intestinal IgA production, we investigated whether lack of CD19 negatively affected mucosal immunity. We found that CD19(-/-) mice have near normal total IgA levels in serum and gut mucosa and, contrary to systemic lymphoid tissues, Peyer's patches (PP) had germinal centers to which also IgA+ B cells localized. However, the mice demonstrated severely impaired responses to oral immunization with keyhole limpet hemocyanin plus cholera toxin adjuvant. Mucosal responses to oral immunization were significantly more impaired than systemic responses. Despite normal specific IL-4 production, a selective defect in Th2-regulated B cell isotypes was observed, with poor or no mucosal IgA, low serum IgG1 and no IgE, but intact serum IgA and IgG2a production. Ex vivo experiments revealed strongly inhibited CD40-stimulated proliferation and IgA differentiation in CD19-deficient PP B cells. Taken together, an impaired CD40 responsiveness selectively affected Th2, but not Th1, coordinated B cell responses. The CD19(-/-) mice provide compelling evidence for the differential regulation of serum and mucosal IgA immunity.     

Oral immunization of interleukin-4 (IL-4) knockout mice with a recombinant Salmonella strain or cholera toxin reveals that CD4+ Th2 cells producing IL-6 and IL-10 are associated with mucosal immunoglobulin A responses.

Mucosal immunoglobulin A (IgA) responses are often associated with Th2-type cells and derived cytokines, and interleukin-4 (IL-4) knockout (IL-4-/-) mice with impaired Th2 cells respond poorly to oral antigens. However, we have noted that IL-4-/- mice have normal mucosal IgA levels, which led us to query whether different oral delivery systems could elicit mucosal immunity. Two oral regimens were used: (i) a live recombinant Salmonella strain which expresses fragment C (ToxC) of tetanus toxin, and (ii) soluble tetanus toxoid (TT) with cholera toxin (CT) as an adjuvant. Oral immunization of IL-4-/- mice with recombinant Salmonella vaccine expressing ToxC induced brisk mucosal IgA and serum IgG (mainly IgG2a) anti-TT antibody responses. TT-specific CD4+ T cells from spleen or Peyer's patches produced gamma interferon, indicative of Th1 responses; however, IL-6 and IL-10 were also seen. Oral immunization of IL-4-/- mice with TT and CT induced weak mucosal IgA to TT; however, brisk IgA anti-CT-B responses and CT-B-specific CD4+ T cells producing IL-6 and IL-10 were also noted. These results show that although IL-4-dependent antibody responses are impaired, mucosal IgA responses are induced in IL-4-/- mice. These result suggest that certain cytokines, i.e., IL-6 and IL-10 from Th2-type cells, play an important compensatory role in the induction and regulation of mucosal IgA responses.     

A novel locus affecting serum levels of IgG2a maps to the murine H2 region.

The effects of genes in the murine H2 region on basal immunoglobulin levels were investigated and ratios of IgG1/IgG2a were calculated, as low ratios indicate a Th1 cytokine mileu. H2b mice with B10 or BALB genetic backgrounds had higher levels of IgG2a than H2k and H2d congenic strains, and hence had low IgG1/IgG2a ratios. B10 (H2b) mice generally had high levels of IgG2b, IgG3, IgA and IgM, but this outcome was more variable. The high IgG2a phenotype was denoted Igis1 (Immunoglobulin isotype-1) and mapped telomeric of IEbeta using B10.A(4R) mice (high IgG2a) and B10.A(3R) and B10.A(5R) mice (low IgG2a). Further mapping in B10.A(2R), B10.A(1R) and B10.A(18R) mice placed Igis1 in the 27kb region between G7c and G7e.     

Short-Term Oral Administration of Ginseng Extract Induces Type-1 Cytokine Production

Ginseng radix (Panax ginseng C.A. Meyer) is a popular herbal medicine used as a major ingredient in tonic recipes in eastern Asian countries. In our study, male BALB/c mice were treated orally with various doses of ginseng root extract for 5 consecutive days. The extract reduced the serum level of IgG but elevated the level of IgA. Under in vitro condition, the lipopolysaccharide-stimulated spleen cells from the ginseng-treated mice also showed a significant decrease in IgG production but an increase in IgA production. The serum level and production of IgM was unaffected. The interleukin-2, interferon-γ (Th1-type cytokines), and interleukin-10 (Tr1-type cytokine) production by Con A-stimulated spleen cells from the ginseng-treated mice showed an upregulation relative to the control group. However, the production of interleukin-4 (Th2-type cytokine) showed no significant change. The activity of natural killer cells was increased in the ginseng group, but the percentages of T-lymphocytes (CD3⁺) and CD4⁺8^?, CD4^?8⁺ subset were reduced. Thus, short-term oral administration of ginseng extract appears to enhance Th1-type cytokine production.     

Mechanisms of monophosphoryl lipid A augmentation of host responses to recombinant HagB from Porphyromonas gingivalis

Porphyromonas gingivalis, a gram-negative, black-pigmented anaerobe, is among the microorganisms implicated in the etiology of adult periodontal disease. This bacterium possesses a number of factors, including hemagglutinins, of potential importance in virulence. Our laboratory has shown the induction of protection to P. gingivalis infection after subcutaneous immunization with recombinant hemagglutinin B (rHagB). The purpose of this study was to determine if humoral antibody responses are induced after intranasal (i.n.) immunization of rHagB and if monophosphoryl lipid A (MPL), a nontoxic derivative of the lipid A region of lipopolysaccharide, acts as a mucosal adjuvant and potentiates responses to rHagB. Further, the effects of MPL on the nature of the response to HagB and on the costimulatory molecules B7-1 and B7-2 on different antigen-presenting cells (APC) were evaluated. Groups of BALB/c mice were immunized three times (2-week intervals) by the i.n. route with HagB (20 microg) alone or with MPL (25 microg). A group of nonimmunized mice served as control. Serum and saliva samples were collected prior to immunization and at approximately 2-week intervals and evaluated for serum immunoglobulin G (IgG) and IgG subclass and for salivary IgA antibody activity by enzyme-linked immunosorbent assay. Mice immunized with rHagB plus MPL had significantly higher salivary IgA (P < 0.05) and serum IgG (P < 0.05) anti-HagB responses than mice immunized with rHagB alone. The IgG1 and IgG2a subclass responses seen in mice immunized with rHagB plus MPL were significantly higher (P < 0.05) than those seen in mice immunized with rHagB only. Further, the IgG2a/IgG1 ratio in the latter group was approximately 1, whereas in mice immunized with rHagB plus MPL the ratio was <1. These results provide evidence for the participation of T helper (Th) 1 and Th2 cells in responses to rHagB and that MPL potentiates a type 2 response to HagB. MPL was also shown to preferentially up-regulate B7-2 expression on B cells, whereas a preferential increase in B7-1 costimulatory molecule was seen on macrophages and dendritic cells. These results provide evidence that MPL exerts a differential regulation in the expression of costimulatory molecules on APC.