High Concentrations Of Lipopolysaccharide Research Articles

Porphyromonas gingivalis lipopolysaccharide stimulation of human monocytes: dependence on serum and CD14 receptor

The purpose of this study was to investigate factors influencing the ability of lipopolysaccharide (LPS) derived from Porphyromonas gingivalis to elicit secretion of tumor necrosis factor-alpha (TNF alpha) from human monocytes (adherent mononuclear cells). The results indicate that P. gingivalis LPS stimulation of TNF alpha from monocytes is comparable to LPS from Escherichia coli. Both LPS, although structurally different, increased TNF alpha secretion in a dose-dependent manner. In serum-free conditions, TNF alpha secretion was relatively low, but it dramatically increased at human serum concentrations as low as 1%. Maximal secretion was observed in the presence of 10% serum, with a slight decrease at higher serum concentrations. The CD14 molecule is a putative monocyte LPS receptor. When cells were pre-incubated with a blocking monoclonal antibody (My4) to CD14, TNF alpha-mRNA accumulation and TNF alpha secretion were reduced to control levels at LPS concentrations of up to 10 ng/ml. At higher LPS concentrations, the blocking effect was only partial, in spite of 50-fold excess antibody concentration. The blocking effect was observed only in the presence of serum. The effect of the CD14 antibody was dose-dependent with saturation at 2.5 micrograms/ml. The results suggest that CD14 is one of the major receptors for P. gingivalis LPS but highlight the necessity to investigate other cell-surface receptors mediating P. gingivalis-LPS interactions. These interactions are believed to be important in the pathogenesis of periodontal destruction.

FK506 and Cyclosporin A Enhance IL‐6 Production in Monocytes: A single‐Cell Assay

The effect of FK506 and cyclosporin A (CsA) on the production of interleukin 6 (IL-6) in adherent monocytes was studied at a single-cell level by the avidinbiotin- peroxidase complex methods. The percentage of IL-6-producing monocytes increased when stimulated with lipopolysaccharide (LPS) at concentrations between 10 ng/ml and 10 μg/ml, in a dose dependent manner. Both FK506 and CsA enhanced the percentage of IL-6- producing monocytes stimulated with 100 pg/ml-1 μg/ml of LPS up to values near those obtained with 10 μg/ml of LPS. The enhancement by FK506 and CsA was not seen when monocytes were stimulated with a high concentration of LPS (10 μg/ml). When monocytes were stimulated with a low concentration of LPS (10 ng/ml), FK506 and CsA enhanced IL-6 production in a dose dependent manner, at a drug concentration of 0.12 nM–1.2 μM (0.1–1 000 ng/ml) for FK506 and 0.83 nM–8.3 μM (1–10 000 ng/ml) for CsA. The optimal effect of FK506 was achieved at a concentration 7-fold lower than that of CsA. In contrast, production of turnout necrosis factor-α (TNFα and interleukin 1β (IL-1β) was slightly suppressed by FK506 and CsA at the concentrations tested. Moreover, pretreatment of monocytes with FK506 and CsA had a significant enhancing effect on LPS-induced IL-6 production, while treatment with FK506 or CsA after LPS stimulation had no effects on IL-6 production, suggesting that the enhancing effect of each drug is exerted before LPS stimulation or at an early stage of the post-receptor pathway after LPS stimulation. These experiments demonstrate that FK506 and CsA can selectively enhance IL-6 production in monocytes under certain conditions in vitro and, possibly, also in vivo.

Inhibition of macrophage inducible protein expression by delta-9-tetrahydrocannabinol

Macrophages have been shown to undergo a sequential process to full activation in response to priming and triggering signals such as gamma interferon (IFN γ) and bacterial lipopolysaccharide (LPS). These cells also may be driven directly to full activation by exposure to relatively high concentrations of LPS. Each of the stages to activation is associated with differential protein expression suggesting that newly synthesized proteins are associated with the functional activities attributable to that activation state. These observations indicate that protein profiles may serve as a barometer of the macrophage activation state. Delta-9-tetrahydrocannabinol (THC), the major psychoactive component in marijuana, was shown to inhibit inducible protein expression in response to the priming agents Concanavalin A (Con A) supernatant and IFN γ. THC also suppressed protein expression in response to LPS. P388D 1 and RAW264.7 macrophage-like cells, treated with Con A supernatant of IFN γ, exhibited restructuring of protein profiles based on iso-Dalt two-dimensional gel electrophoresis. Protein profile restructuring, distinctive from that elicited in response to priming agents, was seen for macrophages treated with LPS. Treatment of macrophages with Con A supernatant, IFN γ, or LPS in concert with THC (10 −7 M to 10 −5 M), resulted in the generation of protein profiles whose patterns reverted approximately to those of unprimed or unactivated macrophages. THC was shown to alter the expression of select proteins whose induction is associated with macrophage priming or activation. The expression of P388D 1 macrophage class II la molecules of the major histocompatibility complex (MHC), in response to Con A supernatant and IFN γ, was inhibited. THC also altered the expression of tumor necrosis factor α (TNF α) elicited by RAW264.7 cells in response to LPS. These results suggest that THC alters macrophage functional activities, at least in part, by suppressing their capacity to express effector molecules elicited in response to priming and activating signals.

Tissue Factor Expression in Bovine Endothelial Cells Induced byPasteurella haemolyticaLipopolysaccharide and Interleukin-1

Pasteurella haemolytica in cattle produces fibrino-hemorrhagic pleuropneumonia characterized by extensive pulmonary microvascular thrombosis and parenchymal necrosis. The purpose of this in vitro study was to determine if P. haemolytica lipopolysaccharide (LPS) promotes vascular thrombosis by inducing a procoagulant state in vascular endothelial cells. After treatment of confluent monolayers of bovine pulmonary artery endothelial cells with various concentrations of either P. haemolytica LPS or Escherichia coli LPS, the procoagulant activity of the endothelial cells was determined using a chromogenic assay dependent on cellular tissue factor expression. The LPS treatment induced significant increases in cellular tissue factor expression in a LPS concentration- and time-dependent manner. Highest levels of tissue factor were present at 22 hours after treatment, although high LPS concentrations induced moderate tissue factor levels at 5 hours after treatment. Interleukin-1 also induced tissue factor expression in endothelial cells and enhanced the LPS-induced effects. This interleukin-1 effect could be diminished by concurrent use of an interleukin-1 receptor antagonist. These results demonstrate that LPS and cytokine promotion of a procoagulant state in endothelial cells occurs in vitro. Similar mechanisms may play a role in P. haemolytica-mediated pulmonary vascular thrombosis.

Neither CD14 nor serum is absolutely necessary for activation of mononuclear phagocytes by bacterial lipopolysaccharide

The stimulation of mononuclear phagocytes by lipopolysaccharide (LPS) is facilitated by the binding of complexes of LPS and LPS-binding protein to CD14. Although it is clear that CD14 is involved in LPS-induced signaling, other investigators have hypothesized the existence of additional signaling pathways in macrophages. We sought to determine whether CD14-independent pathways of monocyte activation might exist. Washed human mononuclear cells responded with reduced sensitivity to LPS in the absence of serum. Anti-CD14 monoclonal antibody (MAb) inhibited the response to LPS in serum-free conditions, but this was easily reversed at higher concentrations of LPS. We established a human monocytic cell line, designated SFM (derived from THP-1), in serum-free medium to examine LPS responses under defined conditions. Differentiation of SFM cells with 1,25-dihydroxycholecalciferol promoted the expression of abundant cell surface CD14. Differentiated SFM cells responded to LPS despite the complete absence of serum proteins for > 20 generations of growth. LPS stimulation of differentiated SFM cells was inhibited by anti-CD14 MAbs only when serum was present. In contrast to anti-CD14 MAb, the LPS antagonists lipid IVa and Rhodobacter sphaeroides lipid A inhibited monocyte activation under serum-free conditions, implying that these compounds compete with LPS at a site distinct from CD14. Undifferentiated SFM cells (expressing minimal CD14) still responded to LPS in serum-free conditions, and anti-CD14 MAb had little inhibitory effect. The addition of purified LPS-binding protein or human serum promoted a CD14-dependent pathway of monocyte activation by LPS in these cells. We conclude that monocytes do not absolutely require serum proteins to be stimulated by LPS and that CD14-independent LPS signaling pathways exist which are inhibitable by lipid IVa and R. sphaeroides lipid A.

Impaired phagocyte responses to lipopolysaccharide in paroxysmal nocturnal hemoglobinuria.

Bone marrow-derived cells from patients suffering from paroxysmal nocturnal hemoglobinuria (PNH) show a defect in the expression of phosphatidylinositol-anchored membrane proteins, including the CD14 molecule. Blocking experiments with anti-CD14 monoclonal antibodies have shown that lipopolysaccharide (LPS)-induced tumor necrosis factor alpha production by monocytes depends on the interaction between CD14 and a complex formed by LPS and LPS-binding protein. We used a whole-blood model to examine the LPS-induced production of tumor necrosis factor alpha and interleukin-6 in PNH patients and healthy volunteers. At low endotoxin concentrations (1 ng/ml), PNH patients displayed a marked defect in the production of both cytokines, whereas at high LPS concentrations (100 ng/ml), cytokine production was similar to that in healthy volunteers. Using flow cytometry, we also studied the expression of the adhesion molecules Mac-1 (CD11b/CD18) and ICAM-1 (CD54) by monocytes and granulocytes after LPS stimulation. Compared with phagocytes from healthy volunteers, CD14-deficient cells showed poor Mac-1 and ICAM-1 upregulation when whole blood was stimulated with LPS (1 ng/ml), whereas their response to higher LPS doses (100 and 1,000 ng/ml) was essentially normal. The importance of the CD14 molecule in the activation of phagocytes by low LPS concentrations was confirmed by the inhibitory effect of an anti-CD14 antibody both in healthy volunteers and in PNH patients. Since these patients produce the soluble form of the CD14 molecule, these data suggest that soluble CD14 could play a role in phagocyte responses to LPS. We conclude that, in whole blood, phagocytes from PNH patients show impaired responsiveness to LPS and this phenomenon is most probably related to their defect in expression of membrane CD14.

Nitric Oxide Production by Murine Macrophages Is Inhibited by Prolonged Elevation of Cyclic AMP

Murine macrophages produce large amounts of nitric oxide (NO) on stimulation by interferon (IFN)-γ and lipopolysaccharide (LPS) or a high concentration of LPS alone. Agents which increase intracellular cAMP levels inhibit cytokine production by macrophages. The effect of increased intracellular cAMP levels on NO production was investigated, using a murine inacrophage cell line, J774. NO production was reduced by prolonged elevation of cAMP, but not by a transient increase.

Pre‐exposure of murine macrophages to lipopolysaccharide inhibits the induction of nitric oxide synthase and reduces leishmanicidal activity

Murine macrophages produce nitric oxide (NO) from L-arginine on stimulation with lipopolysaccharide (LPS), alone or with interferon-gamma (IFN-gamma). The effect of incubation of macrophages with low concentrations of LPS on NO synthesis on subsequent stimulation was investigated, using a murine macrophage cell line, J774, and peritoneal macrophages from CBA mice. Cells which had been incubated with LPS produced significantly lower amounts of NO, and expressed lower levels of NO synthase activity, following stimulation with IFN-gamma and LPS, or with a high concentration of LPS. This effect was not reversed by tumor necrosis factor-alpha. The ability of CBA macrophages to kill the intracellular parasite Leishmania major was markedly reduced by pre-incubation with LPS. Reduced NO production by macrophages previously exposed to LPS is a manifestation of endotoxin tolerance, and may represent an important means of regulation of NO synthesis and thus a survival mechanism for intracellular parasites.

Deregulated c-fos Modulates B Cell Responses to Switch Mediators

We examined effects of deregulated c-fos on the proliferation and differentiation of B cells in vitro, using splenic B cells from H2-c-fos transgenic mice. When these cells were cultured with lipopolysaccharide (LPS) plus recombinant interleukin 4 (rIL-4) (104 U/ml), the proliferative response of the B cells was augmented with any dose of LPS used. This augmented proliferation resulted in an increase in IgG1 production in the culture, at the lower concentrations of LPS (<2.5 μg/ml). However, H2-c-fos B cells did not produce IgG1 in the culture at higher concentrations of LPS (>5 μg/ml) probably due to the perturbation of B cell differentiation to antibody-forming cells. These results suggest that the deregulated expression of c-fos modulates the proliferation and differentiation B cells stimulated with LPS plus rIL-4.

Human Anti-Endotoxin Antibody HA-1A Mediates Complement-Dependent Binding of Escherichia coli J5 Lipopolysaccharide to Complement Receptor Type 1 of Human Erythrocytes and Neutrophils

HA-1A has been shown clinically to decrease mortality in septic patients with gram-negative bacteremia. In this study, the ability of HA-1A to augment the serum complement-dependent immune adherence of 125I-labeled Escherichia coli J5 lipopolysaccharide (LPS) to human erythrocytes (RBC) and polymorphonuclear leukocytes (PMNL) was evaluated. In vitro studies indicated three things: HA-1A mediates immune adherence of 125I-J5 LPS to human RBC and PMNL in a dose-dependent manner; under these conditions, high concentrations of LPS (400 ng/mL) could be specifically bound. Immune adherence occurs via the classical complement pathway as demonstrated by its calcium dependence; HA-1A-J5 LPS-C' immune complexes bound to CR1 on human RBC and PMNL. PMNL binding and internalization of immune complexes was demonstrated by trypsin stripping of externally bound immune complexes. These studies support the proposal that HA-1A can lower the bioavailability of endotoxin by mediating binding and potential clearance of LPS via human RBC through the reticuloendothelial system or via direct internalization by peripheral blood PMNL.

Neutrophil adherence induced by lipopolysaccharide in vitro. Role of plasma component interaction with lipopolysaccharide.

Endotoxemia results in neutrophil localization within a number of microcirculatory beds, reflecting in part an adhesive interaction between neutrophils and the vascular endothelial cell. In previous studies, endotoxin or lipopolysaccharide (LPS) treatment of rabbits resulted in neutrophil sequestration at LPS concentrations well below those effective at increasing neutrophil adherence in vitro. We hypothesized that LPS-induced neutrophil adherence involved a plasma component. In the absence of plasma, high concentrations of LPS (10 micrograms/ml) were required to increase human neutrophil adherence to endothelial cells in vitro. With the inclusion of as little as 1% plasma or serum, however, the LPS dose-response curve was markedly shifted, resulting in increments in adherence at 10 ng/ml, and the time course of enhanced adherence was accelerated. Pretreatment studies suggested that the effect of LPS was on the neutrophil rather than the endothelial cell. Immunoprecipitation of 0111:B4 LPS paralleled the loss of functional activity, suggesting that LPS was an integral part of the active complex, rather than altering a plasma component to make it active. The incubation of plasma with LPS decreased the apparent molecular mass of LPS from 500-1,000 kD to approximately 100 kD. The disaggregated 0111:B4 LPS eluted in the range of albumin and was able to increase adherence in the absence of additional plasma. Plasma depleted of lipoproteins or heat treated retained activity, suggesting that the interaction of LPS with HDL or complement did not account for the observed findings. An LPS-binding protein isolated from rabbit serum enhanced the adherence-inducing effects of both 0111:B4 and Re595 LPS. Furthermore, the activity of rabbit serum was abolished after incubation with an antibody directed against this LPS-binding protein (LBP). An antibody directed against CD14, the putative receptor of the LPS-LBP complex, prevented the adhesive response to LPS. These data suggest that LPS is disaggregated by an LBP in serum and plasma to form an active LPS-plasma component complex. This putative complex then interacts with CD14 on the neutrophil so as to induce an adhesive state.

抗体産生細胞の動態に関する研究

Interleukin-2 (IL-2) is one of the cytokines regulating antibody formations. To examine the effect of IL-2 on lipopolysaccharide (LPS)-specific antibody secretion on the number of antibody secreting cells (ASC) were counted in the presence and absence of IL-2 in rat spleen cells culture. First we established the enzyme-linked immunospot (ELISPOT) assay, which is the method of determinating the number of ASC. Rat spleen cells with high and low concentrations of LPS (10 ng/ml and 10μg/ml) were incubated for 3 days in vitro, and the number of the LPS specific ASC were counted. In the culture incubated with high concentration (LPS 10 ng/ml) the number of ASC is higher than that in the culture with low concentration LPS 10μg/ml, it is obious that the appearance of ASC depended on LPS concentrations. Supprisingly addition of IL-2 decreases the number of ASC at low concentration of LPS (10 ng/ml), but it increases that at high concentration of LPS (10μg/ml). Thus opposite effect of IL-2 was observed at different LPS concentrations. It is well-known that IL-2 enhances antibody formation in the presence of antigen. But it is strange that IL-2 suppressed antibody formation in the presence of antigen, in the case of low concentration of LPS.

Reduced alveolar macrophage production of tumor necrosis factor during sepsis in mice and men

Tumor necrosis factor (TNF) has been implicated as a major humoral mediator of sepsis and endotoxin shock. TNF is secreted by cells of the reticuloendothelial system, including alveolar macrophages. Alveolar macrophage TNF production has been postulated to play a pathogenetic role in the development of adult respiratory distress syndrome (ARDS) in sepsis. To evaluate alveolar macrophage production of TNF during sepsis and endotoxin shock, we studied the effects of sepsis and/or in vivo lipopolysaccharide on the in vitro production of TNF by pulmonary alveolar macrophages. Human pulmonary alveolar macrophages were obtained by bronchoalveolar lavage from six septic and five nonseptic patients, cultured in the presence or absence of lipopolysaccharide (1 ng/mL), and assayed for TNF activity in a bioassay using fibroblast lysis. A murine model of sepsis was also utilized to study pulmonary alveolar macrophage TNF production under more controlled conditions. Normal mice were given ip injections of either lipopolysaccharide or saline. After 2 hrs, pulmonary alveolar macrophages were obtained and cultured in saline or various concentrations of lipopolysaccharide (0.001 to 10 micrograms/mL). There was no difference in baseline TNF activity, expressed as per cent lysis at 1:10 dilution, between pulmonary alveolar macrophages from control and septic patients (35.7 +/- 5.5% vs. 24.4 +/- 9.3%, respectively) (p greater than .05). However, when stimulated with lipopolysaccharide in vitro, the pulmonary alveolar macrophages from nonseptic patients produced significantly (p less than .01) more TNF (82.8 +/- 3.6%) than did pulmonary alveolar macrophages from patients with the septic syndrome (35.2 +/- 3.8%). Similar findings were obtained using the murine sepsis model. The baseline TNF activity in pulmonary alveolar macrophages from control mice was 22.9 +/- 7.0% (mean +/- SEM) and from lipopolysaccharide-injected mice was 26.8 +/- 3.3% (p greater than .05). Stimulation with 1 ng/mL lipopolysaccharide in vitro produced an increase in TNF activity in both groups, but the increase was greater in the control mice (68.1 +/- 5.7%) than in the lipopolysaccharide-injected mice (47.5 +/- 5.3%) (p less than .01). When the murine pulmonary alveolar macrophages were stimulated with higher concentrations of lipopolysaccharide (0.1 to 10 micrograms/mL), pulmonary alveolar macrophages from lipopolysaccharide-injected mice produced less than 25.5% of the TNF produced by pulmonary alveolar macrophages from control mice. These studies indicate that sepsis and endotoxin injection result in a rapid decrease in the ability of pulmonary alveolar macrophages from both humans and mice to produce and secrete TNF in response to lipopolysaccharide. We speculate that a downregulation of TNF production or of macrophage responsiveness to lipopolysaccharide has occurred. These results suggest that sustained TNF production by macrophages is not required for lung injury in sepsis.

Interferon-lnduced Indoleamine 2,3-Dioxygenase Activity in Human Mononuclear Phagocytes

Interferon (IFN)-induced tryptophan degradation, catalyzed by indoleamine 2,3-dioxygenase (IDO), has been shown to mediate antimicrobial activity in epithelial cells. IDO activity has also been augmented in peripheral blood mononuclear cells (PBMC) treated with IFN or interleukin-2 (IL-2). The effector cells in this population have now been further characterized. PBMCs were isolated from normal donors, separated into monocyte and lymphocyte populations by plastic adherence, treated with IFN or IL-2, and cultivated in medium supplemented with [3H]tryptophan. Culture supernatants were collected after a 48-h incubation and fractionated by high-performance liquid chromatography; radioactivity was determined in fractions corresponding to tryptophan and its metabolites. IFN-gamma and IFN-beta induced IDO activity only in monocytes (plastic-adherent, nonspecific esterase-positive PBMCs). The induction of IDO activity by IL-2 required both monocytes and lymphocytes. Interaction was required between these populations for induction of IDO by IL-2, due to production of IFN-gamma by T lymphocytes, with subsequent IFN-gamma-mediated induction of IDO in monocytes. A number of myeloid cell lines as well as monocyte-derived macrophages were also tested for their ability to be induced to degrade tryptophan in response to IFN treatment. Monocyte-derived macrophages were found to retain their capacity to be induced by IFN-gamma and IFN-beta to degrade tryptophan after differentiation, and to possess seven times more IDO activity per cell than IFN-induced monocytes. However, the presence of lipopolysaccharide (LPS) in the culture medium was required for the maximum induction of IDO activity by IFN-beta. Furthermore, higher concentrations of LPS were sufficient to induce IDO activity in macrophages in the absence of exogenous IFN.

Detection of tumour necrosis factor from lipopolysaccharide-stimulated human mononuclear cells by enzyme-linked immunosorbent assay and cytotoxicity bioassay.

Tumour necrosis factor (TNF) or cachectin is an important mediator of endotoxic activity. To investigate the production of TNF from human mononuclear cells (MNC) in response to lipopolysaccharide (LPS), we developed a sensitive and specific enzyme immunoassay (ELISA) and a cytotoxicity bioassay for TNF. The ELISA utilizes the biotin/avidin system and includes four incubation steps. The detection limit was 25 pg recombinant TNF (rTNF)/100 microliter. There was no interference of medium, serum, plasma, spinal fluid, or urine and no cross-reaction with natural or recombinant IL-1-alpha, IL-1-beta, IL-2, IFN-gamma, or lymphotoxin (TNF-beta). Recovery of TNF added to the media was 85-123% (n = 22). The relative standard deviations within and between assays were 7% and 8%, respectively. TNF-induced cytotoxicity was measured on actinomycin-D-treated L-M mouse fibroblasts. The detection limit in this bioassay was 0.5 U/30 microliter or 12.5 pg/30 microliter of rTNF. IL-1-alpha and IL-1-beta slightly inhibited the cytotoxic activity of rTNF. In this bioassay, cytotoxic activity (50-300 U/ml) was detected only when MNC were stimulated with high concentrations of LPS (100-1000 ng/ml). In contrast, using 0.01-100 ng/ml of LPS, the ELISA detected TNF in a dose-dependent manner (0.25 ng/ml to 40 ng/ml). It is concluded that TNF is liberated from human blood MNC if stimulated with minute amounts of LPS. It is suggested that human TNF may be secreted in a relatively inactive form or that inhibitors of TNF are generated along with the monokine. Because of this, and because commonly used bioassays for TNF fail to distinguish between TNF and lymphotoxin, specific ELISA are recommended to supplement TNF bioassays.

A new rapid prospecting method for submarine hydrothermal activity by determining lipopolysaccharide as bacterial biomass.

Submarine hydrothermal activity can hardly be detected by measuring water temperature or found by conventional methods like 3He, CH4 and/or metal ions measurements except through time consuming procedures. We applied a rapid and ultra-sensitive colorimetric method to measure the concentration of lipopolysaccharide (LPS), an indicator of bacterial biomass, in seawater around the Izu-Ogasawara Arc, northwestern Pacific, where the presence of hydrothermal activity is suggested. High anomalies of LPS at ca. 50 m-water layer above the sea bottom were discovered in a submarine caldera along the volcanic front and in a back-arc depression. Many extraordinarily large-sized bacteria were also discovered in the sample containing high LPS concentration. It takes only about an hour to analyze LPS after obtaining a sample, and a mere 100 μl of seawater sample is enough for this method. This method should be very effective for the purposes of prospecting the hydrothermal activity.

L-arginine is required for expression of the activated macrophage effector mechanism causing selective metabolic inhibition in target cells.

L-Arginine is required for expression of the activated macrophage cytotoxic effector mechanism that causes inhibition of mitochondrial respiration, aconitase activity, and DNA synthesis in tumor target cells. This effector mechanism is active in the presence of L-arginine even when the cocultivation medium lacks all other amino acids and serum. Cytotoxic activated macrophage-induced inhibition of mitochondrial respiration in target cells is proportional to the concentration of L-arginine in the medium. L-Arginine must be present during the cocultivation period. Pretreatment of cytotoxic activated macrophages with L-arginine or posttreatment of the target cells after cocultivation is not effective. D-Arginine does not substitute for L-arginine and at high concentrations is a competitive inhibitor of the L-arginine-dependent effector mechanism. Other analogues that could not replace L-arginine include agmatine, argininic acid, arginine hydroxamate, and tosyl-L-arginine methyl ester. L-homoarginine, however, can effectively substitute for L-arginine. NG-monomethyl-L-arginine is a potent competitive inhibitor of this effector mechanism. High concentrations of lipopolysaccharide do not reverse inhibition of the L-arginine-dependent effector mechanism by NG-monomethyl-L-arginine. However, inhibition of the effector mechanism by NG-monomethyl-L-arginine can be overridden by increasing the concentration of L-arginine in the culture medium. We compared NGNG-dimethyl-L-arginine and NGN1G-dimethyl-L-arginine with NG-monomethyl-L-arginine as inhibitors of the L-arginine-dependent effector mechanism. The results show that the inhibitory effect of these guanidino methylated derivatives of L-arginine is highly determined by structure. Guanidine is a weak competitive inhibitor of the L-arginine-dependent effector mechanism. The requirement for L-arginine does not appear to be for protein synthesis, creatine biosynthesis, polyamine biosynthesis, or ADP ribosylation reactions. Bacterial lipopolysaccharide is effective as a second signal only when the cocultivation medium contains L-arginine, and this strict L-arginine dependency is not overridden by increasing the concentration of lipopolysaccharide. Bovine liver arginase, by competing for L-arginine in the cocultivation medium, inhibits the L-arginine-dependent activated macrophage cytotoxic effector mechanism.

Lipopolysaccharide-mediated injury to cultured human glomerular endothelial cells

Lipopolysaccharide induced a dose-dependent detachment of human glomerular cells in vitro. The detachment occurred 24 hr after exposure to endotoxin. Human umbilical vein endothelial cells were not affected by more prolonged exposure or higher concentrations of lipopolysaccharide in the medium. The lipopolysaccharide effect was independent of complement components or leukocytes.

Modulation of cyclic AMP-dependent protein kinase isozyme expression associated with activation of a macrophage cell line.

We have used the RAW 264.7 macrophage (MO) cell line to study cAMPdPK isozymes during activation by lymphokine (LK) and lipopolysaccharide (LPS). Untreated cells were found to have two isozymes of cAMPdPK in their cytosol. PKI and PKII were differentiated based on the Mr of their regulatory subunits (RI, 45,500; and RII, 52,000, respectively) as determined by photoactivated incorporation of the cAMP analog 8-N3-[32P]cAMP. Loss of the RI subunit of PKI occurred in association with activation of the cell line by suboptimal concentrations of LK and LPS (1/40 dilution, 1 ng/ml) or high concentrations of LPS alone (10 ng/ml to 100 micrograms/ml). No modulation of the RII subunit of PKII was observed under these conditions. The loss of RI was dependent on the addition of a triggering signal to the MO. Treatment of RAW 264.7 cells with LK alone at dilutions from 1/10 to 1/1280 was not sufficient to cause a disappearance of the RI subunit from the cytosol or to induce antitumor activity. The addition of a suboptimal concentration of LPS after LK or a high dose of LPS alone was required for acquisition of cytolytic activity and loss of RI. The kinetics for the disappearance of RI from treated cells were found to be identical after activation with either LK and LPS or high concentrations of LPS alone. RI could no longer be detected in the cytosol 8 hr after the addition of activating agents. The antitumor activity of the RAW 264.7 cell line was transiently expressed after activation. Cells no longer exhibited tumoricidal activity 48 hr after the removal of activating agents. It was observed that the loss of cytolytic function was accompanied by the reexpression of RI in the cytosol. This study provides evidence that modulation of cAMPdPK isozymes occurs during activation, suggesting a potential mechanism for controlling the effects of cAMP on the MO.

Role of lipopolysaccharide and complement in susceptibility of Haemophilus ducreyi to human serum.

The role of lipopolysaccharide (LPS) in the susceptibility of Haemophilus ducreyi to human serum and the mechanism of complement activation by serum-susceptible (Sers) strains were investigated. Serum treated with 2 mM Mg2+ and 20 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid was nonbactericidal, but inulin-treated serum remained bactericidal. Absorption of serum with heat-killed whole cells of an Sers strain removed its bactericidal activity against the absorbing strain and also against other Sers strains. LPS obtained from Sers strains inhibited the bactericidal activity of serum against all Sers strains, whereas LPS from serum-resistant (Serr) strains and an Serr isogenic strain did not. However, high concentrations of LPS from the Serr strain inhibited the bactericidal activity of serum, an indication that part of the structural site involved in serum susceptibility is retained in the LPS of this strain. The LPS of Sers strains exhibited higher anticomplement activity than the LPS of Serr strains. These findings suggest that the classical pathway of complement activation is involved in the serum killing of H. ducreyi and that LPS composition may contribute to their susceptibility to complement-mediated serum bactericidal activity.