Lipopolysaccharide-induced selective priming effects on tumor necrosis factor alpha and nitric oxide production in mouse peritoneal macrophages.

Preculture of thioglycollate-elicited C3HeB/FeJ mouse peritoneal macrophages in vitro with subthreshold stimulatory concentrations of lipopolysaccharide (LPS) can induce hyporesponsiveness (desensitization) to both tumor necrosis factor alpha (TNF-alpha) and nitric oxide (NO) production when these cells are subsequently stimulated with 100 ng/ml of LPS. We have established, however, that the primary dose of LPS required for inducing downregulation of NO production is significantly lower than that required for inducing downregulation of TNF-alpha production. Further, when LPS-pretreated macrophages become refractory to subsequent LPS stimulation for NO production, the secondary LPS-stimulated TNF-alpha production is markedly enhanced, and vice versa. These results indicate that LPS-induced TNF-alpha and NO production by macrophages are differentially regulated, and that the observed desensitization process may not reflect a state in which macrophages are totally refractory to subsequent LPS stimulation. Rather, our data suggest that LPS-pretreated macrophages become selectively primed for differential responses to LPS. The LPS-induced selective priming effects are not restricted to LPS stimulation, but extend as well to stimuli such as zymosan, Staphylococcus aureus, and heat-killed Listeria monocytogenes.

Dissociation between mitogenicity and immunogenicity of TNP-lipopolysaccharide, a T-independent antigen.

Two models have been proposed to explain triggering of B cells by so-called "T-independent antigen." Feldmann and Basten (1) proposed that the interaction of multiple repeating determinants on polymeric antigens with specific Ig receptors on the B-cell surface is sufficient to provide the signals for division of these cells and differentiation to antibody-forming cells. In contrast, coutinho et al. (2, and see review, 3) have claimed that there is only one signal, a mitogenic signal, receptors acting merely as passive focusing devices to localize the antigen on specific cells where it delivers a mitogenic signal resulting in differentiation to an antibody-producing cell. This model rests primarily on the demonstration that at high concentration all T-independent antigens they have tested are mitogenic for B cells (4-6). Compatible with this hypothesis are the observations that hydrolysis of lipopolysaccharide (LPS) to remove the ester- linked fatty acids of the mitogenic lipid A component abrogates its mitogenic (7,8) activity as well as its ability, when substituted with the TNP hapten, to induce a T-independent anti- TNP response (9). However, alkali treatment of LPS, although not changing its antigenic component (8), may also modify the molecule physically or chemically which could account for loss of immunogenic properties (10). We therefore investigated other reagents which interact with LPS in a more chemically defined manner in an effort to clarify the relationship between the mitogenic and immunogenic properties of this molecule. Polymyxin B (PB) is one of a family of cyclic peptide antibiotics which are bactericidal for most gram-negative bacteria. It prevents the lethal endotoxic activity of LPS (11, 12) and changes the physical structure of LPS (13). We report here that low doses of PB added to cultures of mouse spleen cells inhibit the mitogenic activity of TNP-LPS, a T- independent antigen, and native LPS, but do not suppress the immune response to TNP-LPS. PB interacts with TNP-LPS and LPS causing a physical change in the molecule. In addition, polymyxin-treated LPS is no longer mitogenic. These results suggest a dissociation between the mitogenic and immunogenic properties of TNP-LPS.

Direct evidence for Hageman factor (factor XII) activation by bacterial lipopolysaccharides (endotoxins).

Purified precursor Hageman factor has been demonstrated to bind to soluble bacterial lipopolysaccharide (LPS, endotoxin) isolated from Escherichia coli 0111:B4, and this complex has been shown to have the capacity to convert prekallikrein to its active form. In addition, LPS-activated Hageman factor substantially reduces clotting times in XII-deficient plasma. The capacity to activate Hageman factor has been demonstrated to reside in the lipid A region of the LPS molecule. Activation of Hageman factor by LPS contrasts with fluid-phase activation (e.g., by kallikrein or trypsin) in that no cleavage to lower molecular weight fragments occurs. High concentrations of LPS inhibit the activity of Hageman factor, probably by a direct LPS-Hageman factor interaction.

Isolation of a lipid A bound polypeptide responsible for "LPS-initiated" mitogenesis of C3H/HeJ spleen cells.

The experiments by Sultzer and Nilsson (1), and later by Watson and Riblet (2), established that spleen cells from the C3H/HeJ strain of mouse were refractory to the mitogenic effects of bacterial lipopolysaccharides (LPS). More recently, however, experiments from our laboratory (3) demonstrated that spleen cells from C3H/HeJ mice were in fact responsive to some preparations of LPS but not to others, and that the method of extraction played a critical role in determining activity. In particular, preparations of LPS prepared by extraction with aqueous butanol had potent mitogenic activity. Our data showed that the mitogenic activity of such positive preparations of LPS coisolated with the LPS during gel filtration chromatography and subsequent equilibrium banding on CsCl. In addition, lipid A isolated from positive preparations of LPS was also capable of stimulating C3H/HeJ spleen cells. Taken together, these experiments provided rather convincing data that it was the LPS (in particular the lipid A) itself, or some contaminant very tightly bound to the lipid A, which was responsible for its biological activity. We further demonstrated that treatment of positive preparations of LPS with hot phenol rendered such preparations nonmitogenic for C3H/HeJ spleens, yet activity for other strains was only moderately decreased. These experiments would suggest either that the phenol treatment chemically alters the lipid A region of the LPS molecule or that such treatment removes the putative tightly bound contaminant responsible for C3H/HeJ mitogenesis. In the experiments reported here, we have explored in greater detail the role of lipid A in the stimulation of C3H/HeJ spleen cells. For these experiments we have utilized our earlier observations that the antibiotic polymyxin B forms a highly stable molecular complex with the lipid A region of LPS (4), and that such polymyxin B-LPS complexes are unable to mitogenically stimulate B lymphocytes (5). In addition, we have attempted to distinguish between the two potential modes of action of phenol on LPS, namely, the chemical alteration of the lipid A or the removal of a tightly bound contaminant by phenol treatment. The results of the experiments we report here support the interpretation that mitogenic activity of positive preparations of LPS is associated with a low mol wt phenol soluble polypeptide of approximately 10,000 mol wt. After partial purification, this polypeptide intitiates a significant mitogenic response at concentrations as low as 10 mug/ml. We conclude that the C3H/HeJ strain of mouse is a true nonresponder to the stimulatory effects of the lipid A region of LPS.

Hydrazine sulfate protects D-galactosamine-sensitized mice against endotoxin and tumor necrosis factor/cachectin lethality: evidence of a role for the pituitary.

In previously published studies, we had demonstrated that hydrazine sulfate pretreatment protected mice against the lethal effects of endotoxin and that this protection was accompanied by a sustained increase in hepatic phosphoenolpyruvate carboxykinase activity (Silverstein, R., C.A. Christoffersen, and D.C. Morrison. 1989. Infect. Immun. 57:2072). The same hydrazine sulfate pretreatment has now been found to protect mice against endotoxin in the D-galactosamine model with an increase in the endotoxin LD50 of approximately four orders of magnitude. Elimination of the pretreatment period, or administration of an additional dose of D-galactosamine at the time of hydrazine sulfate pretreatment, renders the mice refractory to the protection. Given the sensitivity of phosphoenolpyruvate carboxykinase regulation to several hormones, we investigated the possibility that protection may have been hormone mediated. In addition to determining the effect of hydrazine sulfate on the plasma levels of phosphoenolpyruvate carboxykinase regulating hormones, we have investigated the effects of hydrazine sulfate on endotoxin lethality in mice whose capacity to respond hormonally to external stimuli has been compromised by hypophysectomy. Our results show a significant enhancement in circulating levels of plasma corticosterone 30 min after hydrazine sulfate injection. Moreover, hypophysectomy results in a marked increase in sensitivity of mice to endotoxin challenge as well as an abrogation of the protection against endotoxin lethality mediated by hydrazine sulfate. Although hydrazine sulfate protection distinguishes between sensitivity brought on, individually, by D-galactosamine and by hypophysectomy, mice sensitized by both hypophysectomy and D-galactosamine are not protected against endotoxin lethality by hydrazine sulfate. We conclude that hydrazine sulfate protection against endotoxin lethality is endocrine dependent, with the available evidence implicating a pituitary/adrenal axis, with glucocorticoid involvement. In as much as D-galactosamine is known to act directly in the liver in disrupting protein synthesis, it is proposed that events in the liver are critical to the hydrazine sulfate-mediated protection against endotoxin and are possibly the target of the endocrine involvement. Hydrazine sulfate pretreatment also protects D-galactosamine-sensitized mice against the lethal effects of injected tumor necrosis factor/cachectin.

Bactericidal activity of the alternative complement pathway generated from 11 isolated plasma proteins.

Exposure of E. coli K12 W1485 to the cytolytic alternative pathway assembled from the 11 isolated pathway proteins resulted in killing of the bacteria, as evidenced by loss of viability. Lysis of the bacteria required introduction of lysozyme into the reaction mixture. The time-course and dose dependency of bacteriolysis in the isolated system were identical to those in C4-depleted serum. The bacteriolytic activity of the pathway was highly dependent on the concentration of the pathway proteins and became insignificant at 1:16 physiological concentration. Electron microscopic visualization of killed and of lysed bacteria revealed numerous complement membrane lesions and partial disintegration of the outer phospholipid membrane. Scanning electron microscopy showed that killed bacteria were enlarged, partially collapsed and exhibited irregular surface protrusions. Lysed bacteria were fragmented and appeared polymorphic. This study demonstrates that the alternative pathway, in absence of immunoglobulins, has the potential or eradicating gram-negative bacteria.

Macrophage stimulation by bacterial lipopolysaccharides. II. Evidence for differentiation signals delivered by lipid A and by a protein rich fraction of lipopolysaccharides.

Stimulation of macrophages to lyse tumor cells is a property common to lipopolysaccharide (LPS) extracted from a variety of smooth and rough bacterial strains by several different preparative procedures. The relationship between macrophage stimulation and the structural characteristics of LPS is defined. In protein-free LPS, lipid A bears the stimulatory signal which results in the differentiation of elicited macrophages into killer cells. The polysaccharide moiety is neither stimulatory itself nor does it block the activity of complete LPS on macrophages. Extraction of LPS by the butanol or Boivin procedures produces preparations in which LPS is complexed through its lipid A moiety to a protein rich component, LAP. Isolated LAP delivers a macrophage differentiation signal which is independent of lipid A. The presence of these two structurally distinct constituents in the cell walls of gram-negative bacteria broadens the biological environments in which they can stimulate macrophages in vivo.

Immunologic properties of bacterial lipopolysaccharide (LPS). II. The unresponsiveness of C3H/HeJ Mouse spleen cells to LPS-induced mitogenesis is dependent on the method used to extract LPS.

The C3H/HeJ mouse strain, previously shown to be a nonresponder to bacterial lipopolysaccharide (LPS)-induced mitogenesis in vitro, was demonstrated by the present studies to be competent to respond mitogenically to LPS, but only to LPS preparations obtained by selected extraction methods. These preparations appear to be confined to LPS isolated by mild extraction techniques, such as TCA or butanol. In contrast, those obtained by techniques utilizing phenol were only weakly stimulatory or completely nonstimulatory for spleen cells from the C3H/HeJ. All LPS preparations tested, on the other hand, were highly stimulatory for cells from another mouse strain, namely the C3H/St. The critical importance of the method of extraction of LPS on its mitogenic activity for C3H/HeJ cells was stressed by experiments in which LPS was prepared from Escherichia coli K235 using either of two procedures. In these experiments, phenol-extracted LPS, although mitogenic in the C3H/St, was completely nonstimulatory in the C3H/HeJ; whereas, butanol-extracted LPS was highly stimulatory in both strains of mice. This striking difference was attributed to a destructive effect of phenol on LPS, as demonstrated by the fact that treatment of butanol LPS with phenol resulted in a total loss of its mitogenic activity in the C3H/HeJ, but in only a partial loss in the C3H/St. In general, the mitogenic response observed with selected LPS preparations in the C3H/HeJ was quantitatively lower and more transient than that seen with the C3H/St, although qualitatively these responses appeared to be similar. This was evidenced by the observation that in both mouse strains LPS was a specific mitogen for B cells, a property which was also attributed in both strains to the same distinct structural region of the LPS molecule, that is lipid A. A preparation of LPS that failed to stimulate B cells from the C3H/HeJ nonetheless had the capacity to block activation of these B cells by a stimulatory preparation of LPS. These results strongly suggest that mitogenic stimulation of B cells by LPS is a function of the structural integrity of both the LPS molecule and putative B-cell receptors for LPS.

Mediation systems in bacterial lipopolysaccharide-induced hypotension and disseminated intravascular coagulation. I. The role of complement.

We have studied the role of complement in lipopolysaccharide (LPS)-induced hypotension and disseminated intravascular coagulation (DIC) by comparing the effects of injection of three preparations of LPS from E. Coli 0111:B4, S. minnesota Re595, and S. marcescens. Injections of nonlethal doses of these LPS preparations into normal rabbits produced decreases in mean arterial blood pressure during a 5-h period. When rabbits treated with cobra venom factor (CoF) to deplete C3 were injected with the various LPS preparations, mean arterial pressures fell at a rate and extent essentially identical to that observed in normal rabbits. Rabbits genetically deficient in C6 also demonstrated LPS-induced hypotensive changes. Only minimal, or no changes in plasma C3 levels or serum CH50 values were detected in normal rabbits after LPS injection. Hypotensive changes were also induced in rabbits when complement was rapidly activated by intravenous injection of CoF. In contrast to the hypotension induced by LPS, the fall in arterial pressure associated with the consumption of complement was short lived and required the rapid consumption of considerable amounts of C3. The occurrence of DIC noted in normal rabbits injected with each preparation of LPS was not inhibited in either rabbits treated with cobra factor or in C6-deficient rabbits. The DIC was most pronounced after injection of Re595 and S. marcescens LPS. Injection of the various LPS preparations produced a rapid disappearance of circulating neutrophils and mononuclear cells, which occurred with the same kinetics and to the same extent in normal, CoF-treated, and C6-deficient rabbits. Injection of either Re595 LPS or S. marcescens LPS produced a biphasic disappearance of circulating 51Cr-platelets. In contrast, injection of 0111:B4 LPS affected only slightly the rate of disappearance of 51Cr-platelets. Depletion of C3 by cobra factor treatment had no effect on the disappearance of platelets in animals injected with 0111:B4. In marked contrast cobra factor treatment greatly reduced the initial rapid disappearance of platelets in rabbits injected with either Re595 or S. marcescens LPS, but had no effect in the secondary disappearance phase.

Lipopolysaccharide receptors and signal transduction pathways in mononuclear phagocytes.

There is little question but that bacterial lipopolysaccharides (LPS) remain one of the most potent stimuli which can affect macrophage activation. Although the precise biochemical mechanisms responsible for this remain to be fully defined, there is now evidence accumulating from a number of laboratories that functional receptors for these bacterial products do exist and may contribute to the initial triggering event. Unfortunately, there is currently no consensus as to which of the candidate receptors identified to date serves as the primary binding target for LPS, and it is possible that the difference in macrophage cell types, LPS probes, and detection systems will all influence the nature of the binding. At the present time, therefore, macromolecules of 96-kDa, 95-kDa (adhesion beta chain), 80-kDa, 65-kDa, and 55-kDa may be considered as possible LPS targets. With the exception of the 96-kDa protein identified by Hampton and his co-workers, there exists some experimental evidence for a functional role for each of the molecules so far identified. It is apparent that the molecular cloning and sequencing and subsequent biochemical characterization of these LPS receptors will be required to determine unequivocally their role in LPS-mediated triggering events. Such information will be invaluable in sorting out the relevant biochemical second signals involved in macrophage activation. Although much new information has recently been accumulated on potential signaling pathways for LPS, the definitive events remain far from unequivocally established. In view of the obvious importance of LPS-macrophage interactions in the overall capacity of the mammalian host to respond appropriately to the potentially hostile prokaryotic environment, a precise delineation of LPS-mediated macrophage activation is critical to our understanding of this important inflammatory mediator cell.

Lipopolysaccharide structure-function relationship in activation versus reprogramming of mouse peritoneal macrophages.

Lipopolysaccharide (LPS) is one of the most potent stimuli for macrophages. The activities of LPS have been attributed to the lipid A region of the molecule. We have previously shown that pretreatment of macrophages with very low doses of LPS can selectively "reprogram" these cells to respond differentially to subsequent activation, as assessed by tumor necrosis factor-alpha and nitric oxide (NO) production. Here we demonstrate that the relative capacity of various LPS preparations for induction of down-regulation of subsequent LPS-activated NO production correlates well with their relative potency for initiation of NO formation. Although LPS-dependent activation can be regulated by pertussis toxin (PT)-sensitive factor, the LPS pretreatment-induced reprogramming is shown here to be refractory to regulation by PT. These results suggest that, although the structural components of LPS dictating the relative activities of the molecule for activation versus reprogramming are similar, there may exist different pathways in initiation of LPS-induced activation versus reprogramming.

Modulation of murine macrophage metabolism by glycolipids: inhibition of LPS-induced metabolism by specific gangliosides.

We have investigated the ability of gangliosides isolated from murine brain to modulate macrophage metabolism. LPS elicits a profound increase in glucose consumption by cultured resident macrophages. When highly purified mouse brain gangliosides are added to macrophage cultures, a modest inhibition of baseline glucose utilization occurs. Both disialogangliosides and trisialogangliosides mediate this effect. In the absence of serum, these gangliosides may be toxic to cultured macrophages. GT1b is the only brain ganglioside tested that specifically inhibited LPS-induced macrophage metabolism. Sialic acid appears to be a necessary component of the gangliosides, although it is not sufficient to induce inhibition. Asialoganglioside and free sialic acid have no effect on macrophage metabolism in comparison with intact gangliosides. The inhibition of LPS-induced metabolism may be explained by the ability of LPS to form stable molecular complexes with both disialogangliosides and trisialogangliosides. These experiments also suggest that the ganglioside content of macrophages may influence functional responses of macrophages to exogenous stimuli.

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.

Detection and analysis of the 80-kd lipopolysaccharide receptor in macrophages derived from Lpsn and Lpsd mice.

An 80-kd lipopolysaccharide (LPS)-binding protein with specificity for the lipid A region has been identified on lymphocyte and macrophage membranes. In an attempt to gain insight into the hyporesponsiveness of C3H/HeJ (Lpsd) mice to LPS, an affinity-purified rabbit polyclonal IgG with specificity for this receptor was used to compare the expression and distribution of the 80-kd LPS receptor on thioglycollate-elicited peritoneal macrophages derived from normal (Lpsn) and (Lpsd) mice. By enzyme-linked immunosorbent assay, immunofluorescence microscopy, and flow cytometry, macrophages from Lpsn and Lpsd mice showed comparable expression of the 80-kd LPS receptor, although only a subpopulation of macrophages from both strains express it. Macrophages from both strains showed indistinguishable surface distribution of the 80-kd LPS receptor, as determined by confocal microscopy and analysis using an anchored cell analysis station. Treatment of macrophages with interferons, protein-rich LPS, or glucocorticoids, agents known to modulate a variety of macrophage cell surface markers and receptors, failed to alter expression of the 80-kd receptor. These findings support the hypothesis that the hyporesponsiveness of the Lpsd mouse strains to LPS is not due to an absence of receptors but rather is distal to binding of LPS and most likely attributable to a failure of these macrophages to transduce the signal derived at the cell surface to the interior of the cell.

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 O55: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 O55 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 degrees 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.

Activation of human serum complement by bacterial lipopolysaccharides: structural requirements for antibody independent activation of the classical and alternative pathways.

A variety of bacterial lipopolysaccharide (LPS) preparations with highly defined primary polysaccharide chemical structure and/or aggregate macromolecular composition have been employed to examine the molecular requirements for activation of the classical and alternative pathways of human serum complement. Evidence is presented for two independent modes of polysaccharide dependent activation of the APC by LPS. One mechanism is dependent upon specific O-antigen polysaccharides and the second is defined by a specific L-glycero-D-mannoheptose/glucose region of the core oligosaccharide. LPS O-antigen polysaccharide but not core oligosaccharide determinants can convert sheep erythrocytes to cells capable of initiating the APC. The data presented provide convincing evidence that the tertiary assembly of individual LPS subunits into an aggregate macromolecule is a critical determinant in the expression of APC activity by LPS. The results of these studies provide strong evidence that CPC activation by LPS is restricted to the Re-chemotype and isolated lipid A. LPS isolated from other R-chemotypes as well as native wild type LPS preparations do not activate the CPC, in spite of the fact that the former LPS preparations contain more lipid A than polysaccharide on a percentage by wt basis. The presence of core polysaccharide L-glycero-D-mannoheptose, which provides a critical recognition role for activation of the APC, appears to negatively regulate CPC activation in a similar inverse relationship. In addition, the presence of polysaccharide containing LPS subunits in synthetic mixed LPS micellar aggregates can also restrict CPC activation by Re LPS subunits, most probably by steric hindrance at the LPS macromolecular surface. Our data are consistent with the hypothesis that activation of either pathway of human serum complement by a given LPS preparation is a mutually exclusive event dictated by the presence or absence of L-glycero-D-mannoheptose.

Ganglioside modulation of lipopolysaccharide-initiated complement activation.

Highly purified preparations of mouse gangliosides have been demonstrated to bind to purified preparations of lipopolysaccharide (LPS). In some instances, the binding has been demonstrated to be dependent upon the presence of sialic acid in the ganglioside preparation. The binding of gangliosides to LPS from the deep rough Salmonella minnesota Re mutant has suggested that the interaction involves the lipid A-2-keto-3-deoxyoctulosanate region of the LPS macromolecule. The interaction between gangliosides and LPS has been demonstrated to result in an abrogation of lipid A dependent activation of the classical pathway of serum complement by Re LPS. Surprisingly, however, the presence of sialic acid containing glycolipids has been shown to enhance significantly the capacity of LPS to initiate activation of the alternative pathway of complement. These data suggest that sialic acid can enhance as well as inhibit the formation of a stable alternative-pathway C3 convertase.

Hemopoietic effects in mice of a lipid A-associated protein.

The effects of the bacterial cell-wall components (BCWC) lipid A and lipid A-associated protein (LAP) on humoral and cellular hemopoietic parameters were investigated in mice. Both lipid A and LAP increased serum levels of granulocyte-macrophage colony-stimulating factors (CSF) in C57BL/6 mice. In C3H/HeJ mice the CSF responses to lipid A and LAP were 7 and 3 fold less than the corresponding CSF responses found in C3H/GSF mice. Both BCWC increased the numbers of splenic multipotential hemopoietic stem cells (CFUS) as well as colony-forming cells (CFC) for neutrophilic granulocytes, macrophages, eosinophils and megakaryocytes. Lipid A but not LAP caused a marked decrease in the femoral numbers of B lymphocyte colony-forming cells (BL-CFC). The Bl-CFC incidence in the spleen or in the mesenteric lymph node changed little if at all after injection of either of the two BCWC. Morphological analysis of marrow cells showed an increase in the proportion of myeloid cells and a concomitant decrease in the proportion of erythroid precursor cells after injection of both BCWC. In the spleen, lipid A but not LAP caused an increase in the proportion of myeloid cells, erythroid precursor cells and plasma cells. In all experiments where both BCWC showed activity, lipid A was more potent than LAP on a weight basis.

Receptor modulation and mast cell secretion.

Co-workers and I examined the early events in the interaction of rat peritoneal mast cells with a number of noncytotoxic mast cell stimuli. These studies demonstrated that an interaction limited to the cytoplasmic membrane is sufficient to induce a secretory mast cell response. Additional experiments suggested that a continued interaction between the stimulus and the membrane receptor is required for maintenance of the secretory response. We have provided evidence to support the concept that different mast cell activators interact with different receptors on the cell membrane and initiate distinct biochemical pathways leading to secretion. Finally, we have shown that pretreatment of mast cells with concentrations of a stimulus that, by themselves, are incapable of initiating a secretory response can profoundly affect the ability of mast cells to respond fully to a subsequent challenge with an optional concentration of stimulus.

Lipopolyamines as a therapeutic strategy in experimental Gram-negative bacterial sepsis.

Lipopolyamines are a class of polycationic amphiphilic compounds that have been shown to bind with high affinity to polyanionic macromolecules, including both DNA and bacterial lipopoly-saccharide (LPS). One of these compounds, termed DOSPER (1,3-di-oleoyloxy-2-(6-carboxyl-spermyl)- propylamide), is non-cytotoxic and has been shown to inhibit LPS-mediated cytokine release and lethality in endotoxin challenge models. In the study reported here, the activity of DOSPER was tested in neutropenic rats with invasive Gram-negative bacteremia caused by Pseudomonas aeruginosa. DOSPER alone was ineffective (0/8) at influencing mortality, but provided a significant survival advantage if administered in combination with a bactericidal antibiotic, ceftazidime (10/12; P<0.05). Ceftazidime alone was partially protective (6/12) while the control group had no survivors (0/8). DOSPER administration markedly reduced circulating endotoxin levels (P<0.01) and interleukin-6 levels (P<0.05) but had no significant effect on bacteremia and bacterial concentrations of P. aeruginosa in liver or spleen tissue. Lipopolyamines may be potentially valuable as a therapeutic adjunct in treatment of Gram-negative bacterial sepsis.