Comparative evaluation of mono- and neutrophilokins inducing activity of Yersinia pestis antigens

The results of the comparative analysis of the cytokine inducing activity of Yersinia pestis EV antigens are presented. Y. pestis fraction 1A (F1A) and lipopolysaccharide (LPS) were shown to induce mono- and neutrophilokines, regulating cooperative interaction of phagocytes in the process of immunity formation to plague. Neutrophilokines and monokines exceed in their capacity for inducing F1A such acknowledged inductor as Escherichia coli LPS. As revealed by the comparative evaluation of Y. pestis EV LPS and E. coli LPS, neutrophilokines synthesized under the action of the former preparation, have greater influence on the inhibition of the macrophage migration from the infection focus as well as on digestive activity of these cells (in secondary immune response) and on the labilization of the lysosome membranes of macrophages than neutrophilokines induced by E. coli LPS. At the same time they produce a lesser modulating effect on the killer and chemotactic activity of neutrophils, as well as on the expression of FC receptors (FcR) on their surface in comparison with monokines, synthesized under the influence of E. coli LPS.     

Influence of neutrophilokines on the synthesis of lymphokines in the process of the formation of immunity to plague

The evaluation of the complex of neutrophilokines whose synthesis was induced by Yersinia pestis vaccine strain EV on the production of lymphokines in the process of the formation of primary and secondary immunity to plague is presented. As revealed in this study, neutrophilokines regulate the synthesis of IL-2 by T helpers of type 1, IL-4 and IL-5 by T helpers of type 2, IL-1 by B lymphocytes, as well as the expression of receptors IL-2 by immunocompetent cells. The helper effect of neutrophilokines is more pronounced in the secondary immune response.     

Lipopolysaccharides from distinct pathogens induce different classes of immune responses in vivo.

The adaptive immune system has evolved distinct responses against different pathogens, but the mechanism(s) by which a particular response is initiated is poorly understood. In this study, we investigated the type of Ag-specific CD4(+) Th and CD8(+) T cell responses elicited in vivo, in response to soluble OVA, coinjected with LPS from two different pathogens. We used Escherichia coli LPS, which signals through Toll-like receptor 4 (TLR4) and LPS from the oral pathogen Porphyromonas gingivalis, which does not appear to require TLR4 for signaling. Coinjections of E. coli LPS + OVA or P. gingivalis LPS + OVA induced similar clonal expansions of OVA-specific CD4(+) and CD8(+) T cells, but strikingly different cytokine profiles. E. coli LPS induced a Th1-like response with abundant IFN-gamma, but little or no IL-4, IL-13, and IL-5. In contrast, P. gingivalis LPS induced Th and T cell responses characterized by significant levels of IL-13, IL-5, and IL-10, but lower levels of IFN-gamma. Consistent with these results, E. coli LPS induced IL-12(p70) in the CD8alpha(+) dendritic cell (DC) subset, while P. gingivalis LPS did not. Both LPS, however, activated the two DC subsets to up-regulate costimulatory molecules and produce IL-6 and TNF-alpha. Interestingly, these LPS appeared to have differences in their ability to signal through TLR4; proliferation of splenocytes and cytokine secretion by splenocytes or DCs from TLR4-deficient C3H/HeJ mice were greatly impaired in response to E. coli LPS, but not P. gingivalis LPS. Therefore, LPS from different bacteria activate DC subsets to produce different cytokines, and induce distinct types of adaptive immunity in vivo.     

Toll-IL-1 receptor domain-containing adaptor protein is critical for early lung immune responses against Escherichia coli lipopolysaccharide and viable Escherichia coli

Pulmonary bacterial diseases are a leading cause of mortality in the U.S. Innate immune response is vital for bacterial clearance from the lung, and TLRs play a critical role in this process. Toll-IL-1R domain-containing adaptor protein (TIRAP) is a key molecule in the TLR4 and 2 signaling. Despite its potential importance, the role of TIRAP-mediated signaling in lung responses has not been examined. Our goals were to determine the role of TIRAP-dependent signaling in the induction of lung innate immune responses against Escherichia coli LPS and viable E. coli, and in lung defense against E. coli in mice. LPS-induced neutrophil sequestration; NF-kappaB translocation; keratinocyte cell-derived chemokine, MIP-2, TNF-alpha, and IL-6 expression; histopathology; and VCAM-1 and ICAM-1 expression were abolished in the lungs of TIRAP-/- mice. A cell-permeable TIRAP blocking peptide attenuated LPS-induced lung responses. Furthermore, immune responses in the lungs of TIRAP-/- mice were attenuated against E. coli compared with TIRAP+/+ mice. TIRAP-/- mice also had early mortality, higher bacterial burden in the lungs, and more bacterial dissemination following E. coli inoculation. Moreover, we used human alveolar macrophages to examine the role of TIRAP signaling in the human system. The TIRAP blocking peptide abolished LPS-induced TNF-alpha, IL-6, and IL-8 expression in alveolar macrophages, whereas it attenuated E. coli-induced expression of these cytokines and chemokines. Taken together, this is the first study illustrating the crucial role of TIRAP in the generation of an effective early immune response against E. coli LPS and viable E. coli, and in lung defense against a bacterial pathogen.     

TLR2-mediated survival of Staphylococcus aureus in macrophages: a novel bacterial strategy against host innate immunity

TLR2 plays a role as a pattern-recognition receptor in the innate immune response involving secreted proteins against microbial pathogens. To examine its possible involvement in the cellular response, we determined the levels of the engulfment and subsequent killing of bacteria by macrophages prepared from TLR2-deficient and wild-type mice. The level of the engulfment of Staphylococcus aureus or Escherichia coli was almost the same between TLR2-lacking and wild-type macrophages. However, the colony-forming ability of engulfed S. aureus, but not of E. coli, decreased to a greater extent in TLR2-lacking macrophages than in the wild-type control. The incubation with S. aureus caused activation of JNK in wild-type macrophages but not in TLR2-lacking macrophages, and the pretreatment of wild-type macrophages with a JNK inhibitor increased the rate of killing of engulfed S. aureus, but again not of E. coli. In addition, the number of colonies formed by engulfed S. aureus increased in the JNK-dependent manner when TLR2-lacking macrophages were pretreated with LPS. Furthermore, JNK seemed to inhibit the generation of superoxide, not of NO, in macrophages. These results collectively suggested that the level of superoxide is reduced in macrophages that have engulfed S. aureus through the actions of TLR2-activated JNK, resulting in the prolonged survival of the bacterium in phagosomes. The same regulation did not influence the survival of E. coli, because this bacterium was more resistant to superoxide than S. aureus. We propose a novel bacterial strategy for survival in macrophages involving the hijacking of an innate immune receptor.     

The fate of E. coli lipopolysaccharide after the uptake of E. coli by murine macrophages in vitro

The fate of bacterial lipopolysaccharide (LPS) after the uptake of Escherichia coli by macrophages in vitro was studied. The LPS of the galactose epimerase-deficient E. coli J5 mutant was specifically radiolabeled with [3H]galactose by growing the organism in a basic salts medium containing galactose. Control bacteria were uniformly radiolabeled by growth in [14C]glucose and unlabeled galactose-containing medium. Surface constituents of E. coli were also labeled with 125I. After in vitro phagocytosis of labeled E. coli by murine peritoneal exudate macrophages, the rate of exocytosis of LPS, as assessed by release of 3H over a 72-hr period, was considerably reduced in comparison with other bacterial constituents (14C and 125I release). The [3H]galactose-labeled material exocytosed from macrophages and that remaining intracellularly (obtained from macrophage lysates) were isolated by cesium chloride (CsCl) density gradients and were shown to have altered density profiles as compared with purified E. coli LPS. The macrophage-"processed" [3H] galactose-containing fractions from CsCl density gradients of culture supernatants or macrophage lysates were capable of clotting Limulus amebocyte lysate. The [3H]galactose material obtained from 48-hr macrophage lysates and culture supernatants could also induce a lethal response in actinomycin D-treated mice. These data suggest that bacterial LPS may be selectively retained by the macrophage and that the post-phagocytic events that result in bacterial degradation are not accompanied by the degradation of LPS. Furthermore, although the LPS may be modified by the macrophage, it retains its biologic activity.     

Stress-induced extracellular Hsp72 is a functionally significant danger signal to the immune system

Extracellular heat-shock proteins (eHsp) such as those belonging to the 70-kDa family of Hsp (eg, Hsp72) have been hypothesized to act as a "danger signal" to immune cells, promote immune responses, and improve host defense. The current study tested this hypothesis. Adult male F344 rats were exposed to an acute laboratory stressor (100, 5-second, 1.6-mA inescapable tail shocks) and challenged with Escherichia coli. The number of colony-forming units (CFU) of bacteria at the site of injection, the levels of eHsp72, the immune response to eHsp72 and E. coli-derived lipopolysaccharide (LPS), and the amount of time required to recover from in vivo bacterial challenge were measured. CFUs were reduced 2, 4, and 6 hours after injection of E. coli in rats exposed to stress. Rats exposed to stress had elevated eHsp72 that was elevated rapidly (25 minutes) and remained elevated in the circulation and at the inflammatory site (2 hours after stressor termination). Both stressor exposure and eHsp72 administration in the absence of stress resulted in a facilitated pattern of recovery after bacterial inflammation induced by subcutaneous E. coli injection. Rats exposed to acute restraint (100 minutes) did not demonstrate elevated circulating eHsp72 or a facilitated pattern of recovery after bacterial challenge. In vitro stimulation of rat splenocytes and macrophages with eHsp72 elevated nitric oxide (NO), tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-1beta, and IL-6, and this effect was specific to eHsp72 because it was not diminished by polymyxin B and was reduced by earlier heat-denature treatment. Stimulation of cells with eHsp72 combined with LPS resulted in a greater NO and cytokine response than that observed after stimulation with eHsp72 or LPS alone. In vivo, at the inflammatory site, the bacterial-induced NO response was potentiated by stress, and NO inhibition (L-NIO) reduced the stress-induced facilitation but had no effect on the control kinetics of bacterial inflammation recovery. Thus, these results lend support to the hypothesis that intense stressor exposure increases eHsp72, which acts as a danger signal to potentiate the NO response to bacterial challenge and facilitate recovery from bacterial inflammation.     

Correlation of immunogenicity and production of ornithine by peritoneal macrophages

The release of ornithine by macrophages and its correlation with their immunogenicity after treatment with various macrophage-stimulating substances were analyzed. Pristane-elicited peritoneal macrophages (PM) were found to express strong arginase activity and to release L-ornithine into the extracellular space. This activity is strongly reduced within 3 hr after treatment with tetradecanoylphorbol acetate (TPA) but not with lipopolysaccharide (LPS). Resident PM usually express little arginase activity, but this activity is markedly augmented within 24 or 48 hr after treatment with LPS. The release of ornithine by peritoneal cells (PC) (60 to 90% macrophages) was found to be correlated with their immunogenicity as determined by the in vivo immunization for a subsequent in vitro secondary cytotoxic response against minor H antigens. The immunogenicity of pristane-elicited PC is markedly stronger than that of resident PC or TPA-treated, pristane-elicited PC. Moreover, the immunogenicity of the resident PC and TPA-treated elicited PC is substantially augmented by the simultaneous injection of ornithine, whereas the immunogenicity of the untreated elicited PC is not further augmented by exogenous ornithine, indicating that the endogenous production of ornithine by the stimulating cells had a strong influence on the resulting immune response. Injection of glutathione into pristane-treated mice also reduces the ornithine production and immunogenicity of the resulting peritoneal exudate cells. The immunogenicity in this case is at least partly reconstituted by application of exogenous ornithine. Our experiments revealed no correlation between the production of ornithine and prostaglandin E2. Prostaglandin E2 production of resident and pristane-elicited PC is not markedly different and is in either case strongly augmented by TPA. Elicited or resident PM which have been incubated for several days in culture release practically no ornithine; but ornithine production can be induced again by incubation for 24 hr with LPS and to some extent also with interferon-gamma.     

Possible implication of macrophages in the regulation of cytochrome P450 activities in carp (Cyprinus carpio)

Macrophages play a key role in the regulation of cytochrome P450 activity induced by immunostimulants in mammals. We investigated the effects of immunostimulants (LPS, dextran sulfate and tilorone) on biotransformation and macrophage activities in carp. The major effect of LPS was its capacity to inhibit 3-MC-induced cytochrome P450 activities in the liver and head kidney. Basal phase I activities were reduced by tilorone and dextran sulfate in immune organs. Tilorone and dextran sulfate differently modulated total cytochrome P450 contents and P4501A activities suggesting differential sensitivity for P450 classes. In immune organs, tilorone and dextran sulfate inhibited basal EROD activity. Tilorone inhibited 3-MC-induced EROD activity whereas dextran sulfate enhanced this activity. LPS and dextran sulfate increased ROS production by macrophages and all the immunostimulants induced macrophage activating factor (MAF) production. This study demonstrates for the first time in fish the capacity of CYP-regulated immunostimulants to activate macrophages and provides initial insight into the capacity of macrophages to regulate CYP activity induced by immunostimulants in fish.     

Suppression of Splenic Lymphocyte Proliferation by Eucommia ulmoides and Genipin

We investigated the modulation of innate and adaptive immune cell activation by Eucommia ulmoides Oliver extract (EUE) and its ingredient genipin. As an innate immunity indicator, the phagocytic activity of macrophages was determined by measuring engulfed, fluorescently labeled Escherichia coli. As a surrogate marker for the respective activation of cellular and humoral adaptive immunity, concanavalin A (Con A) and lipopolysaccharide (LPS) induction of primary splenocyte proliferation was assayed in in vitro and ex vivo systems. EUE and genipin suppressed the proliferation of primary splenic lymphocytes induced by Con A or LPS, but not macrophage phagocytosis. Oral administration of EUE and genipin to mice decreased splenic lymphocyte proliferation induced by Con A or LPS. These results revealed that E. ulmoides and genipin suppressed cellular and humoral adaptive immunity, and they suggest that E. ulmoides and genipin are promising candidates for immunosuppressive drugs that target diseases that involve excessive activation of adaptive immunity.     

Characterization of biological activities of Brucella melitensis lipopolysaccharide

Biological activities of lipopolysaccharide (LPS) from Brucella melitensis 16M were characterized in comparison with LPS from Escherichia coli O55. LPS extracted from B. melitensis was smooth type by electrophoretic analysis with silver staining. The endotoxin-specific Limulus activity of B. melitensis LPS was lower than that of E. coli LPS. There was no significant production of tumor necrosis factor-alpha and nitric oxide in RAW 264.7 macrophage cells stimulated with B. melitensis LPS, although E. coli LPS definitely induced their production. On the other hand, B. melitensis LPS exhibited a higher anti-complement activity than E. coli LPS. B. melitensis LPS as well as E. coli LPS exhibited a strong adjuvant action on antibody response to bovine serum. The characteristic biological activities of B. melitensis are discussed.     

A requirement for the p85 PI3K adapter protein BCAP in the protection of macrophages from apoptosis induced by endoplasmic reticulum stress

Macrophages are innate immune cells that play key roles in regulation of the immune response and in tissue injury and repair. In response to specific innate immune stimuli, macrophages may exhibit signs of endoplasmic reticulum (ER) stress and progress to apoptosis. Factors that regulate macrophage survival under these conditions are poorly understood. In this study, we identified B cell adapter protein (BCAP), a p85 PI3K-binding adapter protein, in promoting survival in response to the combined challenge of LPS and ER stress. BCAP was unique among nine PI3K adapter proteins in being induced >10-fold in response to LPS. LPS-stimulated macrophages incubated with thapsigargin, a sarcoplasmic/endoplasmic reticulum calcium ATPase inhibitor that induces ER stress, underwent caspase-3 activation and apoptosis. Macrophages from BCAP(-/-) mice exhibited increased apoptosis in response to these stimuli. BCAP-deficient macrophages demonstrated decreased activation of Akt, but not ERK, and, unlike BCAP-deficient B cells, expressed normal amounts of the NF-κB subunits, c-Rel and RelA. Retroviral transduction of BCAP-deficient macrophages with wild-type BCAP, but not a Y4F BCAP mutant defective in binding the SH2 domain of p85 PI3K, reversed the proapoptotic phenotype observed in BCAP-deficient macrophages. We conclude that BCAP is a nonredundant PI3K adapter protein in macrophages that is required for maximal cell survival in response to ER stress. We suggest that as macrophages engage their pathogenic targets, innate immune receptors trigger increased expression of BCAP, which endows them with the capacity to withstand further challenges from ongoing cellular insults, such as ER stress.     

Uptake of LPS/E. coli/latex beads via distinct signalling pathways in medfly hemocytes: the role of MAP kinases activation and protein secretion

In response to LPS/E. coli treatment, extracellular signal-regulated kinase (ERK) is activated in medfly hemocytes. To explore the molecular mechanisms underlying LPS/E. coli/latex beads endo- and phagocytosis, we studied the signalling pathways leading to p38 and c-jun N-terminal kinase (JNK) activation. JNK and p38-like proteins were initially identified within medfly hemocytes. Flow cytometry analysis revealed that mitogen-activated protein kinases (MAPK) are required for phagocytosis. Inhibition of specific MAPK signalling pathways, with manumycin A, toxin A, cytochalasin D and latrunculin A, revealed activation of p38 via Ras/Rho/actin remodelling pathway and activation of JNK that was independent of actin cytoskeleton reorganization. ERK and p38 pathways, but not JNK, appeared to be involved in LPS-dependent hemocyte secretion, whereas all MAPK subfamilies seemed to participate in E. coli-dependent secretion. In addition, flow cytometry experiments in hemocytes showed that the LPS/E. coli-induced release was a prerequisite for LPS/E. coli uptake, whereas latex bead phagocytosis did not depend on hemocyte secretion. This is a novel aspect, as in mammalian monocytes/macrophages LPS/E. coli-triggered release has not been yet correlated with phagocytosis. It is of interest that these data suggest distinct mechanisms for the phagocytosis of E. coli and latex beads in medfly hemocytes.     

N-acetylcysteine improves in vitro the function of macrophages from mice with endotoxin-induced oxidative stress

Reactive oxygen species (ROS) and proinflammatory cytokines produced by immune cells cause the oxidative stress involved in septic shock induced by endotoxin. This oxidative stress can be controlled to a certain degree by antioxidants, which is specially important for a type of immune cell, i.e. the phagocyte, that uses ROS to kill microorganisms and needs antioxidants in order to support its functions. In a previous study we have observed changes in several functions of peritoneal macrophages from BALB/c mice with lethal endotoxic shock caused by intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS) (100 mg/kg), which were associated with a high production of superoxide anion. N-acetylcysteine (NAC) is a thiolic antioxidant that improves the immune response, and we have observed that when administered intraperitoneally (150 mg/kg) at 30 min after LPS injection it counteracts the effects of LPS on macrophages and lymphocytes. In the present work, we have studied the in vitro effect of several concentrations of NAC (0.001, 0.01, 0.1, 1 and 2.5 mM) on the following functions: adherence to substrate, chemotaxis, ingestion of particles, ROS production and the release of tumor necrosis factor (TNFalpha) of peritoneal macrophages from BALB/c mice at 2, 4,12 and 24 h after LPS injection. The results show that the administration of NAC (especially at 0.1 mM) decreases raised adherence, ingestion, ROS production and TNFalpha levels in macrophages from animals injected with LPS, bringing these functions to values near those of control animals. These effects which seem to be linked to a modulation of NF-kappaB, suggest that the improvement of immune functions observed in previous work after injection of NAC to animals with endotoxic shock could be due to a direct action of this thiol antioxidant on immune cells.     

Differential effect of asparaginase on lymphocyte subpopulations and alleviation of the immunosuppression by lipopolysaccharide

Summary The effect of a range of E. coli L-asparaginase (EC2) concentrations on mouse splenocyte blastogenesis was examined at various concentrations of both T- and B-lymphocyte mitogens. At optimal mitogen concentration and with 0.1 unit EC2/ml inhibition of the Con A (T cell) response was 2.5 times as great as inhibition of the LPS (B cell) response. Another T-cell stimulant, periodate, was inhibited in a manner comparable to Con A while the PHA response was inhibited to an intermediate degree. In vivo the PFC response was suppressed to 19% of normal when 100 units EC2 was given with SRBC. However, when T-cell participation in this response was by-passed by simultaneously giving LPS, the residual activity rose from 19% to 52%, again suggesting that T-lymphocytes were the more severely depressed population. These observations demonstrate that a nonspecific stimulant of the immune system may alleviate the depression of immune responsiveness induced by a chemotherapeutic agent.     

Lipopolysaccharide-dependent prostaglandin E(2) production is regulated by the glutathione-dependent prostaglandin E(2) synthase gene induced by the Toll-like receptor 4/MyD88/NF-IL6 pathway

Macrophages produce a large amount of PGE(2) during inflammation. This lipid mediator modulates various immune responses. PGE(2) acts on macrophages and inhibits production of cytokines such as TNF-alpha and IL-12. Membrane-bound glutathione-dependent PGE(2) synthase (mPGES) has been shown to be a terminal enzyme of the cyclooxygenase-2-mediated PGE(2) biosynthesis. Here we identified mPGES as a molecule that is induced by LPS in macrophages. The expression of mPGES was not induced by LPS in mice lacking Toll-like receptor 4 or MyD88. Furthermore, mice deficient in NF-IL6 showed neither induction of mPGES nor biosynthesis of PGE(2) in response to LPS, indicating that mPGES expression in response to LPS is regulated by a Toll-like receptor 4/MyD88/NF-IL6-dependent signaling pathway. We generated mPGES-deficient mice and investigated the role of mPGES in vivo. The mice showed no augmentation of the PGE(2) production in response to LPS. However, they were not impaired in the LPS-induced production of inflammatory cytokines and showed normal response to the LPS-induced shock. Thus, mPGES is critically involved in the biosynthesis of PGE(2) induced by LPS, but is dispensable for the modulation of inflammatory responses.     

Cross-tolerization between Nod1 and Nod2 signaling results in reduced refractoriness to bacterial infection in Nod2-deficient macrophages

Nod2 is an intracellular innate immune receptor that plays a role in host defense and susceptibility to inflammatory disease. We show in this study that macrophages rendered refractory to TLR4 and Nod2 signaling by exposure to LPS and muramyl dipeptide (MDP) exhibit impaired TNF-alpha and IL-6 production in response to pathogenic Listeria monocytogenes and Yersinia pseudotuberculosis as well as commensal bacteria including Escherichia coli and Bacteroides fragilis. Surprisingly, Nod2 deficiency was associated with impaired tolerization in response to pathogenic and commensal bacteria. Mechanistically, reduced tolerization of Nod2-null macrophages was mediated by recognition of bacteria through Nod1 because it was abolished in macrophages deficient in Nod1 and Nod2. Consistently, Nod2-null macrophages tolerant to LPS and MDP showed enhanced production of TNF-alpha and IL-6 as well as increased NF-kappaB and MAPK activation in response to the dipeptide KF1B, the Nod1 agonist. Furthermore, reduced tolerization of Nod2-deficient macrophages in response to bacteria was abolished when mutant macrophages were also rendered tolerant to the Nod1 ligand. Finally, MDP stimulation induced refractoriness not only to MDP, but also to iE-DAP stimulation, providing a mechanism to explain the reduced tolerization of Nod2-deficient macrophages infected with bacteria. These results demonstrate that cross-tolerization between Nod1 and Nod2 leads to increase recognition of both pathogenic and commensal bacteria in Nod2-deficient macrophages pre-exposed to microbial ligands.     

Depression of afferent arc of the in vivo cytotoxic T-cell immunity by bacterial lipopolysaccharides

The afferent arc of the in vivo cytotoxic T-cell immunity assessed by second set rejection of ascitic allogeneic tumors was shown to be depressed by bacterial lipopolysaccharide (LPS) that was administered simultaneously with or 1 day before injection of allogeneic spleen cells as stimulators. Two different LPSs from Escherichia coli O55 and Klebsiella O3 displayed similar activities whereas dextran sulfate, concanavalin A, or poly A:U was not effective. Stimulator activities of allogeneic cells was not directly modified by LPS. Any definite suppressor activity on afferent or efferent arc of the T-cell response was not demonstrable in mice receiving LPS and allogeneic cells. Further, the LPS effect for immune depression was not diminished by whole body X-ray irradiation to the recipient at 300 R, which ablated the B-cell reactivity to LPS for polyclonal activation, or by treatment of the recipient with carrageenan, a known toxic agent to macrophages. It was suggested from these results that LPS suppresses the cytotoxic T-cell immunity by modulating responder T cells to be temporarily refractory to the allogeneic stimulus rather than by activating suppressor cells such as radiation-sensitive lymphocytes and carrageenan-sensitive macrophages.