LPS regulation of the immune response: Bacteroides endotoxin induces mitogenic, polyclonal, and antibody responses in classical LPS responsive but not C3H/HeJ mice

Recent studies have suggested that lipopolysaccharide (LPS) derived from gram-negative organisms such as Bacteroides, which are not members of the Enterobacteriaceae, stimulate B cells from the classic LPS-hyporesponsive C3H/HeJ mouse. In the present study, purified, phenol-water-extracted LPS from Bacteroides fragilis ATCC 25285 (B-LPS) was tested for its ability to induce in vivo and in vitro responses in classic LPS-responsive C3H/HeN, LPS-hyporesponsive C3H/HeJ, and (C3H/HeN X C3H/HeJ)F1 hybrid mice. B-LPS induced mitogenic responses in both C3H/HeN and C3H/HeJ spleen cell cultures when cells were cultured under standard conditions, i.e., 8 X 10(5) cells/well. Interestingly, when lower spleen cell numbers were tested with B-LPS, a typical responsive-nonresponsive pattern developed in which good mitogenic responses were induced by B-LPS in C3H/HeN cultures and in which low responses in C3H/HeJ spleen cell cultures were evident. In vivo immunization of mice with B-LPS resulted in high antibody responses in C3H/HeN, intermediate responses in F1, and low responses in C3H/HeJ mice. When purified splenic B cells were incubated with B-LPS, both mitogenic responses and polyclonal immunoglobulin M (IgM) synthesis occurred in C3H/HeN cultures, whereas intermediate responses were noted in F1 cultures and no response was seen in B cell cultures from C3H/HeJ mice. Furthermore, in vitro TNP-B-LPS responses were induced in C3H/HeN spleen cells or purified B cell cultures, and intermediate anti-TNP PFC responses occurred in F1 spleen cells or purified B cell cultures. The toxicity of B-LPS was tested in galactosamine-sensitized mice. The LD50 values for B-LPS in classic LPS-responsive C3H/HeN and C57BL/6J mice were 0.6 microgram and 1.1 microgram, respectively; F1 hybrid mice were approximately 15-fold more resistant, whereas C3H/HeJ mice gave an LD50 of 1650 micrograms. This study shows that phenol-water preparations of B-LPS are biologically active and induce responses in the classic LPS-responsive but not in the LPS-hyporesponsive C3H/HeJ mouse strain.     

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.     

Immunologic properties of bacterial lipopolysaccharide (LPS). IV. Cellular basis of the unresponsiveness of C3H/HeJ mouse spleen cells to LPS-induced mitogenesis.

Lymphoid cells obtained from the C3H/HeJ mouse strain respond abnormally to LPS in vitro, as shown by the fact that they are unable to make a mitogenic response to some LPS preparations and make only a low mitogenic response to other LPS preparations. In contrast, cells from a closely related C3H substrain, the C3H/St, are highly responsive to both types of LPS preparations. Experiments were carried out to determine the cellular basis of these genetically determined LPS response differences. This question was approached by studying the mitogenic response to LPS in cultures containing mixtures of various combinations of B cells, T cells, and macrophages from C3H/HeJ and C3H/St mice. Experiments utilizing an LPS preparation to which the C3H/HeJ is totally unresponsive (negative LPS) revealed, first, that either spleen cells, or partially purified T cells and/or macrophages obtained from C3H/St, could not restore the ability of C3H/HeJ spleen cells to respond to LPS, indicating that the C3H/HeJ is not deficient in an LPS-specific helper cell population which may be required for mitogenesis. Secondly, the addition of either spleen cells or partially purified T cells or macrophages from the C3H/HeJ to spleen cells from the C3H/St did not inhibit the mitogenic response to LPS, suggesting that the presence of suppressor cell activity is also not involved. Experiments analogous to those described, except utilizing another LPS preparation to which the C3H/HeJ is partially responsive (positive LPS), also failed to demonstrate reconstitutive or suppressive effects when C3H/HeJ and C3H/St spleen cells were admixed. The results obtained indicate that the defect in the C3H/HeJ mouse strain that limits its responsiveness to positive LPS and which renders it totally unresponsive to negative LPS appears to be an intrinsic defect in the capacity of B cells to react to the mitogenic stimulus of LPS.     

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.     

Genetic and biochemical evidence for the involvement of a bacterial component in the mitogenic properties of polyribonucleotides on murine B lymphocytes.

The mitogenic response of C3H/HeJ mice to the B cell mitogens, poly C and poly I, is approximately one-half the response measured in various LPS-responder strains. C3H/HeJ mice respond normally to poly I:C, the heteroduplex polymer. The low responder phenotype of C3H/HeJ mice to poly C and poly I is shown by an analysis of (C3H/HeJ x C57BL/6J-By-Ps)F1 X C3H/HeJ backcross progeny to result from a gene locus that is closely linked or identical to the defective LPS response locus expressed by the C3H/HeJ strain. The entire mitogenic activity in poly C preparations and most of the mitogenic activity in poly I preparations is insensitive to ribonuclease degradation. Hot aqueous phenol extraction of the polynucleotides separates the majority of the mitogenic activity that is soluble in the combined interface and phenol phase fraction from the aqueous soluble polynucleotides. The ribonuclease-insensitive, phenolsoluble contaminant elicits a reduced response in C3H/HeJ mice as compared to an LPS responder strain. We conclude that 1) poly C has no inherent mitogenic activity; 2) poly I preparations contain both ribonucleasesensitive and insensitive mitogenic activities; 3) the ribonuclease-resistant mitogenic activity in polynucleotide preparations has properties unlike those of LPS or lipid A; and 4) the product of LPS response gene has an effect upon the mitogenic stimulation of spleen cells by the contaminant.     

Lack of responsiveness of C3H/HeJ macrophages to lipopolysaccharide: the cellular basis of LPS-stimulated metabolism.

The rate of glucose utilization has been used as a measure of LPS-induced activation of cultures of C3H/HeN and C3H/HeJ spleen cells, peritoneal cells, and purified peritoneal adherent cells. Peritoneal cells utilized 40 to 60 times more glucose than did spleen cells and purified adherent monolayers were more active than mixed peritoneal cells, suggesting that only macrophage metabolism was being measured. The cell preparations for C3H/HeJ mice were not activated by Escherichia coli K235 LPS prepared by extensive phenol extraction, whereas C3H/HeN cells were activated by the LPS. Cells from both strains were activated by a commercially obtained E. coli 0111:B4 LPS and butanol-extracted K235 LPS. The addition of 10% C3H/HeN spleen cells to C3H/HeJ peritoneal cells resulted in a marked enhancement of glucose utilization. These findings suggest that LPS-induced enhancement of macrophage metabolism occurs both by direct action of LPS on macrophages as well as indirectly through activated lymphocytes.     

Immunobiological properties of lipopolysaccharides isolated from Fusobacterium nucleatum and F. necrophorum

Lipopolysaccharides (LPSs) were isolated from Fusobacterium nucleatum ATCC 10953 and F. necrophorum ATCC 25286 by the hot phenol/water procedure. F. nucleatum LPS was composed of 16% (w/w) carbohydrate, 10% (w/w) hexosamine and 40% (w/w) fatty acid, while F. necrophorum LPS was composed of 26% (w/w) carbohydrate, 12% (w/w) hexosamine and 28% (w/w) fatty acid. These LPS preparations induced mitogenic responses in spleen cells of BALB/c, BALB/c (nu/nu) and C3H/HeN mice, and these responses were suppressed by the addition of polymyxin B. The preparations also induced the polyclonal responses of C3H/HeN spleen cells. In addition, enhanced glucose utilization and interleukin-1 production by murine peritoneal macrophages were demonstrated. Neither spleen cells nor macrophages from the 'LPS-nonresponsive' C3H/HeJ mouse were activated by LPSs from the Fusobacterium species.     

The role of HLA-DR determinants in monocyte-macrophage presentation of herpes simplex virus antigen to human T cells

Spleen cell killing of target cells can manifest through spleen cell-target cell interaction in the presence of mitogenic lectin, lectin-dependent cell-mediated cytotoxicity (LDCC). Spleen cells from C57B1/6 mice immunized with C3H mouse cells were found to be capable of cytotoxicity against autologous and other C57B1/6 spleen cells in the presence of Con A. Thus, alloimmune spleen cells are capable of an anti-self cytotoxic response in the presence of mitogenic lectin, antiautologous LDCC. Antiautologous LDCC is blocked by preincubation of cytotoxic cells with colchicine, an inhibitor of the cytotoxic effector mechanism. Analysis of alloimmune spleen cell subpopulations suggests that the antiautologous LDCC cell is an immature alloimmune cytotoxic cell (prekiller cell). Potent LDCC was found in alloimmune spleen cell preparations depleted of alloimmune cytotoxic T cells (killer-depleted) by three passes on allogeneic cell monolayers genetically identical with the immunizing cell. However, some LDCC effectors were also found to adhere to the adsorbing target, suggesting that there is some maturational diversity among LDCC effectors.     

Characterisation of a monoclonal antibody against the fimbrial F8 antigen of Escherichia coli

Abstract A monoclonal IgG1 antibody against F8 fimbriae was obtained with the hybridoma technique using spleen cells from C3H/f mice immunised with a fimbrial preparation of Escherichia coli 2980 (O18ac:K5:H⁻:F1C, F8) and Sp 2/0 Ag8 myeloma cells. The hybrid cells were cloned twice by limiting dilution and grown in tissue culture. The monoclonal antibody was purified from culture supernatants on Protein A Sepharose. It reacted with F8 fimbriae in colony blot, enzyme-linked immunosorbent assay (ELISA) and immunoblot after electrotransfer from sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of fimbrial preparations. The antibody bound to and agglutinated F8-fimbriated bacteria.     

Macrophage-mediated mitogenic suppression induced in mice of the C3H lineage by a vaccine strain of Salmonella typhimurium

Salmonella typhimurium, strain SL3235, an avirulent organism, has been used as a live vaccine in mice of the C3H lineage and has been found to confer high levels of protection. In the present study, it was found that intraperitoneal injection of approximately 5 X 10(5) live SL3235 induced potent suppression of spleen cell mitogenic responses to a panel of B- and T-cell mitogens in the Salmonella-hypersusceptible C3H/HeJ and C3HeB/FeJ, and the inherently resistant C3H/HeNCrlBR mice. Maximal suppression (greater than 99%) was seen at 1 week, and was still significant but waning (50%) at 3 weeks postimmunization. In contrast, cells of mice receiving acetone-killed cells were not suppressed. Removal of macrophages, but not T or B cells, restored responsiveness, indicating that suppression was macrophage mediated. Prostaglandins were not the major mediator of suppression, as in vitro administration of indomethacin failed to abrogate suppression. As mitogenic suppression occurred in mice with high levels of Salmonella immunity, the suppression is interpreted as a marker of a powerful immunomodulatory process induced by live cells, rather than as an indication of poor immune status of the host.     

Suppression of antibody responses in allogeneic mice by products of lymphoid tissue. I. Allogeneic suppressive factor (ASF) from spleens repopulated with thymus cells.

A cellfree extract prepared from the spleen cells of C3H mice is capable of suppressing antibody responses to SRBC when extract material is exposed to alloantigens. The observed immunosuppression was attributed to a soluble factor in the extract. This allogeneic suppressive factor (ASF) was detected in extracts prepared from the spleen cells of unirradiated mice as well as those of irradiated mice repopulated with thymocytes, provided that mice were previously immunized with SRBC. Donors of actively suppressive ASF preparations did not need to be previously exposed to alloantigens. Extracts from thymus and marrow cells of unirradiated mice and the spleen cells of irradiated mice repopulated with marrow cells (or no cells) did not contain ASF. C3H thymocytes stimulated with SRBC generated more ASF activity in spleens of C3BF1 hosts than in those of C3H hosts, indicating that alloantigenic stimulation enhances the production or activity of ASF. Once produced, C3H ASF was able to suppress antibody responses in cell transfer experiments only if exposed to C3BF alloantigens of either donor lymphoid cells or irradiated hosts. Once exposed to alloantigens, ASF appears to be capable of suppressing antibody responses of syngeneic C3H or semi-allogeneic C3BF cells. When both donor lymphoid cells and hosts were syngeneic with the donor of the ASF, there was enhancement of antibody formation in cell transfer experiments. C3H ASF did not interfere with education of C3BF thymocytes to SRBC or with the generation of precursors of anti-SRBC antibody-forming cells by C3BF1 marrow cells. ASF may interfere with cellular cooperative events necessary for humoral immune responses or with terminal differentiation of B cells. Production of ASF could partially account for the suppression of antibody responses observed during graft-vs-host reactions.     

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.     

Mitogenic activity of Actinomyces viscosus. I. Effects on murine B and T lymphocytes, and partial characterization.

Actinomyces viscosus homogenate (AVIS) contins substance(s) which cause spleen cells from conventional and germfree mice to undergo increased DNA synthesis. This mitogenic effect is primarily on B cells since spleen cells from nude mice or T-depleted spleen cells from conventional mice respond as strongly as conventional (T + B) spleen cells. Mouse thymocytes do not respond mitogenically to AVIS. It is unlikely that the mitogenic acitivity is due to the presence of LPS, since A. viscosus is Gram-positive and is not known to have an LPS cell wall component. Also, AVIS is not inactivated by polymyxin B, as are some preparations of LPS, and C3H/HeJ mouse splenocytes respond strongly to AVIS but not to LPS. The activity is heat stable, is not lost upon dialysis, and is not affected by lysozyme. Mitogenic activity is partially lost when AVIS is digested with nonspecific bacterial protease or treated with metaperiodate. Sodium hydroxide treatment completely abolishes mitogenic activity. Actinomycotic lesions are characterized by a long-tern inflammatory response involving a dense plasma cell infiltrate. We suggest that B cell mitogens form Actinomyces may play a role in the elicitation of the plasma cell component of these lesions.     

Specific mitogenic activity of 8-Br-guanosine 3',5'-monophosphate (Br-cyclic GMP) on B lymphocytes.

8-Br-cyclic GMP has been found to be a specific B cell mitogen; it triggers athymic nude mice spleen cells and "B mice" spleen cells, nylon adherent, anti-theta and complement-treated cells to proliferate. It does not stimulate thymocytes or purified T cells. The kinetics of the response to Br-cyclic GMP and LPS are almost identical. The mitogenic effect of LPS and Br-cyclic GMP is additive when the two mitogens are given together to cells. Spleen cells (C3H/HeJ strain) that did not respond to LPS were triggered by Br-cyclic GMP to make DNA. In order to achieve maximal stimulation by Br-cyclic GMP, the drug had to be in contact with the cells for more than 24 hr. Br-cyclic GMP was found to be mitogenic for spleen cells from five different mouse strains, but not for human leukocytes. DB-cyclic AMP was found to inhibit the DNA synthesis of T lymphocytes after they interacted with Con A; DB-cyclic AMP had no effect on the ability of the B lymphocytes to be transformed by LPS. The differential effects of cyclic nucleotides on B vs. T lymphocytes are discussed.     

Stimulation of chicken lymphocytes by T- and B-cell mitogens.

Cultures of chicken spleen, peripheral blood, thymus, and bursal lymphocytes were tested for mitogenic stimulation by phytohaemagglutinin (PHA), concanavalin A (ConA), pokeweed mitogen (PWM), bacterial lipopolysaccharide (LPS), trypsin, and insulin. Spleen and blood leukocytes were stimulated by both the lectins and LPS, and also to some degree by trypsin and insulin as judged by increased incorporation of [³H]thymidine into acid-insoluble material. This was observed in cultures incubated in serum-free medium as well as in the presence of foetal bovine serum or autologous plasma. Thymus cells were reproducibly stimulated by high concentrations of PHA. No significant responses were obtained in bursal cell cultures with any of the compounds tested. Removal of cotton wool-adherent cells from the spleen cell suspensions resulted in a subpopulation of cells which were stimulated by PHA but showed little response to ConA, PWM, or LPS. This procedure did not remove surface immunoglobulin-bearing cells from the original suspension. Both these enriched spleen lymphocytes and the unfractionated spleen, blood and thymus leukocyte cultures were effectively stimulated by a partially purified PHA but with a highly purified PHA preparation only at very high concentrations. These and other results suggest that the mitogenic components in crude PHA preparations are different for chicken and human or mouse cells.     

Mitogenic activity of the outer membrane of Treponema denticola.

The outer membrane (OM) was isolated by detergent extraction from Treponema denticola ATCC 35405, ATCC 33521 and ATCC 35404, representing serovars a, b and c, respectively, as well as from two fresh isolates of T. denticola. Strict precautions were undertaken against the introduction of contaminant lipopolysaccharide when the OM was isolated. The OM was active in mitogenic stimulation of C3H/HeOuJ mouse spleen cultures, but to a somewhat lesser extent than purified lipopolysaccharide (LPS) from Escherichia coli 055:B5. Polymyxin B only partially inhibited the response. Unheated OM abrogated mitogenic activity of E. coli LPS, but heated preparations enhanced the mitogenic activity of E. coli LPS, suggesting the presence of a heat-labile cytolytic factor associated with T. denticola OM in addition to a putative lipopolysaccharide and/or heat-stable lipoprotein.     

In vitro splenic T cell responses of diverse mouse genotypes after oronasal exposure to mouse hepatitis virus, strain JHM.

Mortality rates among BALB/cByJ, A/JCr, C3H/HeSnJ, and C57BL/6NCr mice inoculated oronasally with mouse hepatitis virus (MHV) strain JHM, ranged from 25 to 67%. Spleen cells harvested from the first three genotypes at 5 days postinoculation proliferated poorly in response to concanavalin A stimulation and produced significantly less interleukin (IL) 2 than cells from uninfected control mice. The function of spleen cells harvested at 14 days postinoculation varied and was host genotype-dependent. Despite clinical signs among some infected C57BL/6NCr mice, spleen cell function was relatively unaffected. C57BL/10ScNCr, B10.A, and SJL/JCr mice remained clinically normal after MHV inoculation. Proliferation and IL2 production by cells from inoculated C57BL/10ScNCr and B10.A mice were similar to responses of their respective controls. In contrast, cells from inoculated SJL/JCr mice were hyper-responsive and produced peak levels of IL2 earlier than control cells. Among the seven genotypes tested, only BALB/cByJ and C3H/HeSnJ spleen cells produced detectable IL4 after primary stimulation with concanavalin A or after priming and restimulation. Primary IL4 production by cells from these two genotypes was significantly reduced if donors were inoculated with MHV 5 days prior to spleen harvest. IL4 production by cells from acutely infected BALB/cByJ mice was considerably enhanced by priming and restimulation.     

Lipopolysaccharide-induced suppression of antibody-directed cell-mediated cytotoxicity to chicken red blood cells

Prior treatment with commercially prepared and acetone-extracted lipopolysaccharide (LPS) was found to suppress the expression of antibody-directed, cell-mediated cytotoxicity (ADCC) to chicken red blood cells (CRBC) by spleen cells from C57/BL10, C3H, and BALB/c mice. The in vitro incubation with commercial LPS suppressed ADCC-CRBC activity of spleen cells from both C3H/HeJ and C3H/HeN mice. Only the C3H/HeN strain was suppressed when treated with purified LPS. ADCC-CRBC activity of neonatal spleen cells could be suppressed after a 3-hr in vivo incubation with LPS while adult spleen cells required a minimum of 15 hr preincubation.     

Limiting dilution analysis of TNF producing cells in C3H/HeJ mice

A limiting dilution assay (LDA) that measures the frequency of TNF producing cells is described. LDA determination is based on the inhibition of growth of a highly TNF sensitive subline from the WEHI-164 fibrosarcoma by using a micro assay sensitive to single picogram amounts of recombinant murine TNF. Using such LDA, it was determined that the reported deficiency in LPS-induced TNF production in C3H/HeJ mice is a function of reduced frequency of TNF producing cells rather than a complete lack of responsiveness. In bulk culture, LPS-triggered TNF was produced by Thy-1.2 negative spleen cells with activity recovered in both G10 Sephadex adherent and nonadherent subpopulations. LPS stimulation of spleen cells from C3H/HeJ mice resulted in TNF mRNA expression as shown in both Northern blots and in situ hybridization. The frequency of TNF mRNA bearing cells in control of C3H/HeSnJ mice by in situ hybridization correlated with that found for TNF producing cells in LDA. In C3H/HeJ spleen, significantly higher numbers of TNF mRNA positive cells were found than were shown to produce TNF in LDA.