Identification and characterization of lipopolysaccharide receptor molecules on mammalian lymphoid cells.
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Animals↗
Biomedical subjects
D C Morrison
Publications and source records attributed to D C Morrison.
Monoclonal antibody to mouse lipopolysaccharide receptor protects mice against the lethal effects of endotoxin.
Specific endotoxic lipopolysaccharide (LPS) binding sites on the cell membranes of murine lymphocytes and macrophages that may serve as functional receptors for LPS have recently been identified using photoactivatable cross-linking LPS derivatives. A monoclonal antibody (Mab 5D3) with specificity for this 80-kDa protein has also been generated and characterized. The capacity of MAb 5D3 to protect mice against the lethal effects of endotoxin was investigated. Pretreatment of CF1 mice with as little as 15 micrograms of MAb 5D3 provided virtually complete protection against a dose of endotoxin 10-fold greater than that required to kill all mice in an untreated control group using the galactosamine sensitization model. Significant protection was also afforded normal mice given MAb 5D3 relative to saline. Several lines of evidence suggest that MAb 5D3-mediated protection is due to the agonist properties of this antibody rather than a receptor blockade mechanism.
Animals↗
Lipopolysaccharide/lipid A receptors on lymphocytes and macrophages.
Significant advances have been realized during the past five years in the understanding of the mechanism(s) by which endotoxic LPS interactions with mammalian lymphoreticular cells leads to characteristic cellular responses. There is now strong experimental evidence to support the concept that specific receptors for the lipid A component of LPS do, in fact, exist and are functional on these cells. While the available data do not rule out a potential contribution of nonspecific hydrophobic interactions of lipid A with the membrane bilayer in the cellular activation process, it would appear that interaction with the LPS receptor alone is sufficient to initiate triggering. Whether there exist more than one molecular entity which might function on mammalian cell membranes as a specific receptor for LPS, or whether different cell types may manifest different LPS receptors remains as an interesting area for future research. Further, the concept that molecular complexes of LPS with mammalian host proteins, such as the acute phase LPS binding protein, might trigger additional novel pathways for cell activation is both exciting and of potential importance. The precise mechanism or mechanisms by which LPS-receptor ligand interactions translate into appropriate transmembrane signalling events is currently uncertain. Clearly there exists evidence for contribution of many of the traditional second signals, although at present, the data are incomplete and not always consistent between laboratories. Of potential concern in this respect are the sometimes rather striking differences noted between lipid A and intact polysaccharide containing S-LPS. While such differences may be significant and important, it should be remembered that S-LPS itself is a potent stimulus for many lymphoreticular cell subpopulations, and any postulated pathways must encompass S-LPS as well as lipid A. In any case, it is likely that the further molecular-biochemical characterization of LPS receptors will yield crucial information for the eventual elucidation of the precise pathways for LPS transmembrane signalling. Such information will be invaluable in the future harnessing of the immunostimulatory potential of LPS as well as the abrogation of its profound deleterious pathophysiological effects in endotoxin shock.
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Effects of lipopolysaccharide chemotype structure on binding and inactivation of hen egg lysozyme.
The structural features of lipopolysaccharide [LPS] which influence the binding and inactivation of lysozyme have been examined. Binding of polysaccharide-containing LPS (S-LPS) and Ra-Rc-LPS preparations was independent of temperature between 37-50 degrees C; in contrast, binding of Rd-LPS, Re-LPS and lipid A was temperature-dependent. The binding of lysozyme to Rd-LPS and Re-LPS was increased by treatment with mild alkali, which has little detectable effect on binding of lysozyme to S-LPS and Ra-Rc-LPS preparations. Competitive binding experiments using dansylated lysozyme and/or dansylated polymyxin B indicated independent binding sites on the LPS for lysozyme and polymyxin B. These results indicate significant differences between most LPS preparations and lipid A and glycolipid LPS in their interaction with proteins of mammalian origin.
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Lipopolysaccharide interactions with lysozyme differentially affect lipopolysaccharide immunostimulatory activity.
The effect of complex formation between lysozyme and lipopolysaccharide (LPS) on the immunostimulatory activities of LPS have been investigated in vitro. Three prototype immunostimulatory activities were examined: B-lymphocyte proliferation, B-lymphocyte differentiation and macrophage production of lymphocyte-activating factor activity. Different effects of lysozyme were noted, depending upon the structure of the LPS, even though previous studies have established that all LPS preparations readily bind lysozyme. Both Re-LPS- and lipid-A-dependent immunostimulatory activities were readily inhibited by lysozyme in a dose-dependent fashion. In contrast, S-LPS and Ra-LPS were unaffected in their immunostimulatory activities by lysozyme. These differences were not the result of quantitative differences in LPS binding of lysozyme, or effects of lysozyme on overall binding of LPS to target cells. These data suggest that the factors which dictate the initial interactions between LPS and lymphoreticular cells may not be identical for all LPS preparations and/or purified lipid A.
Adjuvants, Immunologic↗
Products of lipopolysaccharide-activated macrophages (tumor necrosis factor-alpha, transforming growth factor-beta) but not lipopolysaccharide modify DNA synthesis by rat trophoblast cells exhibiting the 80-kDa lipopolysaccharide-binding protein.
Pregnancy losses from gram negative bacterial infections could be caused by direct effects of LPS on placental cells, or indirectly via LPS activation of macrophages in the uteroplacental unit. To evaluate those alternatives, LPS, LPS-activated peritoneal cells, conditioned medium from LPS-activated peritoneal cells, and some purified and recombinant molecules known to be secreted by activated macrophages were tested for their abilities to modify DNA synthesis by rat trophoblast cells. Three trophoblast cell lines derived from midgestation placentas of outbred and inbred rats were used for the experiments. Although the 80-kDa LPS-binding protein was demonstrated on trophoblast cells, LPS alone had no effect on the ability of trophoblast cells to synthesize DNA. In cocultures, trophoblast cell DNA synthesis was slightly enhanced by low concentrations of both unstimulated and LPS-activated peritoneal cells. At higher concentrations, LPS-activated cells caused significant inhibition of DNA synthesis by trophoblast cells. Conditioned media from LPS-activated peritoneal cells were highly inhibitory to trophoblast cell DNA synthesis. When specific molecules likely to be components of those media were tested, IL-1 was found to have a modest but reproducible stimulatory effect and PGE2 did not change trophoblast cell incorporation of [3H]TdR. In contrast, trophoblast cell DNA synthesis was markedly inhibited in a dose-dependent manner by both TNF-alpha and TGF-beta 1. No differences in the sensitivity of trophoblast cells from outbred and inbred rats were observed. Given the limitations of the experimental model system, the results suggest that in cases of infection by gram-negative bacteria LPS may have an adverse effect on pregnancy by stimulating resident macrophages to generate and release molecules that are inhibitory to trophoblast cell DNA synthesis.
Animals↗
Lipopolysaccharide interaction with lysozyme. Binding of lipopolysaccharide to lysozyme and inhibition of lysozyme enzymatic activity.
Experiments have been carried out to characterize the binding of lysozyme (LZM) to bacteriol lipopolysaccharide (LPS). The formation of LPS.LZM complexes can be readily demonstrated using either physical-chemical separation techniques or a radiolabeled photoaffinity LPS probe. The binding affinity of LZM for LPS has been estimated to be approximately 10(8) liters/mol. Binding of LPS results in loss of LZM enzymatic activity by a noncompetitive inhibition, as assessed by either particulate or soluble substrates. This interaction of LPS with LZM is dictated primarily by hydrophobic interactions and appears to be a general property of both constituents. Binding can be demonstrated with LZM of both human and avian sources, as well as with LPS isolated from a variety of Gram-negative organisms. The addition of LPS to biologically relevant fluids containing LZM results in dose-dependent inhibition of LZM enzymatic activity suggesting that such interactions may have relevance in Gram-negative infections. Finally LZM has been shown to reduce the endotoxic activity of LPS as assessed by gelation of Limulus amoebocyte lysates.
Affinity Labels↗
Immunostimulation of C3H/HeJ lymphoid cells by R-chemotype lipopolysaccharide preparations.
Splenocytes from the C3H/HeJ and C57BL/10ScN (nu/nu) inbred mouse strains have been characterized by a genetic defect in their capacity to proliferate in response to purified protein-free LPS preparations. In this manuscript we provide experimental evidence to support the concept that the refractory state of B cells from endotoxin-unresponsive mice to mitogenic stimulation by LPS does not extend to R-chemotype LPS isolated from a variety of rough strains of Salmonella or Escherichia coli. We present several lines of evidence to suggest that the observed mitogenic activity is not the result of contamination of LPS with lipid A-associated proteins. The mitogenic activity of LPS extracted from rough strain mutant bacteria (R-LPS) appears to be dependent upon a structural requirement of the LPS in which the 2-keto-3-deoxyoctulosonate linkage of lipid A with core oligosaccharides is intact. Both alkaline and acid hydrolysis of R-LPS abrogates mitogenic activity in C3H/HeJ splenocytes; only the former is effective in reducing activity of the same LPS preparations in histocompatible normal splenocytes. Finally, we have found that the addition of either polymyxin B or S-chemotype LPS to R-LPS-stimulated C3H/HeJ splenocytes has only minimal effects on the mitogenic activity of the latter. These combined data would indicate that the concept of LPS-unresponsiveness of the C3H/HeJ and C57B1/10ScN inbred mouse strains is not necessarily applicable to all protein-free LPS preparations.
Adjuvants, Immunologic↗
The case for specific lipopolysaccharide receptors expressed on mammalian cells.
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Animals↗
Endotoxic-lipopolysaccharide-specific binding proteins on lymphoid cells of various animal species: association with endotoxin susceptibility.
Endotoxic lipopolysaccharide (LPS), a common structural component of all gram-negative bacteria, is well recognized for its capacity to interact with and perturb immunologically relevant cells. Using a radioiodinated, photoactivatable LPS probe, we have recently identified an 80-kilodalton LPS-specific binding protein on murine B lymphocytes. We now have extended these studies to determine if other mammalian species, as well as representative endotoxin-resistant species (frog and chicken), have a similar LPS-binding protein. We have identified what appears to be a relatively conserved 80-kilodalton LPS-binding protein on mononuclear cells of all mammalian species tested. However, both frog and chicken leukocytes failed to show the presence of a similar LPS-binding protein. It is possible that the presence of specific LPS-binding proteins may be important for endotoxin sensitivity of most mammalian species.
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Modulation of endotoxin lethality in mice by hydrazine sulfate.
Although the precise mechanism of endotoxin lethality has yet to be defined, it is well recognized that the amount of hepatic phosphoenolpyruvate carboxykinase is reproducibly and significantly reduced after challenge with endotoxin. Hydrazine has been shown to be a specific inhibitor of gluconeogenesis, causing a metabolic crossover at the step catalyzed by phosphoenolpyruvate carboxykinase. More recently, it has also been shown that hydrazine sulfate may be of potential therapeutic value against cancer cachexia. The experiments described in this paper demonstrate that treatment of CF1 mice with hydrazine sulfate 5 h prior to challenge with endotoxin from Salmonella enteritidis significantly improved survival. Furthermore, such treatment counteracted the drop in hepatic phosphoenolpyruvate carboxykinase activity in isolated cytosol otherwise evident at 6 h and 12 h after endotoxin challenge. Despite this, there was no corresponding improvement in the plasma glucose, measured at 6, 12, and 24 h following endotoxin challenge. It is suggested that the endogenous response to the metabolic crossover initiated by hydrazine may contribute to the protection. The response to hydrazine sulfate has yet to be fully elaborated but does include the increase in phosphoenolpyruvate carboxykinase activity. In contrast with the protection seen upon hydrazine sulfate pretreatment, injecting a corresponding dose of hydrazine sulfate after the endotoxin resulted in more fatalities.
Animals↗
Marine mammals join the Navy.
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Animal Welfare↗
The physical-chemical characterization and biologic activity of serum released lipopolysaccharides.
As a result of the incubation of Escherichia coli in normal human serum, a finite fraction of LPS is released from the bacterial membrane. Approximately half of the LPS released by the action of serum (S-LPS) exists in association with serum proteins in a lower m.w. form than that manifest in phenol-water extracted LPS preparations. The two major LPS-serum protein complexes have apparent Mr of 68 and 32 kDa. The LPS subunit heterogeneity of S-LPS, however, does not appear to differ significantly from LPS retained on the bacteria after serum treatment, or from LPS derived by lysis of whole cells. The biologic activities of S-LPS and phenol-water extracted LPS examined in these studies, differed significantly. In contrast to phenol-water extracted LPS, S-LPS was 1) reduced in lethal toxicity for sensitized mice; 2) reduced in Limulus reactivity; 3) a more potent murine splenocyte mitogen; 4) reduced in the capacity to elicit extracellular, but not membrane-associated IL-1; and 5) reduced in the ability to mediate TNF production. These data suggest that humoral "detoxification" of LPS may involve, in part, the formation of LPS-serum protein complexes with reduced capacities to elicit extracellular cytokine production, whereas the immunomodulatory effects of LPS appear to be enhanced.
Adjuvants, Immunologic↗
Specific endotoxic lipopolysaccharide-binding proteins on murine splenocytes. II. Membrane localization and binding characteristics.
We have characterized the binding of LPS to an 80-kDa LPS-binding protein detected by an LPS photoaffinity probe to be present on murine splenocytes. Specific binding of LPS to the 80-kDa protein is directly proportional to LPS concentration at low concentrations of LPS and is saturable at high concentrations of LPS. Binding is inhibited by both homologous and heterologous underivatized LPS as well as by polysaccharide-free lipid A, indicating a specificity for the biologically active component of LPS. Analysis of the kinetics of binding indicate a time-dependent increase over the first 15 min, but increases are not detected after this time. Binding of LPS to the 80-kDa LPS-binding protein is reduced but still readily detectable at 4 degrees C in the presence of azide. The presence of the 80-kDa LPS-binding protein in an isolated cytoplasmic membrane fraction of murine splenocytes as well as its release from intact splenocytes by octylglucoside suggest that this LPS-binding protein is membrane localized. The results are consistent with, but do not establish unequivocally, the identity of the 80-kDa LPS-binding protein as a specific membrane receptor for lipid A.
Animals↗
Specific endotoxic lipopolysaccharide-binding proteins on murine splenocytes. I. Detection of lipopolysaccharide-binding sites on splenocytes and splenocyte subpopulations.
Experiments have been carried out using a unique radio-iodinated, disulfide-reducible, photoactivatable LPS derivative (ASD-LPS) to detect specific LPS-binding proteins on murine splenocytes. Fractionation of LPS-photo-cross-linked, reduced, and solubilized splenocyte extracts on two-dimensional polyacrylamide gels has allowed the identification of an 80-kDa LPS-binding protein with approximate pI of 6.5. This LPS-binding protein is present on partially purified populations of splenic B lymphocytes, T lymphocytes, and macrophages. It is also the dominant LPS-binding protein on the murine 70Z/3 B cell line and the YAC-1 and EL4 T cell lines but is not detectable on the undifferentiated murine Sp2/0 myeloma cell line. Of potential importance is the fact that the 80-kDa protein appears to be indistinguishable when photolabeled extracts of splenocytes from the C3HeB/FeJ (lpsn) and LPS-nonresponder C3H/HeJ (lpsd) mice are compared.
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Tumor cell killing and cytostasis by C3H/HeJ macrophages activated in vitro by lipid A-associated protein and interferon gamma.
Experiments have been carried out to assess the ability of purified protein-free lipopolysaccharide (LPS) and lipid A-associated protein (LAP) containing LPS to activate macrophages from C3H/HeJ endotoxin-unresponsive mice. Assays for in vitro activation have included cytotoxic and cytostatic effects on a simian virus 40 (SV40)-transformed fibroblast cell line. While neither preparation of LPS would effect C3H/HeJ macrophage activation for either cytostasis or cytolysis, the addition of murine interferon gamma to cultures of LAP-LPS stimulated C3H/HeJ macrophages resulted in cells which were both cytotoxic and cytostatic. These results suggest that LAP can provide one component of the triggering mechanism but, of itself, is insufficient to effect full activation. A second signal, which can be provided by murine interferon gamma, appears also to be required.
Animals↗
The interaction of Escherichia coli with normal human serum: factors affecting the capacity of serum to mediate lipopolysaccharide release.
We previously demonstrated that incubation of E. coli in normal human serum (NHS) resulted in the release of a finite fraction (approximately 30%) of LPS from the bacterial outer membrane. In experiments reported here, we examined factors which may enhance or diminish the capacity of NHS to mediate this limited LPS release. Both the susceptibility to serum killing and LPS release were dependent on growth phase. Optimal killing and release coincided with the midlogarithmic growth phase. The composition of LPS subunits in the outer membrane appeared to influence serum-mediated LPS release. Serum treated E. coli enriched for Rc-chemotype LPS released less LPS from their outer membrane than the wildtype 'smooth' bacteria during exponential growth. LPS fractions released by NHS or EDTA appeared to a large degree to overlap, suggesting that NHS-mediated LPS release may involve the action of a serum chelator. A serum-resistant mutant failed to release LPS in either NHS or EDTA. This latter observation suggests that LPS release may be a relevant event in serum killing. We did not detect any modulation of LPS release when E. coli were pre-incubated with a series of antibiotics prior to treatment with NHS.
Anti-Bacterial Agents↗
Determinants of immunity to murine salmonellosis: studies involving immunization with lipopolysaccharide-lipid A-associated protein complexes in C3H/HeJ mice.
We have earlier demonstrated that the C3H/HeJ Salmonella hypersusceptible mouse can be protected against infection with this organism by prior immunization with lipopolysaccharide (LPS)-lipid A-associated protein (LAP) complexes, but not with LPS alone. In the current studies, protection has been shown to correlate with the induction of LPS-specific antibody in immunized mice. LPS was demonstrated to be a relevant target antigen for Salmonella immunity since C3H/HeJ mice were afforded higher survival rates when they were challenged with Salmonella that shared the same LPS O-antigen as the vaccine. Although low levels of LPS-specific antibody can be detected 14 days after immunization with LAP-LPS, significant antibody is present only after 21-28 days. In addition, anti-LAP specific antibodies can be detected after 14 days of immunization with LAP-LPS. Adoptive transfer of either day 28 anti-LAP-LPS immune serum or day 28 LAP-LPS immune splenocytes alone to naive recipients affords mice minimal, if any, survival against lethal S. typhimurium LT2 challenge. In contrast, transfer of day 28 anti-LAP-LPS immune serum and day 28 LAP-LPS immune splenocytes together is able to transfer Salmonella immunity to naive C3H/HeJ mice. Further, equivalent transfer of only day 28 anti-LAP-LPS immune serum to C3H/HeJ mice immunized 7 days previously with LAP-LPS provides protection similar to that found in mice adoptively transferred with immune cells and serum. These results suggest that a host cellular factor or factors responsive to LAP-LPS, in addition to day 28 anti-LAP-LPS immune serum, may contribute to the protection afforded C3H/HeJ mice following immunization with LAP-LPS.
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