Potent adjuvant action of lipopolysaccharides possessing the O-specific polysaccharide moieties consisting of mannans in antibody response against protein antigen

It was previously reported that Klebsiella O3 lipopolysaccharide (LPS) exhibits extraordinarily strong adjuvant activity in augmenting antibody response against protein antigens in mice compared with other kinds of LPS, for example, LPS from Escherichia coli O55, O111, and O127 and Salmonella enteritidis. The present study was undertaken to clarify the relationship between the strong adjuvant activity in augmenting antibody response against deaggregated bovine gammaglobulin and the chemical structure of LPS. Among LPS from Klebsiella O1, O4, O5, and O7, only O5 LPS exhibited nearly the same degree of the strong adjuvant activity as did O3 LPS. The adjuvant activity of the other LPS was very weak in a degree similar to that of LPS from E. coli O55 and O127. Even when the natural forms of Klebsiella O3 LPS and O1 LPS were converted to various defined uniform salt forms, their adjuvant activity did not significantly differ from that of the respective natural forms. It is therefore unlikely that the difference in strength of the adjuvant activity between Klebsiella O3 LPS and O1 LPS is due to the difference in their salt forms. The common feature in the structures of Klebsiella O3 LPS and O5 LPS is their O-specific polysaccharide chains consisting of the mannose homopolysaccharides (mannans). LPS from E. coli O8 and O9, the O-specific polysaccharide chains of which consist of the mannans, also exhibited much stronger adjuvant activity than do LPS from E. coli O55 and O127, and the strength of the adjuvant activities of the former two was comparable to that of LPS from Klebsiella O3 and O5. On the other hand, LPS from Klebsiella O3 and O5 and E. coli O8 and O9 showed the ability to activate B lymphocytes polyclonally in vivo in a degree similar to that of the other kinds of LPS. From the present results it can be concluded that LPS possessing the O-specific polysaccharide moieties consisting of the mannans exhibit extraordinarily strong adjuvant activity in augmenting antibody response against protein antigen.     

Minimal concentration of human IgM and IgG antibodies necessary to protect mice from challenges with live O6 Escherichia coli

This work evaluated the ability of human anti-lipopolysaccharide O6 IgM and IgG antibodies to protect mice challenged with Escherichia coli serotype O6 : K2ac. Purified IgM-effluent, purified IgG, pools of normal human serum (NHS), or control group were injected into mice 18 h before challenges with O6 E. coli. Interleukin 6 and tumor necrosis factor alpha were quantified in the sera of test and control groups. All mice receiving purified IgM-effluent (66.6 mg L(-1) of anti-lipopolysaccharide O6 IgM antibodies) and NHS survived. Purified IgG (1.1 mg L(-1) of anti-lipopolysaccharide O6 IgG antibodies) protected 87.5% of the animals. The control group showed no protective ability. The minimal concentration of anti-lipopolysaccharide O6 IgM antibodies, able to protect 50% of the animals was 33.3 mg L(-1) of purified IgM-effluent, whereas purified IgG was able to protect 50% of the animals with only 1.1 mg L(-1) of anti-lipopolysaccharide O6 IgG antibodies. Serum from animals pretreated with purified IgM-effluent and purified IgG before challenges with lipopolysaccharide O6 did not have detectable pro-inflammatory cytokines. Hepatocytes of the control group were completely invaded by bacteria, whereas none was found in animals pretreated with purified IgM-effluent and purified IgG. Higher concentrations of anti-lipopolysaccharide O6 IgM antibodies as compared to anti-lipopolysaccharide O6 IgG antibodies were needed to protect mice from challenges with E. coli O6 serotype.     

A possible correlation between histological changes in regional subcutaneous tissue induced by bacterial lipopolysaccharides and their adjuvant activities

Previously it was demonstrated that Klebsiella pneumoniae O3 lipopolysaccharide (KO3 LPS) exhibited much stronger adjuvant action on antibody response to subcutaneously (s.c.) injected sheep red blood cells or deaggregated bovine serum albumin than did other kinds of LPS, the R-form LPS lacking the O-specific polysaccharide chain of KO3 LPS (R-LPS), and the lipid A fractionated from KO3 LPS. We compared histological changes in the regional subcutaneous tissues of mice injected subcutaneously (s.c.) with KO3 LPS, the lipid A, and R-LPS. At the early stage after injection, KO3 LPS induced the infiltration of a large number of inflammatory cells, mainly polymorphonuclear leukocytes (PMN), at the site of injection. Neither R-LPS nor the lipid A induced the accumulation of PMN so much as KO3 LPS did. When injected s.c. with LPS from Escherichia coli O111 (EO111 LPS) and O55 (EO55 LPS), and Salmonella enteritidis (Sent LPS), the appearance of PMN at the regional site was much less than KO3 LPS. KO3 LPS could accumulate more 51Cr-labeled leukocytes at the injection site than EO111 LPS and Sent LPS. Administration of acetylsalicylic acid, which can inhibit leukocyte migration in inflammatory lesions, suppressed its adjuvant action. It was therefore suggested that the strong adjuvant action of KO3 LPS in s.c. injection might be dependent on its potent capability of accumulating PMN at the regional subcutaneous tissue. Furthermore, at the late stage after injection, the formation of several lymphoid follicles at the regional site was seen only in mice injected with KO3 LPS. It might be also related to the strong adjuvant action of KO3 LPS.     

Effects of Bordetella pertussis components on IgE and IgG1 responses

The effect of dermonecrotic toxin (DNT), fimbrial hemagglutinin (FHA), K-agglutinogen, lipopolysaccharide (LPS), and pertussigen from Bordetella pertussis on the production of IgE and IgG1 antibodies to hen egg albumin (Ea) was investigated in C57BL/6 mice. The IgE antibody contents were determined by passive cutaneous anaphylaxis (PCA) in the skin of Lewis rats, while the IgG1 antibody contents were determined by PCA reactions on the skin of mice using sera that had been heated for 3 hr at 56 C to destroy the IgE antibodies. Among the B. pertussis components tested, pertussigen was the most effective adjuvant for increasing the IgE and IgG1 antibodies to Ea. LPS also moderately increased both types of antibodies, and FHA slightly increased the IgG1 titers. When LPS was given 5 days before Ea, it suppressed both IgE and IgG1 titers while FHA had only slight adjuvant action on both type of antibodies. When each of the components was tested for its ability to modify the adjuvant action of pertussigen, it was found that only DNT interfered significantly with the adjuvanticity of pertussigen when given on the day of immunization with Ea. When the components were given 5 days before Ea, DNT produced significant suppression of only the IgG1 response. LPS, FHA, and K-agglutinogen did not significantly affect the adjuvant action of pertussigen.     

Long-term systemic and mucosal antibody responses measured in BALB/c mice following intranasal challenge with viable enterotoxigenic Escherichia coli

The immunogenicity induced in BALB/c mice following intranasal challenge with a viable nonlethal dose (1.2 x 10(8) CFU) of enterotoxigenic Escherichia coli (ETEC) strain E23477A (O139:H28:CS1:CS3:LT+:ST+) was studied over a 140-day period. Serum IgG and IgM antibodies against coli surface antigen 3 (CS3), O139 lipopolysaccharide and heat-labile enterotoxin were measured by day 14 and remained at elevated levels out to day 140. The serum IgG response to the somatic antigens (CS3 and O139 lipopolysaccharide) was significantly greater (P < 0.05) than the IgG response to heat-labile enterotoxin, and the serum IgG response to CS3 was significantly greater (P < 0.05) than the IgG response to O139 lipopolysaccharide. The predominant serum IgG subclasses to CS3 were IgG1 and IgG2a, and they were significantly greater (P < 0.05) than IgG2b and IgG3. The predominant serum IgG subclass response to O139 lipopolysaccharide was initially IgG3 until day 56, after which IgG1 was predominant. The serum subclass response to CS3 indicated a mixed T helper 1/2 (Th1/Th2) profile, whereas the response to O139 lipopolysaccharide was primarily that of a Th2-type, at least over time. Fecal IgG and IgA responses to CS3 and O139 lipopolysaccharide were detected by day 14 and were measured out to day 140, with the CS3 fecal antibody responses being significantly greater (P < 0.05) than the O139 lipopolysaccharide and heat-labile enterotoxin fecal antibody responses. The aim of this study is the development of the intranasal mouse model that can aid in better understanding the immunopathology of ETEC infection and in screening of vaccine candidates prior to volunteer trials.     

Preterm and term neonates transplacentally acquire IgG antibodies specific to LPS from Klebsiella pneumoniae, Escherichia coli and Pseudomonas aeruginosa

High incidences of Gram-negative bacteria are found in neonatal nosocomial infections. Our aim was to investigate placental transmission of immunoglobulin G (IgG) reactive with lipopolysaccharide from Klebsiella pneumoniae, Pseudomonas aeruginosa and Escherichia coli O111, O6 and O26. The total and lipopolysaccharide-specific IgM and IgG were determined in 11 maternal/umbilical-cord sera aged ≤33 weeks (GI); 21 aged >33 and <37 weeks (GII); and 32 term newborns (GIII). The total and lipopolysaccharide-specific IgM concentrations were equivalent in maternal sera. The total IgG concentrations were equivalent in maternal and newborn sera, with the exception of GIII newborns as compared with their mothers (P<0.0001) and with neonates from GI and GII (P<0.05). Lipopolysaccharide-specific IgG concentrations were lower in GI neonates than in their mothers (P<0.01) and lower in GII (P<0.05). Lower lipopolysaccharide-specific IgG levels were observed among neonates only for O111 in GI (P<0.05) and for O26 and Pseudomonas in GII, both as compared with GIII (P<0.05). The anti-lipopolysaccharide IgG transfer ratios were lower in GI (except for O26) and in GII (except for Klebsiella and O111) as compared with GIII (P<0.05). Our results suggest that the greater susceptibility to infections in preterm infants is influenced (besides the humoral response) by factors intrinsic and extrinsic to the condition of prematurity.     

Inhibition by succinyl concanavalin A of strong adjuvant activity of lipopolysaccharides which possess mannans as the O-specific polysaccharide chains

It has been reported that lipopolysaccharides (LPS) from Klebsiella O3 and O5 and Escherichia coli O8 and O9 exhibit extraordinarily strong adjuvant activity in augmenting antibody responses against protein antigens in mice as compared with other kinds of LPS. These four kinds of LPS all possess homopolysaccharides consisting of mannose (mannans) as the O-specific side chains. When these kinds of LPS were mixed in vitro with succinyl concanavalin A (Con A) which is known to bind specifically to alpha-mannoside and alpha-glucoside, their strong adjuvant activity was inhibited. Degree of the inhibition of the adjuvant activity of Klebsiella O3 LPS by succinyl Con A was dependent upon the dose of succinyl Con A. However, phytohemagglutinin, which is known to bind specifically to N-acetyl-D-galactosamine, did not inhibit the adjuvant activity of Klebsiella O3 LPS and O5 LPS. When Klebsiella O3 LPS was mixed with succinyl Con A in the presence of excess amounts of alpha-methyl mannoside or the polysaccharide fraction isolated from Klebsiella O3 LPS, the inhibitory effect of succinyl Con A on the adjuvant activity of Klebsiella O3 LPS was blocked. By contrast, the activity of Klebsiella O3 LPS as a polyclonal B-cell activator was not affected by treatment with succinyl Con A. From these results it is concluded that the mannans, as the O-specific polysaccharide chains of the LPS, significantly contribute to expression of their strong adjuvant activity.     

Monoclonal antibodies against O and K antigens of uropathogenic Escherichia coli O4 : K12 : H− as opsonins

Abstract Monoclonal antibodies of subclasses IgG1 and IgG2b and specific for the O4 antigen of Escherichia coli 20025 (O4 : K12 : H⁻) and the capsular K12 polysaccharide of the same strain (IgM) were obtained with the hybridoma technique using spleen cells from Balb/c mice, immunized with a crude bacterial extract, and Sp2/O-Ag8 myeloma cells. The anti-O4 antibodies reacted exclusively with the O4 lipopolysaccharide and not with those from serologically O-cross reactive E. coli . The anti-K12 antibodies recognized as epitope (part of) the KDO moiety of the capsular K12 polysaccharide. Not only anti-K12, but also anti-O4 antibodies effectively phagoopsonized encapsulated E. coli 20025. The opsonized bacteria were killed in subsequent in vitro phagocytosis by human leokocytes in the presence of human serum complement.     

Homologous and heterologous antibody responses to lipopolysaccharide after enterohemorrhagic Escherichia coli infection

To evaluate antibody responses against lipopolysaccharide (LPS: O157, O26, and O111) in enterohemorrhagic Escherichia coli(EHEC) infection, sera of 24 schoolchildren associated with the Morioka outbreak in 1997 and of 74 sporadic patients suspected of having EHEC infection were examined. Using a positive standard serum, quantitative evaluation of LPS antibodies by an enzyme-linked immunosorbent assay (ELISA) was established. High levels of specific IgM and IgA antibodies against homologous E. coli LPS were present in the acute period and are characteristic of EHEC. This could be used for the serological diagnosis of EHEC infection, except for early infants and the elderly. In addition to the specific homologous response, multiple antibody responses against different serotypes other than those isolated were demonstrated in many cases by qualitative analysis using Western blotting.     

Immunoprotective murine monoclonal antibodies specific for the outer-core polysaccharide and for the O-antigen of Escherichia coli 0111:B4 lipopolysaccharide (LPS)

The antigen specificity of two immunoprotective monoclonal antibodies derived from mice immunized with Escherichia coli 0111:B4 bacteria and boosted with purified lipopolysaccharide (LPS) were investigated. One of the antibodies, B7, was shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunostaining to bind to the O-antigen containing LPS species, whereas the other antibody, 5B10, reacted with both O-antigen containing homologs and the O-antigen-deficient LPS. 5B10 did not bind to LPS from E. coli J5, an Rc mutant of E. coli 0111:B4 that lacks both the O-antigen and outer core sugars. 5B10 did not cross-react with LPS from several other E. coli strains. Thus 5B10 appeared to recognize a type-specific epitope in the outer core of LPS exclusive of Rc determinants. The monoclonal antibody specific for the polymeric O-antigen is of the IgG3 subclass, and the monoclonal antibody 5B10 specific for the outer core of LPS is an IgG2a. Although B7 and 5B10 were equally able to protect mice from a lethal challenge of E. coli 0111:B4 organisms, the outer core-specific IgG2a antibody was much more efficient at mediating the binding of human complement C3 than the O-antigen-specific IgG3 monoclonal antibody.     

B subunit of Escherichia coli heat-labile enterotoxin as adjuvant of humoral immune response in recombinant BCG vaccination

The B subunit of Escherichia coli heat-labile enterotoxin (LTB), a nontoxic molecule with potent biological properties, is a powerful mucosal and parenteral adjuvant that induces a strong immune response against co-administered or coupled antigens. In this paper, the effect of LTB on the humoral immune response to recombinant BCG (rBCG) vaccination was evaluated. Isogenic mice were immunized with rBCG expressing the R1 repeat region of the P97 adhesin of Mycoplasma hyopneumoniae alone (rBCG/R1) or fused to LTB (rBCG/LTBR1). Anti-R1 systemic antibody levels (IgG1, IgG2a, IgG2b, IgG3, IgM, and IgA) were measured by ELISA using recombinant R1 as antigen. With the exception of IgM, LTB doubled the anti-R1 antibody levels in rBCG vaccination. The IgG1/IgG2a mean ratio showed that both rBCG/LTBR1 and rBCG/R1 induced a mixed Th1/Th2 immune response. Interestingly, anti-R1 serum IgA was induced only by rBCG/LTBR1. These results demonstrate that LTB has an adjuvant effect on the humoral immune response to recombinant antigens expressed in BCG.     

Serum and Egg Antibody Responses in Chickens to Escherichia coli

The effects of Freund’s adjuvants on antibody production in chickens against E. coli whole cells were examined. The levels of anti-E. coli IgG antibodies in serum were higher when Freund’s complete (FCA) or incomplete adjuvant (FIA) was administered than that without adjuvant. Production of antibodies recognizing E. coli cells and their lipopolysaccharide was enhanced by FIA, while both FIA and FCA enhanced production of antibodies recognizing outer membrane components. In contrast, serum IgM antibody levels were higher when no adjuvant was used. Anti-E. coli IgG antibodies in serum were efficiently transferred to egg yolk, giving antibody activity in egg yolk similar to that in serum. However, anti-E. coli IgM antibodies were not detected in the egg, suggesting that egg (white) IgM was not influenced by antigenic stimulation of the humoral immune system. Antimicrobial activity of the egg yolk IgG was highest when the bacteria antigen was injected with FIA.     

Intranasal administration of a detoxified endotoxin vaccine protects mice against heterologous Gram-negative bacterial pneumonia

When given passively or elicited actively, antibodies induced by a detoxified Escherichia coli J5 mutant lipopolysaccharide (J5dLPS)-group B meningococcal outer membrane protein (-OMP) vaccine previously protected animals from lethal sepsis. To assess the use of this vaccine for the treatment of Gram-negative bacillary pneumonia, we vaccinated mice, with or without the adjuvant CpG, by intranasal (i.n.) or intraperitoneal (i.p.) routes of administration. Local and systemic IgG levels were 2-3 logs higher following i.p. immunization compared to i.n. However, i.n. immunization elicited both local and systemic IgA, unlike i.p. administration. The addition of CpG to the vaccine, by either route of administration, elicited greater levels of antibody. Intranasal immunization protected mice against lethal heterologous Gram-negative bacillary pneumonia and post-immunization serum and broncho-alveolar lavage fluid mediated enhanced bacterial killing with peritoneal and alveolar macrophages in vitro. We conclude that further studies on the use of J5dLPS-OMP for the prevention of nosocomial pneumonia are warranted.     

Further Studies of the Polysaccharide of Klebsiella pneumoniae Possessing Strong Adjuvanticity: III. Augmentation of the Antibody Response to Subcutaneously Injected Sheep Red Blood Cells by the Adjuvant Polysaccharide

The adjuvant action of the O3 antigen of Klebsiella (KO3) on the antibody response to sheep red blood cells (SRBC) was elucidated by injecting both KO3 and SRBC subcutaneously at the right inguinal region of SMA mice. We demonstrated that KO3 exhibits a novel ability to augment anti-SRBC plaque-forming cell responses in both the local lymph node and the spleen at a relatively late stage of immunization. Escherichia coli lipopolysaccharide, dextran sulfate and concanavalin A showed such an action only minimally. In parallel with the development of the adjuvant action, KO3 definitely activated B cells in the local lymph node polyclonally for either IgM or IgG synthesis, suggesting that the mechanism of the adjuvant action includes direct stimulation of B cells by KO3 at the late stage. Neither increase in trapping of lymphocytes in the local lymph node nor change in tissue distribution of antigen was shown to be primarily involved in the mechanism of the adjuvant action.     

Heterogeneity of antibody response to Salmonella lipopolysaccharide measured by passive hemagglutination and hemolysis in mice

The complement-requiring passive hemolysis test with Salmonella typhimurium lipopolysaccharide-coated sheep erythrocytes is more sensitive for antibodies directed against the lipopolysaccharide than is the passive hemagglutination test. The hemagglutinating and hemolyzing antibodies produced in Swiss mice by hyperimmunization, either with or without Freund's adjuvant, were distributed in both the light and heavy fractions isolated by sucrose density gradient fractionation and gel filtration. IgM fractions, whether tested by hemagglutination or hemolysis, were sensitive to 2-mercaptoethanol (0.15 m). On the other hand, IgG hemolytic antibodies were more sensitive to 2-mercaptoethanol than were IgG hemagglutinating antibodies. The resistance of IgG hemagglutinating activity amounted to about 72 to 95% of the total IgG recovered, whereas the resistant portion of the IgG hemolytic activity was approximately 40 to 53%. It is suggested that, although mercaptoethanol sensitivity is not a definitive test for IgM antibody, its use in connection with the hemagglutination test gives at least an approximation of the IgG antibody, whereas the hemolysis test gives a better approximation of maximal measurable antibody against Salmonella lipopolysaccharides.     

The diacylated lipopeptide FSL-1 induces TLR2-mediated Th2 responses

The diacylated lipopeptide FSL-1 enhanced the generation of IgG antibodies in TLR2(+/+) mice, but not in TLR2(-/-) mice, when administered together with hen egg lysozyme as an antigen. Escherichia coli lipopolysaccharide enhanced the generation of antigen-specific antibodies in both TLR2(-/-) and TLR2(+/+) mice. In TLR2(+/+) mice, the level of enhancement due to FSL-1 was similar to that caused by lipopolysaccharide. Analysis of the IgG antibodies subclass demonstrated that the level of Th2-type IgG1 antibodies was higher than that of Th1-type IgG2a antibodies. Both FSL-1 and lipopolysaccharide induced production of IL-10 and IL-6 by splenocytes from TLR2(+/+) mice. Lipopolysaccharide also induced production of these cytokines by splenocytes from TLR2(-/-) mice, but FSL-1 did not. Neither FSL-1 nor lipopolysaccharide induced IL-12p70 production by splenocytes from either type of mice. FSL-1 upregulated B7.2 expression in B220(+) cells from TLR2(+/+) mice but not those from TLR2(-/-) mice, whereas lipopolysaccharide upregulated B7.2 expression in B220(+) cells from both types of mice. FSL-1 and, to a lesser extent, lipopolysaccharide activated mitogen-activated protein kinases in splenocytes. FSL-1 and, to a lesser extent, lipopolysaccharide induced the expression of c-Fos, which is known to be involved in Th2-type responses, in splenocytes. Thus, this study demonstrated that FSL-1 possessed TLR2-mediated Th2-type responses in vivo.     

Further studies of the polysaccharide of Klebsiella pneumoniae possessing strong adjuvanticity. II. Serological relationship between the adjuvant polysaccharide and O3 antigen of Klebsiella

The serological specificity of the neutral polysaccharide possessing extraordinarily strong adjuvanticity originally isolated from the culture supernatant of Klebsiella K1 strain Kasuya has been investigated. Among all of the reference strains (K1-K82) of Klebsiella obtained from the International Escherichia and Klebsiella Center, Statens Seruminstitut, Copenhagen, only 13 strains have been shown to produce the adjuvant polysaccharide by the passive hemagglutination inhibition test. All of these 13 strains belong to the O3 group, and the strains which belong to other O groups of which were not identifiable did not produce it. The gel precipitation test has demonstrated that the adjuvant polysaccharide is antigenically identical to O3 antigen isolated from the cells of the decapsulated mutant (strain LEN 1) of Klebsiella K1 strain Kasuya and to O9 antigen of Escherichia coli isolated from either the culture supernatant or the cells, which has already been shown to be antigenically and structurally identical to the O3 antigen of Klebsiella.     

IgG-mediated systemic anaphylaxis to protein antigen can be induced even under conditions of limited amounts of antibody and antigen

Systemic anaphylaxis is an acute, severe, and potentially fatal allergic reaction. Two classes of antibodies, IgE and IgG, contribute to the development of anaphylaxis in mice, through different mechanisms with distinct usage of effector cells and chemical mediators. Larger quantities of antibody and antigen are reportedly required to induce IgG-mediated anaphylaxis than IgE-mediated one, suggesting that the former may not happen as frequently as the latter in real life. To readdress this issue, we established in the present study a novel mouse model of passive IgG-mediated systemic anaphylaxis to a native protein antigen, ovalbumin (OVA), rather than artificially haptenated protein antigens used in previous studies. Passive sensitization of mice with a cocktail of but not individual IgG1 mAbs specific to distinct OVA epitopes elicited systemic anaphylaxis in response to OVA challenge. Importantly, much smaller doses of antibody and antigen than previously reported were sufficient for the induction of IgG-mediated systemic anaphylaxis. Moreover, a relatively small dose of antigen could induce severe anaphylaxis through both IgE- and IgG-mediated mechanisms when mice had been passively sensitized with antigen-specific IgE and IgG. These results strongly suggest that IgG-mediated systemic anaphylaxis is not rare among antibody-mediated systemic anaphylaxis, in contrast to previous thought, and significantly contributes to active systemic anaphylaxis in real life, at least in mice.     

Cellular basis of in vitro anti-DNA antibody production: evidence for T cell dependence of IgG-class anti-DNA antibody synthesis in the (NZB X NZW)F1 hybrid

An in vitro system was designed to measure anti-DNA antibody synthesis, and the cellular basis of this autoantibody production in NZB X NZW (B/W)F1 (B/W F1) mice was analyzed. The spleen cells from old B/W F1 mice contained a number of B cells that spontaneously produced anti-DNA antibodies of both IgM and IgG classes in the absence of stimulants, thereby demonstrating that these B cells had been activated in vivo. These activated B cells could be removed by Sephadex G-10 column (G-10) filtration. Such G-10-passed, homogeneously small B cells were activated by the stimulant lipopolysaccharide (LPS) and produced both IgM and IgG class anti-DNA antibodies. The G-10-passed cells contained both B and T cells, and the cytotoxic treatment of the cells with monoclonal antibodies to T cells, anti-Thy-1 and anti-L3T4, abolished the LPS-induced IgG class, but not IgM class, anti-DNA antibody syntheses. Thus, the LPS-induced production of IgG class anti-DNA antibodies in B/W F1 mice is regulated by T cells. Reconstitution experiments revealed the requirement of T-B cell contact but not of the proliferative response of T cells. Moreover, there was no apparent adherent cell requirement. Such IgG class anti-DNA antibodies were produced only by spleen cells from old B/W F1 mice, but not from young B/W F1, NZB, NZW, and C57BL/6 mice. Like IgM class anti-DNA antibodies, LPS-induced synthesis of polyclonal IgM was T cell-independent. Only a slight reduction in the polyclonal IgG synthesis was observed after the G-10-passed cells had been treated with anti-Thy-1 antibody plus complement. This study should facilitate investigation of cell to cell interactions in the formation of autoantibodies and their correlations to immunologic abnormalities in autoimmune disease.     

Defective oral tolerance promotes nephritogenesis in experimental IgA nephropathy induced by oral immunization.

Oral tolerance, an important feature of the mucosal immune system, appears to protect against immune-mediated disease by blunting production of systemic IgG and IgM antibody directed toward immunogens chronically present at mucosal surfaces. In this study, we explored the role of oral tolerance and mucosal immunoregulation in an experimental model of IgA nephropathy (IgAN), an important form of nephritis in humans. Cyclophosphamide and estradiol were used to inhibit the expression of oral tolerance, which otherwise develops after chronic oral presentation of Ag. BALB/c mice given drinking water containing 0.1% bovine gamma globulin (BGG) continuously for 14 wk were randomly assigned to groups given either 2 mg of cyclophosphamide i.p., 2 mg of estradiol s.c. or both drugs. Groups of control mice received neither BGG nor drugs. In three separate experiments, a low percentage of saline-treated orally immunized mice had microscopic hematuria (0 to 20%), as did nonimmunized controls (0 to 20%). However, 58 to 83% of mice given estradiol and/or cyclophosphamide at appropriate times developed significant hematuria. If drugs were given at suboptimal times, only 25 to 56% of mice developed hematuria. Drug-treated immunized mice also had more serum IgG and IgM anti-BGG antibodies than control and saline groups. Immunofluorescence showed significantly more glomerular deposits of IgG, IgM, and C3 in drug-treated immunized mice compared to saline-treated immunized and normal untreated control mice. Hematuria and glomerular deposits of IgG, IgM, and C3 paralleled serum IgG and IgM antibody. All immunized mice showed significant mesangial IgA and BGG deposits and there were no differences in such deposits between saline- and drug-treated immunized mice. We suggest that blunting of oral tolerance with promotion of systemic IgG and IgM antibody production leads to nephritis in chronically orally immunized mice and that glomerular immune complexes containing IgG and/or IgM promote complement deposition and hematuria in IgAN. Analogous defects in oral (or more generally mucosal) tolerance could play a role in the genesis of symptomatic human IgAN.