LPS Responder Research Articles

A fetal sheep liver extract containing immunostimulatory substances including LPS acts as leukocyte activator in cells of LPS responder and non responder mice

Purified fractions from a fetal sheep liver extract (FSLE) were investigated, in a murine model, for induction of leukocyte stimulating activities. The fractions FSLE-1 and FSLE-2 induced splenocyte proliferation in vitro in C57Bl/10ScSn (LPS responder) mice comparable to LPS, and in C57Bl/10ScCr (LPS non responder) mice. They also stimulated the release of nitrogen radicals in bone marrow-derived macrophages (BMDM) from several mouse inbred strains including both C57Bl/10ScSn and C57Bl/10ScCr mice. Stimulation of NO production could be blocked by L-NMMA, an inhibitor of iNOS, and enhanced by the simultaneous addition of IFN- γ. Moreover, stimulation of macrophages by FSLE-1 and FSLE-2 induced a cytostatic effect of the activated macrophages for Abelson 8-1 tumor cells. The stimulatory activity of the purified fractions is partially due to trace amounts of LPS derived from the fetal liver extract which was enriched during purification. Our results may help to explain the beneficial effect of the extract in patients which has been observed clinically.

Activation of innate immune responses by IL-2-expressing Salmonella typhimurium is independent of Toll-like receptor 4

We previously demonstrated that an attenuated strain of Salmonella enterica serovar Typhimurium, engineered to express IL-2 (strain GIDIL2), is cleared more rapidly than its parental, non-cytokine-expressing, strain (designated BRD509) from the reticuloendothelial system of susceptible BALB/c mice. This early clearance correlated with the induction of a strong innate immune response within a few hours of administration of GIDIL2 organisms. In the present study, we wished to assess the contribution of LPS recognition to GIDIL2-induced immune responses using Toll-like receptor 4 (TLR4) mutant C3H/HeJ mice. In contrast to LPS responder mice, both BRD509 and GIDIL2 strains persisted at higher levels in LPS non-responder animals. However, the GIDIL2 bacterial loads in the peritoneal cavity and spleen, recovered over a period of 21 days post infection, were consistently lower than the corresponding CFUs of the BRD509 strain. Direct evidence for the induction of innate immunity was shown by demonstrating increased NK cell cytotoxicity, NOS2 gene expression, and nitric oxide synthesis by peritoneal cells obtained as early as 2 h after infection with GIDIL2, but not BRD509, organisms. Unlike BALB/c mice, however, these responses failed to afford any protection against virulent challenge in C3H/HeJ mice. Taken together, our data demonstrate that despite the induction of innate immune responses by IL2-expressing organisms, this was not sufficient to induce protection in TLR4 mutant mice.

Role of interferons in LPS hypersensitivity

The innate immune response to Gram-negative bacteria depends mainly on the ability of the host to respond to the LPS component. Consequently, the state of LPS sensitivity at the time of infection and the numbers of invading bacteria ( i.e. the amounts of LPS) are primary factors determining the innate responses provoked by Gram-negative pathogens. LPS sensitivity increases following treatment of mice with live or killed micro-organisms. Two types of sensitization have been recognized, strong, IFN-γ-dependent and moderate IFN-γ-independent. IL-12 and IL-18 are intimately involved in the induction of IFN-γ by bacteria. We showed that Gram-negative bacteria induce IFN-γ in mice also by an IFN-β-dependent pathway that requires IL-18 and is independent of IL-12 signaling. This pathway is STAT4 dependent, the activation of which is directly linked to IFN-β. Further, IFN-β can be replaced by IFN-α. While different components of Gram-negative bacteria induce IL-12 and IL-18, LPS seems to be the only component in these bacteria capable of inducing IFN-β. Therefore, the IFN-β pathway of IFN-γ induction, unlike the IL-12 pathway, proceeds only in LPS responder mice. The IFN-α/β-dependent pathway is expected to play a role whenever IFN-α or IFN-β, and IL-18 are produced concomitantly during infection.

Role of interferons in LPS hypersensitivity.

The innate immune response to Gram-negative bacteria depends mainly on the ability of the host to respond to the LPS component. Consequently, the state of LPS sensitivity at the time of infection and the numbers of invading bacteria (i.e. the amounts of LPS) are primary factors determining the innate responses provoked by Gram-negative pathogens. LPS sensitivity increases following treatment of mice with live or killed micro-organisms. Two types of sensitization have been recognized, strong, IFN-gamma-dependent and moderate IFN-gamma-independent. IL-12 and IL-18 are intimately involved in the induction of IFN-gamma by bacteria. We showed that Gram-negative bacteria induce IFN-gamma in mice also by an IFN-beta-dependent pathway that requires IL-18 and is independent of IL-12 signaling. This pathway is STAT4 dependent, the activation of which is directly linked to IFN-beta. Further, IFN-beta can be replaced by IFN-alpha. While different components of Gram-negative bacteria induce IL-12 and IL-18, LPS seems to be the only component in these bacteria capable of inducing IFN-beta. Therefore, the IFN-beta pathway of IFN-gamma induction, unlike the IL-12 pathway, proceeds only in LPS responder mice. The IFN-alpha/beta-dependent pathway is expected to play a role whenever IFN-alpha or IFN-beta, and IL-18 are produced concomitantly during infection.

Lipopeptide adjuvants in combination treatment

Synthetic lipopeptides derived from the N-terminus of bacterial lipoprotein constitute potent immunoadjuvants for parenteral and mucosal immunization. When combined with tetanus toxoid (TT) or gliadin as antigens, the lipopeptide N-palmitoyl- S-[2,3-bis(palmitoyloxy)-(2 RS)-propyl]-( R)-cysteinyl-seryl-(lysyl) 3-lysine (P 3CSK 4) markedly enhanced the specific antibody levels. Lipopeptides also act as macrophage/monocyte activators: P 3CSK 4 induced nitric oxide release from bone marrow-derived macrophages (BMDM) of LPS responder and nonresponder mice. The antitumoral effect of the lipopeptide was demonstrated by a strong cytostatic activity of the lipopeptide-treated macrophages against the murine B-cell lymphoma cell line Abelson 8-1. The chemically well-defined lipopeptides can be synthesized with high purity and reproducibility and constitute ideal agents to be combined with antigens/vaccines or antitumor treatment.

The involvement of Ran GTPase in lipopolysaccharide endotoxin-induced responses

By functional cloning, we have established that Ran GTPase is involved in LPS-induced signal transduction. This has been accomplished by several functional comparisons of the two cDNAs, Lpsn/Ran (or RanT/n) and Lpsd/Ran (or RanC/d), which were isolated from cDNA libraries of LPS responder and hyporesponder mice, respectively. The letter n refers to the `normal' phenotype and the letter d refers to the `deficient' phenotype. Consistent with our previous results, more animal studies indicated that adenoviral transduction of RanC/d cDNA, but not RanT/n cDNA, into sensitive mice conferred significant resistance against endotoxin challenge. Thus the incorporation of RanC/d cDNA into gene therapy protocols as a therapeutic sequence remains very attractive. At steady state, hematopoietic cells transduced with RanC/d cDNA led to about a 10-fold increase in exogenous Ran protein compared with RanT/n cDNA. Furthermore, our cumulative data suggest that a slight elevation of Ran protein in B cells enhances LPS responsiveness, but the same elevation of Ran in macrophages does not. On the other hand, a high level of overexpression of Ran in both macrophages and B cells down-regulates LPS signal transduction. Thus LPS-induced signal transduction in macrophages and B cells is likely to occur via different signaling pathways.

5403919 Method to control leukocyte extravasation

Protein I from the outer membrane of Escherichia coli is a B-lymphocyte mitogen in mice. Polyclonal activation of mouse splenocytes was demonstrated by 3H-thymidine incorporation into DNA, 3H-uridine incorporation into RNA, and by a hemolytic plaque assay in three inbred mouse strains. B-lymphocytes from LPS responder mice (C57B1/10, STU/nu/nu) and LPS non-responder mice (C3H/HeJ) both responded well to protein I. The presence of serum was not necessary for mitogenicity; bovine serum albumin exhibited a beneficial effect on serum-depleted cultures. Thymocytes of C3H/HeJ mice were not activated by protein I. Protein II* from E. coli was also tested in our systems and showed a weak B-lymphocyte stimulatory activity. Human peripheral blood lymphocytes were not activated.

Immunopharmacological activities of the nontoxic monophosphoryl lipid A of Porphyromonas gingivalis

The low endotoxic lipid A derived from Porphyromonas gingivalis 381, 1-phospho β(1–6)-linked glucosamine disaccharide with 3-hydroxy-15-methylhexadecanoyl and 3-hexadecanoyloxy-15-methylhexadecanoyl groups at the 2- and 2′-positions, respectively, induced mitogenic responses in LPS low responder C3H HeJ as well as LPS responder C3H HeN mouse splenocytes. The mitogenic activities of P. gingivalis lipid A in splenocytes of LPS responder mice were comparable to that of the synthetic Escherichia coli-type lipid A (compound 506). The addition of polymyxin B resulted in the inhibition of mitogenic activity. P. gingivalis lipid A also stimulated strongly nonspecific host resistance against Pseudomonas aeruginosa infection in BALB c mice, and specific immune response in guinea pigs against infection with P. gingivalis. Furthermore, P. gingivalis lipid A demonstrated antitumour activity against MH134 hepatoma in C3H HeN mice. These life-preserving with tumour regression properties were comparable to those of monophosphoryl lipid A derived from Salmonella minnesota Re 595. Thus, P. gingivalis lipid A appears to have beneficial properties as an immunopharmacological agent.

Lipopolysaccharide nonresponder cells: The C3H/HeJ defect

Since its initial discovery as endotoxin resistant, the C3H/HeJ mouse has been extensively studied and used as a comparative model to help reveal the mechanism under genetic control which governs host responses to endotoxin. Most of the research has focused on the B lymphocyte and macrophage of this strain which fail to be activated by LPS. Recently, specific LPS binding proteins have been isolated on lymphocytes and other cells; however a receptor which transduces an activation signal has not been isolated as yet from responder cells which is missing or altered on C3H/HeJ nonresponder cells. Investigations into the signal transduction pathways used by C3H/HeJ B cells when they are activated by a protein mitogen have been found to be similar to those used by LPS responder cells when activated by LPS. Protein kinase C and tyrosine kinase, which phosphorylate signal proteins in cells have been found to be operative in C3H/HeJ and C3H/OuJ B cells. In both cases, DNA synthesis is shut off by either PKC or PTK blockade; however, PTK inhibition will also block activation of PKC stimulated DNA synthesis, indicating tyrosine kinase initiated phosphorylation may regulate the PKC signal pathway. Further analysis of the proteins that are phosphorylated in LPS responder and LPS nonresponder B cells is needed before conclusions can be drawn as to whether the defect in C3H/HeJ cells resides in the signal pathway leading to gene activation and proliferation. Nevertheless, the notion of a missing or defective signal receptor still remains as a working hypothesis to explain C3H/HeJ cell hyporesponsiveness to LPS. Isolation of the Lps n gene and its product will provide the evidence needed for a clearer understanding of how LPS reacts with cells.

Population kinetics of peritoneal LPS-reactive B lymphocytes

The dynamic behaviour of isolated populations of peritoneal LPS-reactive (LPSr) B lymphocytes was studied upon transfer of peritoneal cells (PerC) from C57BL/6 LPS responder into C57BL/10ScCr LPS non-responder mice. We have followed the persistence and life-span of the transferred LPSr donor B cells in the spleen and peritoneal cavity of both intact and X-irradiated adult hosts after i.v. or i.p injection and neonatal 1-day-old recipients after i.v. transfer. We have found that lymphocyte life-spans can be influenced by local host environments, as the transferred PerC LPSr cells showed different kinetics according to their route of injection, organ localization, and age or state of the recipients. Thus, while in intact hosts most of the transferred peritoneal LPSr cells decayed with time, following transfer into X-irradiated recipients the same cells were able to expand and replenish the lymphoid tissues of the host. Moreover, upon transfer into intact hosts, the kinetic properties of peritoneal LPSr cells from adult mice differ from splenic LPSr cells of age-matched animals, but mimic those of spleen cells from young, 1-to 2-week-old donors. These findings may reflect the different phenotype composition of adult spleen cells (poor in Ly1 B cells) and peritoneal and neonatal spleen cells (both rich for Ly1 B cells), or may be the result of selective events leading to the peritoneal accumulation of cells with different population dynamics.

Neutrophil recruitment and bacterial clearance correlated with LPS responsiveness in local gram-negative infection.

The inflammatory response to Gram-negative infection was studied in LPS responder and nonresponder C3H mice. Twenty-four hours after ascending E. coli urinary tract infection, an influx of neutrophils into the urine was observed in C3H/HeN mice (Lpsn,Lpsn); no significant neutrophil influx occurred in C3H/HeJ mice (Lpsd,Lpsd) at this time. A second peak of urinary neutrophil excretion was observed in both strains of mice approximately 6 days post-infection. The first, but not the second peak was inducible by inoculation with formalin-killed E. coli but not by Gram-positive bacteria. This finding suggested that the first peak is triggered by LPS, whereas the second peak emanates from other bacterial components which activate both LPS responder and nonresponder mice. The first peak of the inflammatory response was inversely related to bacterial clearance. C3H/HeJ mice (Lpsd,Lpsd) retained about 2000-fold more E. coli in the kidneys than C3H/HeN mice (Lpsn,Lpsn). The infection persisted despite the late-occurring influx of neutrophils in C3H/HeJ mice. These results suggest that an inflammatory response to LPS is required for the elimination of a local Gram-negative infection.

Lipopolysaccharide receptors on lymphocytes. I. Lack of immunologic recognition of a putative LPS receptor on LPS-responder lymphocytes by LPS-nonresponder mice.

We have examined the potential immunogenicity of viable lymphocytes from C3HeB/FeJ responder mice adoptively transferred into congenic nonirradiated C3H/HeJ nonresponder mice. Immunologic rejection or acceptance of donor cells was employed as indirect evidence for the presence or absence of an antigenically distinct "LPS receptor" present on donor lymphocytes. Immunogenicity was evaluated by in vitro assessment of the subsequent proliferative response of recipient splenocytes to protein-free LPS after multiple i.p. injections of responder lymphocytes. Control experiments have made use of syngeneic donor lymphocytes differing immunologically by the presence of the H-Y minor histocompatibility antigen. The results of these experiments provide evidence for the concept that if the phenotypic difference between LPS responder and nonresponder mice is also expressed antigenically in the form of an LPS receptor, that antigenic difference is significantly less immunogenic than a minor histocompatibility antigen.

Selective association of lipid-rich R-like lipopolysaccharide subunits with murine spleen cells

SDS-PAGE was used to analyze the subunit composition of 125I-lipopolysaccharide (LPS) in the cell-associated and supernatant fractions of murine spleen cells after culture with radiolabeled LPS from the smooth strain of E. coli 055:B5. Quantitative estimates from densitometric scans of autoradiographs indicated that certain R-like subunits were selectively enriched in cell-associated fractions by a factor of 2.8 as compared to native 055 LPS. Coincident with this selective enrichment was a 57% decrease in these subunits in supernatant fractions. In contrast, the level of polysaccharide-containing subunits in cell-associated fractions was equivalent to or less than the corresponding subunit in native LPS. LPS bound at 37°C was capable of eliciting a significant B-lymphocyte proliferative response in responder spleen cells. However, this selective binding of lipid-rich R-like subunits to splenocytes is insufficient, by itself, to initiate a triggering event since it is both quantitatively and qualititatively indistinguishable in lymphoid cells from the LPS responder ( C3HeB FeJ ) mouse and the LPS non-responder ( C3H HeJ ) mouse.

The role of interleukin 1 in acute phase serum amyloid A (SAA) and serum amyloid P (SAP) biosynthesis.

The acute phase SAA and SAP profiles have been compared for localized and endotoxin induced inflammation in LPS responder and nonresponder strains of mice. The SAP profile can reflect a delay with respect to the start of the increase. Its maximum is on the order of ten times the nonacute phase concentration and elevated concentrations are sustained 24 to 48 hours after SAA concentration is rapidly decreasing to normal. The role of Interleukin 1, known to have an essential role in SAA production, was investigated for SAP production. Purified mouse IL 1 and rabbit IL 1 produced a minimal elevation of SAP concentration above normal values, especially when compared with their effects on SAA concentration. BCG infection was shown to synergistically augment SAA induction by LPS and was shown to enhance IL 1 production by macrophages in response to LPS. Unlike SAA synthesis, BCG-preinfection fails to synergistically augment the LPS-induced SAP response. BCG infection alone produced highly elevated and sustained increases in SAP concentration, whereas, the effect on SAA concentration was minimal. Macrophages appear to play an important role in SAP acute phase elevation, but the mechanism in different from that of SAA elevation.

Protein I and Protein II from the Outer Membrane of Escherichia Coli are Mouse B-Lymphocyte Mitogens

Protein I from the outer membrane of Escherichia coli is a B-lymphocyte mitogen in mice. Polyclonal activation of mouse splenocytes was demonstrated by 3H-thymidine incorporation into DNA, 3H-uridine incorporation into RNA, and by a hemolytic plaque assay in three inbred mouse strains. B-lymphocytes from LPS responder mice (C57B1/10, STU/nu/nu) and LPS non-responder mice (C3H/HeJ) both responded well to protein I. The presence of serum was not necessary for mitogenicity; bovine serum albumin exhibited a beneficial effect on serum-depleted cultures. Thymocytes of C3H/HeJ mice were not activated by protein I. Protein II* from E. coli was also tested in our systems and showed a weak B-lymphocyte stimulatory activity. Human peripheral blood lymphocytes were not activated.

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.

Chemical and Biologic Properties of A Protein-Rich Fraction of Bacterial Lipopolysaccharides

Abstract Butanol LPS preparations from three serotypes of Escherichia coli lose their ability to stimulate C3H/HeJ lymphocytes after the removal of a protein-rich fraction (LAP) by extraction with phenol. This loss of mitogenic activity for LPS nonresponder spleen cells is accompanied by an increase in equilibrium density of approximately 0.03 g/cm3 by each of the three lipopolysaccharides. The LAP preparations exhibited potent mitogenic activity for both LPS responder C3H/St and nonresponder C3H/HeJ lymphocytes. Each LAP produced an optimal response at approximately 10 µg/ml and each was toxic at a 10-fold higher concentration. Like LPS, LAP is mitogenic for congenitally athymic nude mice. Although LAP is isolated in association with LPS and shares certain of its biologic properties, the two B cell mitogens may be distinguished by several criteria. Of primary importance is the ability of LAP to stimulate LPS-nonresponder lymphocytes. In addition, these experiments demonstrate that the LAP-induced response by C3H/HeJ lymphocytes is not blocked by preincubation with high concentrations of LPS. The response to LAP by C3H/St spleen cells is not blocked by polymyxin B, an inhibitor of the lipid A-induced mitogenic response. Furthermore, such LAP activity is destroyed by mild alkaline hydrolysis under conditions that do not decrease LPS activity. Finally, we observed that the peak level of 3H-thymidine incorporation that results from stimulation with LPS is consistently lower than the level which is obtained with optimal LAP doses. The experiments presented here also demonstrate that isolated LAP and protein-free LPS can reassociate in vitro to generate an LPS-LAP complex with mitogenic activity for C3H/HeJ lymphocytes.

Sera and the in Vitro Induction of Immune Responses

Abstract We have extended our studies on the role of bacterial contamination in the generation of fetal calf sera which support primary in vitro humoral responses by cultured mouse spleen cells. Gram-negative, gliding bacteria were isolated from a strongly supportive sample of fetal calf sera. Medium conditioned by the growth of these microorganisms had strong adjuvant effects in cultures of mouse spleen cells supplemented with a non supportive, deficient sera. The adjuvant and mitogenic activities of the bacterial conditioned medium were then compared to those of bacterial lipopolysaccharide from Salmonella typhosa 0901 in cultures of LPS responder and LPS nonresponder spleen cells. From the results of these comparative studies, we conclude that the active factor(s) obtained from the gliding bacteria is an adjuvant with properties very distinct from those of highly purified enteric bacterial LPS.

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.