Induction of phenotypic lymphocyte differentiation in LPS unresponsive mice by an LPS-induced serum factor and by lipid A-associated protein.

Spleen cells from C3H/HeJ mice are known to be unresponsive to mitogenic stimulation by LPS. We show here that T and B cell precursors of C3H/HeJ mice are unresponsive to induction of differentiation by LPS. Phenotypic differentiation of C3H/HeJ lymphocytes can be induced with DB-cAMP, Lipid A-associated protein, and with a serum factor induced by LPS in LPS responder strains.     

Neointimal formation in two apolipoprotein E-deficient mouse strains with different atherosclerosis susceptibility

C57BL/6 (B6) and C3H/HeJ (C3H) are two commonly used mouse strains that differ markedly in atherosclerosis susceptibility. In this study, we determined plaque formation after removal of the endothelium in the two strains carrying the mutant apolipoprotein E gene (apoE(-/-)). At 10 weeks of age, male B6.apoE(-/-) and C3H.apoE(-/-) mice underwent endothelial denudation of the left common carotid artery. Two weeks after injury, B6.apoE(-/-) mice developed significantly larger neointimal lesions in the vessel than their C3H.apoE(-/-) counterparts, although they had comparable plasma cholesterol levels on a chow diet. Feeding of a Western diet aggravated lesion formation in both strains, but the increase was more dramatic in B6.apoE(-/-) mice than in C3H.apoE(-/-) mice. Immunohistochemical and histological analyses demonstrated the presence of macrophage foam cells in neointimal lesions. We then compared neointimal growth in F1 mice reconstituted with bone marrow from B6.apoE(-/-) and C3H.apoE(-/-) mice. No significant lesions were observed 2 weeks after endothelial denudation in the mice reconstituted with bone marrow from either donor. Thus, these data indicate that foam cell formation contributes to neointimal growth in the hyperlipidemic apoE(-/-) model and that neither endothelial cells nor blood cells alone explain the dramatic difference between B6 and C3H mice in plaque formation.     

Autoreactive antibody-forming cells directed against thymocytes and thymus-derived lymphocytes.

Antibody-forming cells with specificities against syngeneic and allogeneic thymocytes are detected in the spleens of normal mice after activation in vitro or in vivo with lipopolysaccharide (LPS). The activity of such cells was measured in a complement-dependent plaque assay employing trypan blue dye to assess zones of lysis. Plaques were rarely seen in the absence of LPS treatment. Anti-immunoglobulin added to the plaque assay abrogated the appearance of plaques, but the addition of LPS had no effect. Furthermore, plaque formation was 2-mercaptoethanol sensitive indicating that the antibody responsible was of the IgM class. Plaque forming cells (PFC) were also detected against syngeneic and allogeneic lymph node cells and to a much lesser extent against splenocytes. The numbers of PFC found against syngeneic, allogeneic, or a mixture of thymocytes was similar and ranged from 1000 to 3000 PFC/10(8) viable spleen cells tested. All murine strains tested, including congenitally athymic nude mice, exhibited anti-thymocyte PFC after LPS activation. C3H/HeJ mice, genetically unresponsive to LPS, did not respond mitogenically to LPS and no anti-thymocyte plaques were observed. These findings suggest that clones of autoreactive B cells are present in normal mice and can be activated by LPS.     

Suppressive effect of interferon-beta-activated natural killer cells on lipopolysaccharide-induced B cell differentiation of MRL/Mp-lpr/lpr mice

The suppressive effects of mouse recombinant interferon-beta (IFN-beta) on B cell differentiation of MRL/Mp-lpr/lpr (MRL/1) mouse, a model of autoimmune diseases, and C3H/H2 mice, a normal situation, were investigated. Spleen mononuclear cells were cultured in the presence of lipopolysaccharide (LPS), and the suppressive effect of IFN-beta was examined on differentiation of B cells to plaque-forming cells (PFCs) by highly sensitive reversed hemolytic plaque assay. IFN-beta (5,000-10,000 units/ml) suppressed more than 50% of PFCs of both MRL/1 and C3H/H2 mice. This suppressive activity as well as the cytotoxicity of natural killer (NK) cells enhanced by IFN-beta was abrogated by treatment of the spleen cells with anti-asialo GM1 antibody in the presence of complement. This suppressive activity was also abrogated by intravenous administration of 20 microliter/mouse of anti-asialo GM1 12 hr before cultivation of spleen cells. These results suggest that NK cells activated by IFN might be responsible for the immunoregulation in autoimmune diseases.     

Differentiation of B lymphocytes in C3H/HeJ mice: the induction of Ia antigens by lipopolysaccharide.

The lipid A moiety of bacterial lipopolysaccharide (LPS) elicits several types of responses in murine B lymphocytes. First, lipid A induces the nonproliferative expression of cell surface antigens in more immature cell types. Second, lipid A induces a mitogenic response in more mature B cell types. Lipid A induces the expression of Ia antigens on bone marrow cells from C3H/DiSn but not C3H/HeJ mice. The Ia-inducible cells possess surface immunoglobulin. Agents that elevate intracellular levels of adenosine 3',5'-cyclic monophosphate (cyclic AMP) induce the appearance of Ia antigens on B lymphocytes from both C3H/HeJ and C3H/DiSn mice, suggesting that lipid A exerts its inductive effects by increasing cyclic AMP levels in cells. In contrast to what is observed by using other strains of mice, mature B lymphocytes from C3H/HeJ mice do not support a mitogenic response to lipid A. The subpopulation of B lymphocytes in C3H/HeJ mice that normally respond mitogenically to LPS not only appear to lack an LPS-response mechanism utilized in the mitogenic pathway, but they lack the LPS-response pathway of the immature B cell types. A lipid A-bound protein (LAP) induces both the expression of Ia and a mitogenic response in the different subpopulations of B lymphocytes from C3H/HeJ and C3H/DiSn mice. The genetic defect in C3H/HeJ mice that limits responses to lipid A may be associated with a receptor that is normally expressed on many different cell types.     

Intrinsic B lymphocyte and macrophage defects in C3H/HeJ mice

C3H/HeJ mice are genetically defective in their responsiveness to lipopolysaccharide (LPS). Their B cells also have a characteristically low cloning efficiency in semisolid agar cultures, where LPS does not seem to be required. Adherent macrophages facilitate clonal proliferation in such cultures via diffusible substances. C3H/HeJ macrophages functioned poorly in this respect, and addition of normal C3HeB/FeJ macrophages to cultures of C3H/HeJ B cells did not lead to normal colony numbers. Although immune interferon can stimulate normal resident peritoneal macrophages to function well in semisolid agar cultures, it did not improve the cloning efficiency of C3H/HeJ cells. Similarly, addition of indomethacin or interleukin 1 to the cultures did not reveal that abnormally elevated production of prostaglandins or a deficiency in interleukin 1 are responsible for poor C3H/HeJ colony formation. These results indicate that C3H/HeJ mice have defects intrinsic to both B cells and macrophages that are not overcome by interferon. Purified B cells from these mice cloned poorly and did not respond to stimulation in liquid cultures with anti-mu-coated beads plus factors. There was a tendency for the poor cloning of C3H/HeJ B cells to improve with age.     

Frequency analysis of functional immunoglobulin C and V gene expression in murine B cells at various ages

The frequencies of lipopolysaccharide- (LPS) reactive B cells and their antibody specificity repertoire have been determined in the spleen and bone marrow (BM) of mice at different ages. A limiting dilution culture system was employed that allows the growth and development of every LPS-reactive B cell into a clone an IgM-secreting cells that are capable of switching to other Ig heavy chain isotypes (C gene expression). The secretion of IgM and IgG1 was assessed in the protein A plaque assay, whereas specific IgM antibody-secreting cells (V gene expression) were detected with the use of plaque assays specific for various heterologous erythrocytes and sheep red blood cells (SRBC) coupled with a number of different haptens. The frequencies of LPS-reactive B cells in the spleen and BM of C3H/Tif, C57BL/Ka, BALB/c, and CBA/Rij mice appeared to be similar in 6- to 12- and 100-wk-old animals, as was the switch frequency to IgG1 secretion in three strains tested. Moreover, no age-related changes were observed in the frequencies of antigen-specific B cells within the pool of LPS-reactive B cells in the spleen and BM of C57BL/Ka mice. The frequencies ranged from 1 in 10 to 1 in 20 for NIP4- and NNP2-SRBC, from 1 in 50 to 1 in 100 for TNP30-SRBC, and from 1 in 1000 to 1 in 4000 for SRBC, HRBC, and GRBC. The specificity repertoire of the "spontaneously" occurring ("background") IgM-secreting cells in the spleen and BM, on the other hand, did differ between young and old C57BL/Ka mice. During aging the frequencies of the tested specificities decreased in the spleen but increased in the BM. Our data indicate that in unintentionally immunized mice the clonal selection of B lineage cells by antigen takes place at the level of the mature, antigen-reactive B cell.     

LPS regulation of the immune response: separate mechanisms for murine B cell activation by lipid A (direct) and polysaccharide (macrophage-dependent) derived from Bacteroides LPS

Lipopolysaccharide (LPS) derived from Bacteroides fragilis has been reported to stimulate mitogenic responses in spleen cell cultures from the classical LPS-hyporesponsive C3H/HeJ mouse strain; however, we have shown that purified splenic B cells from C3H/HeJ mice are hyporesponsive to phenol-water extracted LPS from B. fragilis ATCC 25285 (B-LPS). In the present study, B-LPS and its purified lipid A and polysaccharide components were tested for their ability to induce mitogenic and polyclonal IgM synthesis in spleen cell and purified splenic B cell cultures from classical LPS-responsive and -hyporesponsive mice. Mitogenic responses to B-LPS and E. coli K235 LPS(Ph) of whole spleen cells (2 X 10(5) cells/culture) or purified B cells (5 X 10(5) cells/culture) from classical LPS-responsive mouse strains (C3H/HeN, BALB/c, C57BL/6J, C57BL/10Sn, and DBA/2), F1 mice (derived from crosses between LPS responsive and C3H/HeJ mice), and classical LPS-hyporesponsive mice (C3H/HeJ and C57BL/10ScN) were high, intermediate, and low, respectively. When a higher number of whole spleen cells (5 X 10(5) cells/well) were cultured, B-LPS induced high mitogenic responses in C3H/HeN, intermediate responses in F1, and lower but significant responses in C3H/HeJ cultures. Similar results were obtained when polyclonal IgM synthesis was assessed in cultures containing 1 X 10(6) cells/culture. In contrast, the purified lipid A component of B-LPS failed to induce mitogenic responses in either whole spleen or purified B cell cultures. The addition of purified splenic B cells from C3H/HeJ mice to C3H/HeN or C3H/HeJ splenic adherent cells resulted in mitogenic responses to B-LPS, implying that the hyporesponsiveness to B-LPS seen in whole spleen cell cultures from C3H/HeJ mice at the lower cell concentration was due to limiting numbers of M phi. When splenic B cells and M phi from either C3H/HeN or C3H/HeJ mice were incubated with the lipid A or the polysaccharide moiety of B-LPS, lipid A induced mitogenic responses only in C3H/HeN cultures, whereas the polysaccharide moiety induced similar responses in both C3H/HeN and C3H/HeJ cultures. These results suggest that Bacteroides lipid A does not stimulate B cells from the classical LPS-hyporesponsive C3H/HeJ mouse strain, whereas the polysaccharide moiety of B-LPS is biologically active and mediates B cell stimulation via M phi.     

Relationship between immune system and gram-negative bacteria. IV. T lymphocytes from Lpsd mice possess binding site(s) for Rb Salmonella

We have previously shown that Salmonella minnesota R345 (Rb) spontaneously binds to 50 to 55% of human peripheral blood mononuclear cells (PBMC). In the present study, we have compared Rb cytoadherence to lymphoid cells from various tissues of lipopolysaccharide (LPS) hyporesponsive (Lpsd) and LPS responsive (Lpsn) mouse strains. A higher number of spleen cells from Lpsd mice (C3H/HeJ and C57BL/10ScN) bound Rb bacteria (22 to 30%) than cells from Lpsn mice (4 to 9%). Rb bound mainly to T cells, and cytoadherence occurred in both Lyt-1+ and Lyt-2+ T cell subsets. By contrast, purified splenic B cells from Lpsd and Lpsn mice gave less than 4% Rb cytoadherence. In both mouse strains, cytoadherence was mediated by the homologous LPS structure, because purified Rb-LPS blocked Rb Salmonella binding to T cells. On the other hand, smooth Salmonella typhimurium LT-2 LPS (S-LPS) and Salmonella R595 (Re) LPS (Re-LPS), which contain mainly lipid A, were without effect on Rb binding. Increased Rb binding was seen with T cells from Peyer's patches (PP), mesenteric lymph nodes (MLN), and peripheral blood than from spleen of C3H/HeN (Lpsn) mice; however, greater cytoadherence was always seen with T cells of these tissues from C3H/HeJ mice. Interestingly, treatment of whole spleen or purified T cells from C3H/HeN mice with neuraminidase enhanced cytoadherence to levels seen with C3H/HeJ cells. The observed Rb binding to PP, MLN, and PBMC cells in both mouse strains suggests that gut microbial environment may play an important role in Rb cytoadherence. This is also supported by the evidence that when spleen cells of germfree and conventional mice were tested for Rb binding, higher cytoadherence was observed in conventional mice only. Taken together, these results indicate that T cells of Lpsd mice express binding site(s) for Salmonella, whereas Lpsn mice have T cells with these structure(s) in a cryptic configuration.     

Serological properties and immunobiological activities of lipopolysaccharides from black-pigmented and related oral Bacteroides species

Lipopolysaccharides (LPS) from five species of oral Bacteroides, B. gingivalis strains 381 and ATCC 33277, B. oralis ATCC 33269, B. loescheii ATCC 15930, B. intermedius ATCC 25611 and B. corporis ATCC 33547, were extracted from whole cells by the phenol/water procedure, and subsequently purified by treatment with nuclease and ultracentrifugation. The LPS were composed of hexoses, glucosamine, fatty acids and phosphorus. Heptose and 2-keto-3-deoxyoctonate were not detected. The LPS preparations from B. gingivalis strains 381 and ATCC 33277 presented very similar SDS-polyacrylamide gel electrophoresis patterns when stained with ammoniacal silver. They produced a fused precipitin band against an antiserum to B. gingivalis 381 LPS in immunodiffusion tests. Antisera raised against the LPS from B. loescheii and B. intermedius reacted with the LPS prepared from all the oral Bacteroides strains except those of B. gingivalis. All the LPS preparations were mitogenic for spleen cells of BALB/c (nu/nu) mice, but not for thymus cells from C3H/HeN mice. The LPS induced marked mitogenic responses and polyclonal B cell activation for spleen cells of not only C3H/HeN (LPS responder) mice, but also C3H/HeJ (LPS nonresponder) mice. The mitogenic responses were not suppressed significantly upon addition of polymyxin B to the reaction mixture. These LPS also enhanced interleukin-1 production by murine peritoneal macrophages and mouse cell line J744. 1 macrophages. Hydrolysis of B. gingivalis ATCC 33277 LPS in 1 m-HCl at 100 degrees C for 1 h yielded lipid and polysaccharide. The lipid portion was largely composed of fatty acids and glucosamine, and was mitogenic for spleen cells from C3H/HeJ as well as C3H/HeN mice, while the polysaccharide portion induced no significant mitogenic responses under similar experimental conditions.     

Partial restoration of the lipopolysaccharide-induced proliferative response in splenic B cells from C3H/HeJ mice

C3H/HeJ mice are hyporesponsive to the biologic effects of bacterial lipopolysaccharide (LPS), and their splenic B cells do not proliferate after exposure to LPS. The molecular basis of this hyporesponsiveness is unknown but it may result from defective membrane signal transduction after LPS binding. To examine this possibility, we added bioactive compounds in combination with LPS to C3H/HeJ B cell cultures in order to bypass the putative defect. The addition of PMA, monensin, or ionomycin, either alone or in combination, had no effect on C3H/HeJ B cell responses to LPS. In contrast, the addition of trypsin together with LPS resulted in a partial restoration of the proliferative response in C3H/HeJ splenic B lymphocytes. The maximal C3H/HeJ B cell response varied from 25 to 60% of the C3Heb/FeJ (LPS responder) B cell response. The trypsin-mediated enhancement of the LPS response was abrogated by pretreatment of the trypsin with the trypsin inhibitors DFP or TLCK. Pretreatment of the LPS with polymyxin B, which blocks lipid A-dependent reactions, also abrogated the trypsin effect. Because the C3H/HeJ B cell responds to all other B cell mitogens, we suggest that the defect is in an LPS-specific step and that the action of trypsin results in the restoration of the missing signal. At the present time the identity of this signal is not known, but the experiments described in this report provide a unique model to elucidate the basis of LPS hyporesponsiveness in splenic B cells from C3H/HeJ mice.     

Cellular mechanism of endotoxin unresponsiveness in C3H/HeJ mice.

B cells from C3H/HeJ mice fail to respond to an endotoxin (LPS K235) which is mitogenic for normal mice including the closely related C3H/HeN strain. The cellular basis for this unresponsive state has been investigated. The C3H/HeJ mice have normal numbers of B cells, which are capable of normal responses to other B cell mitogens, such as polyinosinic acid (Poly I). Addition of normal macrophages or spleen cells fails to reconstitute the normal response. Furthermore, neither macrophages nor spleen cells from the C3H/HeJ strain suppress the normal C3H/HeN spleen cells. Finally, spleen cells enriched for B cells by the removal of macrophages or T cells demonstrate the same differences in responsiveness to LPS. These results indicate that LPS unresponsiveness is a defect of the B cell itself and not due to suppressor cells or the absence of helper cells. When LPS is added to Poly I-stimulated cultures, there is additional enhancement of the response of normal C3H/HeN spleen cells. However, LPS causes a dose-dependent suppression of the Poly I response of C3H/HeJ spleen cells. This suppression is dependent on the time of addition of LPS to the Poly I-stimulated cultures. These data are interpreted as indicating that the binding of LPS to the membrane of C3H/HeJ B cells results in their inactivation or suppression, and that this is the basis of LPS unresponsiveness in this mouse strain.     

C57BL/10/CR mice: nonresponders to activation by the lipid a moiety of bacterial lipopolysaccharide.

C57BL/10/CR mice do not respond to the lipid A moiety of LPS. This defect was demonstrated in vivo by a decreased production of the acute phase serum amyloid protein SAA. In addition, the defect was demonstrated in vitro by using cultures of spleen cells and peritoneal cells. No mitogenesis of spleen cells or enhanced glucose utilization by either spleen cells or peritoneal cells was observed when the cells were stimulated by lipid A or phenol-extracted Escherichia coli K235 LPS. The response of these mice to PHA, Con A, Poly I:C, 8BrcGMP, and butanol extraced E. coli K235 LPS was normal. Thus, the inability of lipid A to stimulate B lymphocyte mitogenesis and activate peritoneal cells in vitro may correlate with its inability to induce the acute phase SAA in vivo. B10/CR mice represent another strain, similar to the C3H/HeJ, which is nonresponsive to lipid A and should be useful in eludcidating the mechanism by which bacterial LPS activates cells.     

Regulatory effects of Spirulina complex polysaccharides on growth of murine RSV‐M glioma cells through Toll‐like receptor 4

This study is the first to report that Spirulina complex polysaccharides (CPS) suppress glioma growth by down‐regulating angiogenesis via a Toll‐like receptor 4 signal. Murine RSV‐M glioma cells were implanted s.c. into C3H/HeN mice and TLR4 mutant C3H/HeJ mice. Treatment with either Spirulina CPS or Escherichia coli (E. coli) lipopolysaccharides (LPS) strongly suppressed RSV‐M glioma cell growth in C3H/HeN, but not C3H/HeJ, mice. Glioma cells stimulated production of interleukin (IL)‐17 in both C3H/HeN and C3H/HeJ tumor‐bearing mice. Treatment with E. coli LPS induced much greater IL‐17 production in tumor‐bearing C3H/HeN mice than in tumor‐bearing C3H/HeJ mice. In C3H/HeN mice, treatment with Spirulina CPS suppressed growth of re‐transplanted glioma; however, treatment with E. coli LPS did not, suggesting that Spirulina CPS enhance the immune response. Administration of anti‐cluster of differentiation (CD)8, anti‐CD4, anti‐CD8 antibodies, and anti‐asialo GM1 antibodies enhanced tumor growth, suggesting that T cells and natural killer cells or macrophages are involved in suppression of tumor growth by Spirulina CPS. Although anti‐interferon‐γ antibodies had no effect on glioma cell growth, anti‐IL‐17 antibodies administered four days after tumor transplantation suppressed growth similarly to treatment with Spirulina CPS. Less angiogenesis was observed in gliomas from Spirulina CPS‐treated mice than in those from saline‐ or E. coli LPS‐treated mice. These findings suggest that, in C3H/HeN mice, Spirulina CPS antagonize glioma cell growth by down‐regulating angiogenesis, and that this down‐regulation is mediated in part by regulating IL‐17 production.     

Restoration of LPS responsiveness of C3H/HeJ mouse lymphocytes by microinjection of cytoplasmic factor(s) from LPS-stimulated normal lymphocytes

Cytosol fractions of lymphocytes from LPS-responder mice (C3H/HeN) were prepared and injected into B lymphocytes of LPS-nonresponder mice (C3H/HeJ) by a microinjection technique utilizing polyethyleneglycol-mediated cell fusion. The B lymphocytes of C3H/HeJ mice microinjected with cytosol prepared from LPS-stimulated C3H/HeN cells became normally responsive to LPS. Microinjection of cytosol itself did not stimulate C3H/HeJ cells to proliferate or differentiate into immunoglobulin-producing cells, and the cells injected with cytosol had to be restimulated with LPS in order to proliferate and differentiate. These data suggested that C3H/HeJ B cells acquired LPS responsiveness by microinjection of cytoplasmic factor(s) from LPS-stimulated C3H/HeN cells and that these factor(s) may be one of the components involved in normal signal transmission from cell surface to nucleus in the early stages of the LPS response. The apparent m.w. of the cytoplasmic factor(s) is 100,000 by gel filtration. Chromatofocusing analysis suggested that these factors may consist of two components with the same m.w.     

Frequency analysis of functional immunoglobulin C- and V-gene expression by mitogen-reactive B cells in germfree mice fed chemically defined ultra-filtered "antigen-free" diet

The frequencies of lipopolysaccharide (LPS)-reactive B cells and their antibody specificity repertoire have been determined in the spleen and bone marrow (BM) of conventional (CV) and "antigen-free" C3H/HeCr mice of various ages. The antigen-free mice were germfree (GF)-raised and were fed an ultrafiltered solution of chemically defined (CD) low m.w. nutrients, and were thus devoid of exogenous antigenic stimulation. Spleen and BM cells were grown in a limiting dilution culture system that allows the growth and development of every newly formed LPS-reactive B cell into a clone of IgM-secreting cells which are capable of switching to other immunoglobulin (Ig) heavy chain isotypes (C-gene expression). The secretion of IgM and IgG1 was determined in the protein A plaque assay, whereas specific IgM antibody-secreting cells (V-gene expression) were detected in plaque assays specific for various heterologous erythrocytes and sheep red blood cells (SRBC) coupled with a number of different haptens. The absolute frequency of LPS-reactive B cells and their capacity to switch to IgG1-secretion was not significantly different in 8- to 12-wk-old and 52-wk-old GF-CD mice and their age-matched CV controls. Moreover, no differences were observed in the frequencies of antigen-specific B cells within the pool of LPS reactive B cells. These frequencies ranged from 1 in 20 to 1 in 50 for NIP4-SRBC and NNP2-SRBC, from 1 in 100 to 1 in 150 for NIP0.4-SRBC, from 1 in 50 to 1 in 100 for TNP30-SRBC, and from 1 in 1000 to 1 in 2000 for SRBC and horse red blood cells. Within the limitations of having determined the switching capacity of IgM to IgG1 only and having assessed only a minor fraction of the total B cell antibody-specificity repertoire, the data indicate that young and old GF-CD mice, although devoid of exogenous antigenic and/or mitogenic stimulation, generate B cells with a similar switching capacity and a similar IgM antibody specificity repertoire as CV mice.     

A reappraisal of "T-independent" antigens. II. Studies on single, hapten-specific B cells from neonatal CBA/H or CBA/N mice fail to support classification into TI-1 and TI-2 categories

Spleen cells from adult CBA/H or CBA/N mice, or from neonatal CBA/H mice, were fractionated on thin layers of fluorescein (FLU)-gelatin to yield FLU-specific B lymphocytes. A single cell, or small numbers ranging from 1 to 10, were cultured in 10-microliter microcultures together with various antigens and mitogens. The results were compared with those of bulk culture or limiting dilution cultures supported by thymus filler cells. B cell growth and differentiation-promoting conditioned media (BGDA) were added to some cultures. The CBA/N results gave no support to the commonly used classification of T cell-independent (TI) antigens into TI-1 and TI-2 categories. A typical supposed TI-1 antigen, FLU-LPS, strongly stimulated normal adult single FLU-specific B cells to proliferate and form antibody, but virtually failed to trigger CBA/N B cells of comparable antigen-binding avidity. The same was true of LPS or LPS plus dextran sulfate acting as mitogens. The allegedly TI-2 antigen FLU-Ficoll, although still triggering comparatively poor responses, was actually marginally more active than FLU-LPS. FLU-Brucella abortus (FLU-BA) + BGDA gave the best results with single CBA/N B cells, but still induced only 1.27% of cells to develop into antibody-forming clones vs 12.2% with CBA/H cells. The results obtained with single neonatal B cells also lent no support to the distinction between TI-1 and TI-2. Both "TI-1" and "TI-2" stimuli caused adequate proliferation, one "TI-2" antigen stimulating 23.2% of the cells. None of the antigens caused good antibody formation, however, probably because multivalent antigens can deliver signals impeding the differentiation of immature B cells. It is therefore suggested that the classification of TI-1 antigens into two subcategories be abandoned, at least for the time being.     

LPS regulation of the immune response: suppression of immune responses to orally administered T-independent antigen

The regulation of immune responses to gastrically administered TI antigens has been investigated, and the characterization of a regulatory cell population has been performed. Intragastric administration of TNP-haptenated homologous erythrocytes (TNP-MRBC) induced splenic IgM anti-TNP PFC responses in LPS nonresponsive C3H/HeJ mice that were higher than those in LPS-responsive C3H/HeN mice and similar to those noted in athymic (nu/nu) C3H/HeN animals. The simultaneous intragastric administration of LPS with TNP-MRBC augmented immune responses in a manner similar to that previously reported for parenterally administered LPS and antigen. Further, LPS-induced augmentation of TNP-MRBC responses was greater in athymic mice. These findings were substantiated using in vitro spleen cultures. Intragastric challenge with a 2nd TI antigen, TNP-LPS, induced approximately 8-fold higher splenic anti-TNP PFC responses in athymic C3H/HeN mice compared with those in euthymic littermates. By admixture of B and T cell populations, it was demonstrated that the host responsiveness to TNP-LPS was negatively regulated by suppressor cells. Suppressive activity resided in a Thy 1.2-bearing, irradiation-resistant, nylon wool-nonadherent cell population. These cells could be demonstrated in spleen and Peyer's patches from young or old LPS-responsive C3H/HeN mice, but not in tissues from LPS nonresponsive C3H/HeJ mice. The specificity of the regulator cells was not limited to TNP-LPS responses, since immune responsiveness to another TI antigen, TNP-dextran, was also under the control of this cell population. These studies confirm the TI nature of TNP-MRBC and indicate that immune responses to gastrically administered antigens such as TNP-LPS, TNP-dextran, and possibly TNP-MRBC are negatively regulated by a suppressor T cell population. A role for endogenous LPS in the generation of regulator cells and the effect of these cells on host responses to gut-derived antigens is discussed.     

Mucosal immunoregulation: environmental lipopolysaccharide and GALT T lymphocytes regulate the IgA response

In this review, we have emphasized: 1) bacterial lipopolysaccharide (LPS) involvement in IgA responses to orally administered thymic-dependent (TD) antigens; 2) characterization of Peyer's patch (PP) lymphoreticular cells; and 3) gastrointestinal immunization with gram negative pathogens and anti-LPS immunity to infection. Gut LPS, which interacts with PP lymphoreticular cells, is a major determinant for host responses to orally administered TD antigens. Bacteroides species are the principal microflora present in the gastrointestinal tract and our studies with phenol-water LPS extracts from Bacteroides fragilis indicate that both polysaccharide and lipid A activate lymphoreticular cells. The B. fragilis lipid A moiety, like that derived from E. coli and Salmonella LPS, induces B cell mitogenic responses in cultures from LPS responsive mice, but does not stimulate C3H/ H3J B cells. The inability of lipid A to stimulate gut-associated lymphoreticular tissue (GALT) cells of C3H/HeJ mice results in the induction of greater T helper cell activity in this tissue in response to orally administered TD antigens and ultimately results in an elevated IgA response pattern. Murine PP contain accessory cells (approximately 1% dendritic cells and 6-8% macrophages) and lymphocytes T (35-38%) and B (40-42%). Recent studies with antigen-specific T cell clones from C3H/ H3J PP have resulted in the isolation of IgA isotype-specific T helper cells (PP Th A cells). PP Th A cells are antigen-specific, bear Fc alpha receptors, and require H-2 histocompatibility with B cells for helper activity. PP Th A cells most effectively collaborate with surface IgA (sIgA)-bearing B cells (IgA committed B cells) for IgA isotype responses. Other studies have shown that PP dendritic cells and T cells form clusters when stimulated in vitro with sodium periodate and that these clusters promote polyclonal IgA responses in B cell cultures. Polyclonal IgA responses in cultures containing PP cell clusters from C3H/ H3J mice are considerably higher than those in identical cultures from LPS responsive mice. In other studies, the environmental influence on GALT B cells and their resultant commitment to IgA isotype is under investigation. CBA/N, X-linked immunodeficient (xid) mice possess an immature splenic B cell population which cannot respond to thymic-independent class-2 (TI-2) or certain TD antigens. However, GALT B cells of xid mice possess a mature Lyb-5+ B cell subpopulation capable of both TI-2 and TD responses.(ABSTRACT TRUNCATED AT 400 WORDS)