Early-phase endotoxin tolerance: induction by a detoxified lipid A derivative, monophosphoryl lipid A.

After a sublethal exposure to lipopolysaccharide (LPS) or to lipid A, which is that portion of the LPS molecule associated with endotoxicity, a transient period ensues during which a normally responsive individual is rendered hyporesponsive to LPS-induced toxicity. This period has been defined as early-phase endotoxin tolerance. Recently, a nontoxic derivative of lipid A from Salmonella typhimurium, monophosphoryl lipid A (MPL), was isolated and purified. In this study, we assessed the ability of MPL to induce early endotoxin tolerance. Initial injection of MPL resulted in a dose-dependent stimulation of both serum colony-stimulating factor and serum interferon, indicators of in vivo LPS responsiveness. In contrast, MPL failed to induce the symptoms of endotoxicity which are normally seen after injection of even sublethal amounts of intact endotoxin or lipid A preparations. Injection of MPL on day 0 reduced significantly the amount of LPS-induced serum colony-stimulating factor and interferon produced upon challenge with Escherichia coli LPS 3 days later and also mitigated toxic manifestations, as evidenced by a marked increase in the 50% lethal dose. Like the early tolerance induced by wild-type (toxic) LPS, MPL-induced tolerance was characterized by an accompanying elevation in the number of bone marrow-derived macrophage progenitor cells and by an alteration in bone marrow cell sizing profiles. These results indicate that MPL is effective in inducing a state of LPS-hyporesponsiveness without the toxic side effects of endotoxin and that the structural component(s) necessary for induction of early-phase endotoxin tolerance is contained within MPL.     

Dissociation of lipopolysaccharide (LPS)-inducible gene expression in murine macrophages pretreated with smooth LPS versus monophosphoryl lipid A.

Lipopolysaccharide (LPS) and the nontoxic derivative of lipid A, monophosphoryl lipid A (MPL), were employed to assess the relationship between expression of LPS-inducible inflammatory genes and the induction of tolerance to LPS in murine macrophages. Both LPS and MPL induced expression (as assessed by increased steady-state mRNA levels) of a panel of seven "early" inflammatory genes including the tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta, type 2 TNF receptor (TNFR-2), IP-10, D3, D8, and D2 genes (the last four represent LPS-inducible early genes whose functions remain unknown). In addition, LPS and MPL were both capable of inducing tolerance to LPS. The two stimuli differed in the relative concentration required to induce various outcome measures, with LPS being 100- to 1,000-fold more potent on a mass concentration basis. Characterization of the tolerant state identified three distinct categories of responsiveness. Two genes (IP-10 and D8) exhibited strong desensitization in macrophages pretreated with tolerance-inducing concentrations of either LPS or MPL. In macrophages rendered tolerant by pretreatment with LPS or MPL, a second group of inducible mRNAs (TNF-alpha, interleukin-1 beta, and D3) showed moderate suppression of response to secondary stimulation by LPS. The third category of inducible genes (TNFR-2 and D2) showed increased expression in macrophages pretreated with tolerance-inducing concentrations of either LPS or MPL. All of the LPS-inducible genes examined exhibited modest superinduction with less than tolerance-inducing concentrations of either stimulus, suggesting a priming effect of these adjuvants at low concentration. The differential behavior of the members of this panel of endotoxin-responsive genes thus offers insight into molecular events associated with acquisition of transient tolerance to LPS.     

Recombinant interleukin-1 alpha and recombinant tumor necrosis factor alpha synergize in vivo to induce early endotoxin tolerance and associated hematopoietic changes.

Endotoxin, the lipopolysaccharide (LPS) derived from gram-negative bacteria, invokes a wide range of responses in susceptible hosts. It is known that virtually all responses to LPS are mediated by the action of macrophage-derived cytokines (such as interleukin-1 [IL-1], tumor necrosis factor [TNF], and others) which are produced principally by macrophages and maximally within several hours of LPS administration. One manifestation of LPS administration which is not well understood is the phenomenon of "early endotoxin tolerance." In response to a single sublethal injection of LPS, experimental animals become refractory to challenge with a homologous or heterologous LPS preparation 3 to 4 days later. Animals rendered tolerant exhibit mitigated toxicity and a reduced capacity to produce circulating cytokines (i.e., colony-stimulating factor or interferon) in response to the challenge LPS injection. Previous studies have also shown that this state of transient, acquired hyporesponsiveness to LPS is accompanied by a marked increase in the size of cells in the bone marrow which are enriched in numbers of macrophage progenitors. In this study, we examined the capacity of recombinant IL-1 or recombinant TNF or both to induce early endotoxin tolerance and its associated hematopoietic changes. Neither cytokine alone was able to mimic LPS for induction of tolerance. Combined administration of recombinant IL-1 and recombinant TNF doses which were not toxic when administered individually led to synergistic toxicity (as assessed by death or weight loss). However, within a nontoxic range, the two cytokines synergized to induce a significant reduction in the capacity to produce colony-stimulating factor in response to LPS, as well as the characteristic increase in bone marrow cell size and macrophage progenitors shown previously to be associated with LPS-induced tolerance.     

Pulmonary and Hepatic Gene Expression following Cecal Ligation and Puncture: Monophosphoryl Lipid A Prophylaxis Attenuates Sepsis-Induced Cytokine and Chemokine Expression and Neutrophil Infiltration

Polymicrobial sepsis induced by cecal ligation and puncture (CLP) reproduces many of the pathophysiologic features of septic shock. In this study, we demonstrate that mRNA for a broad range of pro- and anti-inflammatory cytokine and chemokine genes are temporally regulated after CLP in the lung and liver. We also assessed whether prophylactic administration of monophosphoryl lipid A (MPL), a nontoxic derivative of lipopolysaccharide (LPS) that induces endotoxin tolerance and attenuates the sepsis syndrome in mice after CLP, would alter tissue-specific gene expression post-CLP. Levels of pulmonary interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), granulocyte colony-stimulating factor (G-CSF), IL-1 receptor antagonist (IL-1ra), and IL-10 mRNA, as well as hepatic IL-1β, IL-6, gamma interferon (IFN-γ), G-CSF, inducible nitric oxide synthase, and IL-10 mRNA, were reduced in MPL-pretreated mice after CLP compared to control mice. Chemokine mRNA expression was also profoundly mitigated in MPL-pretreated mice after CLP. Specifically, levels of pulmonary and hepatic macrophage inflammatory protein 1α (MIP-1α), MIP-1β, MIP-2, and monocyte chemoattractant protein-1 (MCP-1) mRNA, as well as hepatic IFN-γ-inducible protein 10 and KC mRNA, were attenuated in MPL-pretreated mice after CLP. Attenuated levels of IL-6, TNF-α, MCP-1, MIP-1α, and MIP-2 in serum also were observed in MPL-pretreated mice after CLP. Diminished pulmonary chemokine mRNA production was associated with reduced neutrophil margination and pulmonary myeloperoxidase activity. These data suggest that prophylactic administration of MPL mitigates the sepsis syndrome by reducing chemokine production and the recruitment of inflammatory cells into tissues, thereby attenuating the production of proinflammatory cytokines.     

Differential regulation of cytokine production in lipopolysaccharide tolerance in mice.

We investigated the pattern of down-regulation of cytokine production in endotoxin (lipopolysaccharide [LPS]) tolerance. A 4-day treatment with LPS (35 micrograms per mouse) was followed by a challenge on day 6 with one more injection of LPS. Circulating tumor necrosis factor (TNF) and interleukin-6 (IL-6) could not be induced (> 99% inhibition) by LPS in LPS-tolerant mice; colony-stimulating factor (CSF) was also down-regulated by more than 95%, whereas interferon (IFN) and IL-1 syntheses were only partially inhibited. To study the mechanism of cytokine down-regulation in tolerance, we attempted to reverse the tolerant state by pretreatment with phorbol 12-myristate 13-acetate (PMA) (4 micrograms per mouse) 10 min before the LPS challenge. PMA completely restored IL-6 production and partially that of CSF. PMA had no effect on IFN production and inhibited the induction of IL-1. TNF production was also not restored by PMA. To investigate the role of endogenously produced cytokines in the development of LPS tolerance, we administered IL-6, TNF, or IL-1 alpha, using the same treatment schedule as that for LPS. Whereas IL-6 had no effect, IL-1 alpha or TNF induced partial tolerance to LPS in terms of inhibition of LPS-stimulated TNF and IL-6 production. However, a full LPS-tolerant state could not be induced by administration of recombinant cytokines, suggesting the existence of additional mechanisms, such as a loss of LPS receptors or changes in release of soluble binding proteins.     

Stimulation of the ceramide pathway partially mimics lipopolysaccharide-induced responses in murine peritoneal macrophages.

Recent studies have suggested that lipolysaccharide (LPS) stimulates cells by mimicking the second-messenger function of ceramide, a lipid generated in the cell by the action of sphingomyelinase (SMase). To examine this possibility further, we compared the abilities of LPS, SMase, and/or ceramide analogs to induce cytokine secretion, modulate gene expression, and induce endotoxin tolerance in macrophages. SMase and LPS induced secretion of tumor necrosis factor alpha (TNF-alpha) to comparable degrees; however, unlike LPS, SMase failed to stimulate detectable interferon activity. Cell-permeable analogs of ceramide induced the expression of many LPS-inducible genes; however, the expression of interferon-inducible protein 10 (IP-10) and interferon consensus sequence-binding protein (ICSBP) mRNAs was significantly lower than that induced by LPS. Both SMase-induced TNF-alpha secretion and LPS-induced TNF-alpha secretion were inhibited by pretreatment with a serine/threonine phosphatase inhibitor, calyculin A. Macrophages preexposed in vitro to LPS to induce a well-characterized state of endotoxin tolerance secreted little or no TNF-alpha upon secondary challenge with either LPS or SMase, whereas macrophages preexposed to SMase secreted high levels of TNF-alpha upon secondary stimulation with LPS or SMase. Collectively, these results suggest that ceramide activates a subset of LPS-induced signaling pathways in murine peritoneal exudate macrophages.     

Rhodopseudomonas sphaeroides lipid A derivatives block in vitro induction of tumor necrosis factor and endotoxin tolerance by smooth lipopolysaccharide and monophosphoryl lipid A.

Rhodopseudomonas (Rhodobacter) sphaeroides diphosphoryl lipid A is a relatively inert species of lipid A but has been shown to antagonize the effects of toxic lipopolysaccharide (LPS) both in vivo and in vitro. The antagonist and its monophosphoryl derivative were examined for the ability to block tumor necrosis factor synthesis and reverse tolerance induction in vitro in macrophage cultures stimulated with bioactive preparations of smooth LPS, rough LPS, diphosphoryl lipid A, and monophosphoryl lipid A. Inhibition of agonist activity and reversal of tolerance by these novel penta-acylated lipid A antagonists provides new insight into macrophage-LPS interactions.     

Induction of long-term lipopolysaccharide tolerance by an agonistic monoclonal antibody to the toll-like receptor 4/MD-2 complex

We have established an agonistic monoclonal antibody, UT12, that induces stimulatory signals comparable to those induced by lipopolysaccharide (LPS) through Toll-like receptor 4 and MD-2. UT12 activated nuclear factor kappaB and induced the production of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) in peritoneal exudative cells. In addition, mice injected with UT12 rapidly fell into endotoxin shock concomitant with the augmentation of serum TNF-alpha and IL-6 levels, followed by death within 12 h. On the other hand, when the mice were pretreated with a sublethal dose of UT12, the mice survived the subsequent lethal LPS challenges, with significant suppression of serum TNF-alpha and IL-6, indicating that UT12 induced tolerance against LPS. This effect of UT12 was maintained for at least 9 days. In contrast, the tolerance induced by LPS continued for less than 3 days. These results illuminate a novel potential therapeutic strategy for endotoxin shock by the use of monoclonal antibodies against the Toll-like receptor 4/MD-2 complex.     

Restoration of normal febrile response to endotoxin in pyrogen-tolerant rabbits by injection with human beta interferon.

Early-phase pyrogen tolerance was induced in rabbits by two consecutive daily injections of 125 ng of endotoxin per kg of body weight. The second injection of the same dose of endotoxin evoked only a monophasic fever with a peak response 1.5 h after the injection; no second peak was observed. The rabbits were released from the tolerance to develop a typical biphasic fever by an injection of 125 ng of endotoxin along with human beta interferon (HuIFN-beta), although the tolerance-inducing amount of endotoxin alone could not. The profile of the febrile response of tolerant rabbits injected with both endotoxin and HuIFN-beta could not be distinguished from that of normal rabbits. There was no essential difference between natural and recombinant HuIFN-beta in breaking tolerance. Heat-stable (70 degrees C, 30 min) endogenous pyrogen or tumor necrosis factor was increased significantly in concentration in the serum of tolerance-broken rabbits. These results suggest that HuIFN-beta stimulates the production of tumor necrosis factor in tolerant rabbits to elicit the second peak of febrile response.     

Endotoxin tolerance: independent regulation of interleukin-1 and tumor necrosis factor expression.

The injection of lethal or sublethal doses of bacterial lipopolysaccharide (LPS) into mice results in transient increases in both serum tumor necrosis factor (TNF) and interleukin-1 (IL-1). The peak in serum TNF was detected prior to maximal elevation in endogenous corticosterone and was no longer apparent 3 to 4 h post-LPS injection, a point at which corticosterone and IL-1 levels had significantly increased. The initial increase in serum IL-1 may, in part, be modulated by the preceding TNF peak, as pretreating animals with a monoclonal antibody against murine TNF resulted in a significant decrease in IL-1 levels 3 h post-LPS injection. A second injection of LPS at 20 h failed to result in a secondary TNF peak, suggesting an endotoxin-tolerant state. However, in contrast to TNF, significant increases in serum IL-1 were detected in the endotoxin-tolerant animals following a repeated LPS stimulus. This secondary increase in IL-1 occurred despite the elevation in serum corticosterone. While peritoneal macrophages from endotoxin-tolerant mice demonstrated only a modest 10 to 15% increase in TNF and IL-1 mRNA relative to the levels after the primary 1-h LPS stimulus, a secondary increase in IL-1 but not TNF mRNA in the spleen was apparent following a second LPS injection. The spleen, however, was not essential for the increase in serum IL-1, as endotoxin-tolerant splenectomized mice had comparable increases in IL-1 following a repeated LPS stimulus. These results demonstrate the differential regulation of IL-1 and TNF in vivo during endotoxin tolerance.     

Superantigen and endotoxin synergize in the induction of lethal shock

Endotoxin (lipopolysaccharide; LPS) and superantigens (exotoxins) have been identified as potent inducers of lethal shock. While endotoxin primarily interacts with CD 14 receptors on macrophages, superantigens like the staphylococcal enterotoxin B (SEB) preferentially activate T cells. Both cell types are triggered to release pro-inflammatory cytokines that in turn induce lethal shock. We analyzed whether endotoxin and superantigen interact during the induction phase of lethal shock. We report that LPS and SEB operate synergistically. Lethal doses of both inducers were reduced 100-fold when given in combination. The induced serum levels of tumor necrosis factor, interleukin-6, and interferon-γ (IFN-γ) were elevated and remained high for a prolonged period. Moreover, synergistic action of LPS and SEB induced lethal toxic shock even without presensitization of mice with D -galactosamine (D -GalN). Opposed to D -GalN-pretreated mice, mice injected with LPS and SEB showed less liver damage, but rather apoptosis of epithelial cells in the bowel. Cyclosporin A and treatment with anti-IFN-γ monoclonal antibody blocked the synergistic action of LPS and SEB, indicating that T cell-derived IFN-γ is the mediator of the observed synergism. Concomitant injection of LPS and SEB had no influence on SEB-induced T cell deletion and anergy induction. Since Gram-positive and Gram-negative bacteria can be recovered from septic blood samples, the synergistic action of endotoxin and superantigens might be relevant during lethal septicemia.     

Activation of macrophages from aging mice by detoxified lipid A.

A detoxified derivative of endotoxic lipopolysaccharides (LPS), monophosphoryl lipid A (MPL), which is capable of inducing nonspecific resistance against several infectious organisms, was tested for its capacity to activate peritoneal macrophages (M phi) from young and immunodeficient aging BALB/c and C3H/HeN mice. Superoxide generation and hydrogen peroxide release by M phi from aging mice were elevated following intraperitoneal injection with 25 micrograms of LPS or MPL, although they did not reach the peak levels achieved in LPS or MPL-treated young mice. Nitroblue tetrazolium reduction (NBT) by peritoneal M phi from aging C3H/HeN mice treated with MPL was higher than that in control aging mice, equalling that from MPL-treated young mice. LPS, its toxic counterpart, however, failed to increase NBT reduction in either group. MPL enhanced lysozyme activity in M phi from both aging and young C3H/HeN mice above initial control levels. On the other hand, LPS suppressed lysozyme activity in M phi from young, but not aging mice. Phagocytosis of Candida albicans by M phi from BALB/c mice was increased in both groups when stimulated by MPL, but not LPS. Similarly, MPL enhanced the ability to kill Candida in both aging and young BALB/c mice. This effect was not seen with LPS. Thus, a detoxified derivative of LPS was found capable of activating the respiratory burst, NBT reduction, elevating lysozyme activity, as well as phagocytosis and killing of Candida in murine peritoneal M phi from both young and aging mice.     

Detection of circulating tumor necrosis factor after endotoxin administration

Cytokines, products of stimulated macrophages, are thought to mediate many host responses to bacterial infection, but increased circulating cytokine concentrations have not been detected consistently in infected patients. We measured plasma concentrations of circulating tumor necrosis factor alpha (cachectin), interleukin-1 beta, and gamma interferon, together with physiologic and hormonal responses, in 13 healthy men after intravenous administration of Escherichia coli endotoxin (4 ng per kilogram of body weight) and during a control period of saline administration. Eight additional subjects received ibuprofen before receiving endotoxin or saline. Plasma levels of tumor necrosis factor were generally less than 35 pg per milliliter throughout the control period, but increased 90 to 180 minutes after endotoxin administration to mean peak concentrations of 240 +/- 70 pg per milliliter, as compared with 35 +/- 5 pg per milliliter after saline administration. Host responses were temporally associated with the increase in circulating tumor necrosis factor at 90 minutes, and the extent of symptoms, changes in white-cell count, and production of ACTH were temporally related to the peak concentration of tumor necrosis factor. Ibuprofen pretreatment did not prevent the rise in circulating tumor necrosis factor (mean peak plasma level, 170 +/- 70 pg per milliliter) but greatly attenuated the symptoms and other responses after endotoxin administration. Concentrations of circulating interleukin-1 beta and gamma interferon did not change after endotoxin administration. We conclude that the response to endotoxin is associated with a brief pulse of circulating tumor necrosis factor and that the resultant responses are effected through the cyclooxygenase pathway.     

Effect of monophosphoryl lipid A on the in vitro function of peritoneal leukocytes from uremic patients on continuous ambulatory peritoneal dialysis.

We analyzed the effects of monophosphoryl lipid A (MPL), a relatively nontoxic immunostimulant derived from bacterial endotoxin, on the depressed in vitro immune function of leukocytes derived from six patients undergoing continuous ambulatory peritoneal dialysis and who had histories of recurrent bacterial peritonitis. MPL was also tested for its capacity to stimulate the proliferation of peritoneal fibroblasts, as determined by [3H]thymidine incorporation. In vitro incubation of peritoneal lymphocytes and macrophages (PM phi) with increasing amounts of MPL, up to 5 micrograms/ml, resulted in a dose-dependent enhancement of gamma interferon and interleukin-2 production by peritoneal lymphocytes and interleukin-1 release by PM phi. In vitro incubation of PM phi with MPL also resulted in an increase of PM phi bacterial killing and membrane Fc receptor number, although no change in peritoneal fibroblast proliferation was seen with any of the MPL concentrations tested. These results suggest that the peritoneal leukocyte dysfunction observed in patients undergoing continuous ambulatory peritoneal dialysis and who have high rates of peritonitis may be alleviated, to some degree, by MPL, without directly inducing a potentially deleterious fibrotic lesion.     

Tumor necrosis factor mediates endotoxic effects in mice.

Endotoxic reactions induced in mice by recombinant human tumor necrosis factor (TNF) were examined. Mice showed a dose-dependent hypothermia after intravenous TNF injection which was similar to a reaction to lipopolysaccharide injection. Plasma glucose levels were decreased, and plasma lactate levels were increased. Blood hematocrit levels were increased after TNF injection. No interleukin-1 activity was detected in the plasma of TNF-treated animals. The number of leukocytes was reduced 30 min after TNF injection and returned to normal within 24 h. Thus, the data demonstrate that the pathophysiological effects induced by TNF were similar to the effects induced by bacterial endotoxin. Since lipopolysaccharide is a very potent agent for eliciting TNF release from activated macrophages, these results suggest that TNF could act as an endogenous mediator of endotoxin effects.     

Interleukin-1beta induces in vivo tolerance to lipopolysaccharide in mice

Endotoxin or lipopolysaccharide (LPS) tolerance may be partially due to the secretion of potent anti-inflammatory cytokines following severe Gram-negative infections, or by low doses of LPS. In this work, we describe the effects of interleukin-1 (IL-1) and tumour necrosis factor alpha (TNF-), two early cytokines secreted after LPS exposure, in the induction of LPS tolerance. Our results demonstrate that mice treated with three daily doses of 100 ng of IL-1 were tolerant to LPS-induced shock. However, TNF- was unable to induce an LPS refractory state. Given the fact that 100 ng of IL-1 increase the plasma levels of glucocorticoids, we evaluated whether a daily injection of dexamethasone (DEX) alone was able to reproduce the LPS-like tolerant state. However, no signs of LPS refractoriness were detected, except when DEX was administered concomitantly with a dose of IL-1 that does not induce corticosterone secretion (12 ng/mouse). This dose was found to induce in vitro up-regulation of the glucocorticoid receptors (GcR) of peritoneal macrophages following 24 h of treatment. In addition, we demonstrate that IL-1 is capable of inducing the down-regulation of Toll-like receptor 4 (TLR4), a crucial molecule in the signal transduction of LPS. Taken together, our results indicate that IL-1 can generate tolerance to LPS in vivo, and suggest that the regulation of mechanisms of the down-regulation of TLR4, as well as those involved in the expression of GcR and/or in the secretion of glucocorticoids, would be crucial for these effects.     

Pertussis toxin-induced alterations of murine hepatic drug metabolism following administration of diphtheria and tetanus toxoids and pertussis vaccine adsorbed.

Administration of pertussis toxin (PT) in combination with diphtheria and tetanus toxoids adsorbed (DT vaccine) or with acellular pertussis vaccine adsorbed and diphtheria and tetanus toxoids (APDT) elicits dose- and time-dependent alterations in hepatic drug metabolism in mice. Cytochrome P-450 (P-450) levels were inhibited more than 50% at 7 days following a single injection of PT mixed with either vaccine. When combined with DT vaccine, 125 ng of PT was required to produce this effect, while as little as 16 ng of PT combined with APDT vaccine inhibited P-450 levels. The inhibition of P-450 levels is similar to that observed after a single injection of diphtheria and tetanus toxoids and pertussis vaccine adsorbed (DTP). Alterations of P-450 levels were accompanied by increased activities of quinone reductase but not with changes in plasma interleukin-6 or tumor necrosis factor levels. Other Bordetella pertussis virulence factors, such as filamentous hemagglutinin, fimbriae and pertactin, were also tested but had no significant effect on hepatic drug metabolism. Endotoxin or preparations containing endotoxin caused alterations in hepatic drug metabolism within 24 h, concomitant with increased interleukin-6 and tumor necrosis factor levels, but these effects had resolved by 1 week. DTP vaccine and preparations containing PT caused a marked induction of gamma interferon coincident with the maximal inhibition of P-450 levels. This effect was not present with DT or APDT vaccine alone, nor with endotoxin or any combination of factors that did not contain PT. These results demonstrate that PT is a necessary component for the sustained effects of DTP vaccine on hepatic drug metabolism and suggest a role for gamma interferon in this process.     

Lipopolysaccharide and monophosphoryl lipid A differentially regulate interleukin-12, gamma interferon, and interleukin-10 mRNA production in murine macrophages.

Monophosphoryl lipid A (MPL) is a nontoxic derivative of the lipid A region of lipopolysaccharide (LPS) that is being developed as both an adjuvant and prophylactic drug for septic shock. We compared the ability of LPS and MPL to induce interleukin-10 (IL-10), IL-12 p35, IL-12 p40, gamma interferon (IFN-gamma), glucocorticoid receptor (GR), IL-1 receptor antagonist (IL-1ra), and inducible nitric oxide synthase mRNA expression in murine peritoneal macrophages. These genes were chosen for their ability to positively or negatively regulate the host immune response and thus for their potential involvement in MPL-induced adjuvanticity or in its ability to protect against sepsis. LPS was a more potent inducer of IL-12 p35, IL-12 p40, and IFN-gamma mRNA, as well as of IL-12 protein, than MPL. In contrast, MPL induced higher levels of IL-10 mRNA than did LPS from 1 to 1,000 ng/ml. In general, MPL was not a more potent inducer of negative regulatory genes, since MPL and LPS induced similar levels of GR and IL-1ra mRNA. Addition of anti-IL-10 antibody to cultures increased the induction of MPL-induced IL-12 p35, IL-12 p40, and IFN-gamma mRNA, suggesting that the enhanced production of IL-10 by MPL-stimulated macrophages contributes to decreased production of mRNA for IL-12 (p35 and p40) and IFN-gamma. Conversely, the addition of exogenous IL-10 to LPS-treated macrophages reduced the mRNA expression of these cytokine genes. These studies suggest that enhanced production of IL-10 by MPL-stimulated macrophages may contribute to the reduced toxicity of MPL through its negative action on induction of cytokines shown to enhance endotoxicity.     

T‐helper 1 and T‐helper 2 adjuvants induce distinct differences in the magnitude,quality and kinetics of the early inflammatory response at the site of injection

Vaccine adjuvants activate the innate immune system and thus influence subsequent adaptive T‐cell responses. However, little is known about the initial immune mechanisms preceding the adjuvant‐induced differentiation of T‐helper (Th) cells. The effect of a T‐helper 1 (Th1) adjuvant, dimethyldioctadecylammonium liposomes with monophosphoryl lipid‐A (DDA/MPL), and a T‐helper 2 adjuvant, aluminium hydroxide [Al(OH)₃], on early, innate chemotactic signals and inflammatory cell influx at the site of injection was therefore investigated. Injection of the adjuvants into the peritoneal cavity of mice demonstrated distinct differences in the magnitude, quality and kinetics of the response. The inflammatory response to DDA/MPL was prominent, inducing high local levels of pro‐inflammatory cytokines, chemokines and a pronounced inflammatory exudate consisting of neutrophils, monocytes/macrophages and activated natural killer cells. This was in contrast to the response induced by Al(OH)₃, which, although sharing some of the early chemokine signals, was more moderate and consisted almost exclusively of neutrophils and eosinophils. Notably, Al(OH)₃ specifically induced the release of a significant amount of interleukin (IL)‐5, whereas DDA/MPL induced high amounts of tumour necrosis factor‐α (TNF‐α), IL‐1α and IL‐6. Finally, a microarray analysis confirmed that the effect of DDA/MPL was broader with more than five times as many genes being specifically up‐regulated after injection of DDA/MPL compared with Al(OH)₃. Thus, the adjuvants induced qualitatively distinct local inflammatory signals early after injection.     

Prevention of endotoxin-induced acute lethality in Propionibacterium acnes-primed rabbits by an antibody to leukocyte integrin beta 2 with concomitant reduction of cytokine production.

Acute lethality was induced in rabbits by the sequential injection of Propionibacterium acnes and lipopolysaccharide (LPS). P. acnes induced the infiltration of inflammatory cells into the liver lobules during the early phase, and LPS in the late phase caused death in association with pathological changes mimicking hepatocellular necrosis or degeneration around infiltrated mononuclear cells and fibrin deposition in the liver, lung, and kidney, suggestive of a systemic Schwartzman-like reaction. These pathological changes were accompanied by the elevation of plasma tumor necrosis factor (TNF) and interleukin-8 (IL-8) levels. A neutralizing antibody to a leukocyte adhesion molecule, integrin beta 2 (CD18), administered at the time of LPS challenge, prevented reduced the elevation of plasma TNF and IL-8 levels. An anti-TNF alpha antibody but not an anti-IL-8 mediator in this model. These results indicate that CD18 is critically involved in vivo in activating leukocytes to produce cytokines in response to LPS.