Suppression of macrophage responses to bacterial lipopolysaccharide by a non-secretory form of secretory leukocyte protease inhibitor.

Expression of secretory leukocyte protease inhibitor (SLPI) suppresses the ability of macrophages to respond to bacterial lipopolysaccharide (LPS). Here, addition of recombinant or native SLPI to the extracellular medium was non-suppressive, while transfection with a non-secretory form of SLPI was fully suppressive, an effect overcome by treatment with interferon-gamma. A portion of the SLPI produced by untransfected macrophages was localized in the cytosol. Thus, SLPI can act intracellularly to block macrophage activation by LPS.     

Effects of macrophage colony-stimulating factor on microglial responses to lipopolysaccharide and beta amyloid

A challenge for studies involving microglia cultures is obtaining sufficient cells for downstream experiments. Macrophage colony-stimulating factor (M-CSF) has been used to improve yield of microglia in culture. However, the effects of M-CSF on activation profiles of microglia cultures are still unclear. Microglia activation is characterised by upregulation of co-stimulatory molecules and an inflammatory phenotype. The aim of this study is to demonstrate whether M-CSF supplementation alters microglial responses in resting and activated conditions. Microglia derived from mixed glia cultures and the BV-2 microglia cell line were cultivated with/without M-CSF and activated with lipopolysaccharide (LPS) and beta amyloid (Aβ). We show M-CSF expands primary microglia without affecting microglial responses to LPS and Aβ, as shown by the comparable expression of MHC class II and CD40 to microglia grown without this growth factor. M-CSF supplementation in BV-2 cells had no effect on nitric oxide (NO) production. Therefore, M-CSF can be considered for improving microglia yield in culture without introducing activation artefacts.     

Suppressor of cytokine signaling-1 has IFN-gamma-independent actions in T cell homeostasis

Suppressor of cytokine signaling (SOCS)-1 is a member of a family of proteins that negatively regulate cytokine signaling pathways. We have previously established that SOCS-1 is a key regulator of IFN-gamma signaling and that IFN-gamma is responsible for the complex inflammatory disease that leads to the death of SOCS-1-deficient mice. In this study, we provide evidence that SOCS-1 is also a critical regulator of IFN-gamma-independent immunoregulatory factors. Mice lacking both SOCS-1 and IFN-gamma, although outwardly healthy, have clear abnormalities in their immune system, including a reduced ratio of CD4:CD8 T cells in lymphoid tissues and increased expression of T cell activation markers. To examine the contribution of TCR Ag specificity to these immune defects, we have generated two lines of SOCS-1-deficient mice expressing a transgenic TCR specific for an exogenous Ag, OVA (OT-I and OT-II). Although TCR transgenic SOCS-1(-/-) mice have a longer lifespan than nontransgenic SOCS-1(-/-) mice, they still die as young adults with inflammatory disease and the TCR transgenic SOCS-1(-/-) T cells appear activated despite the absence of OVA. This suggests that both Ag-dependent and -independent mechanisms contribute to the disease in SOCS-1-deficient mice. Thus, SOCS-1 is a critical regulator of T cell activation and homeostasis, and its influence extends beyond regulating IFN-gamma signaling.     

Innate IFNs and plasmacytoid dendritic cells constrain Th2 cytokine responses to rhinovirus: a regulatory mechanism with relevance to asthma

Human rhinoviruses (RV) cause only minor illness in healthy individuals, but can have deleterious consequences in people with asthma. This study sought to examine normal homeostatic mechanisms regulating adaptive immunity to RV in healthy humans, focusing on effects of IFN-αβ and plasmacytoid dendritic cells (pDC) on Th2 immune responses. PBMC were isolated from 27 healthy individuals and cultured with RV16 for up to 5 d. In some experiments, IFN-αβ was neutralized using a decoy receptor that blocks IFN signaling, whereas specific dendritic cell subsets were depleted from cultures with immune-magnetic beads. RV16 induced robust expression of IFN-α, IFN-β, multiple IFN-stimulated genes, and T cell-polarizing factors within the first 24 h. At 5 d, the production of memory T cell-derived IFN-γ, IL-10, and IL-13, but not IL-17A, was significantly elevated. Neutralizing the effects of type-I IFN with the decoy receptor B18R led to a significant increase in IL-13 synthesis, but had no effect on IFN-γ synthesis. Depletion of pDC from RV-stimulated cultures markedly inhibited IFN-α secretion, and led to a significant increase in expression and production of the Th2 cytokines IL-5 (p = 0.02), IL-9 (p < 0.01), and IL-13 (p < 0.01), but had no effect on IFN-γ synthesis. Depletion of CD1c(+) dendritic cells did not alter cytokine synthesis. In healthy humans, pDC and the IFN-αβ they secrete selectively constrain Th2 cytokine synthesis following RV exposure in vitro. This important regulatory mechanism may be lost in asthma; deficient IFN-αβ synthesis and/or pDC dysfunction have the potential to contribute to asthma exacerbations during RV infections.     

IFN-gamma enhances bovine macrophage responsiveness to Mycobacterium bovis: Impact on bacterial replication, cytokine release and macrophage apoptosis

We sought to determine the impact of bovine IFN-gamma on the interaction between Mycobacterium bovis and bovine macrophages. Bovine macrophages released small amounts of nitric oxide (NO), TNF-alpha, IL-1beta and IL-12 upon infection with bacille Calmette-Guérin (BCG). Prior pulsing of cells with IFN-gamma significantly enhanced the release of NO and IL-12. Infection of bovine macrophages with virulent M. bovis led to the release of higher levels of pro-inflammatory mediators, compared to levels released upon BCG infection. IFN-gamma treatment of macrophages enhanced the release of pro-inflammatory mediators, but did not modify bacterial replication in M. bovis-infected macrophages. Treatment of macrophages with a combination of IFN-gamma and LPS led to a reduction in bacterial replication. Infected cells treated with IFN-gamma/LPS progressed mostly through an apoptotic pathway, whereas untreated infected cells eventually died by necrosis. Agents that prevented the acquisition of bacteriostatic activity by activated macrophages also prevented the induction of apoptosis in infected macrophages (IL-10 and neutralizing anti-TNF-alpha). We conclude that virulent M. bovis is a major determinant of release of pro-inflammatory cytokines by macrophages. IFN-gamma amplifies the macrophage cytokine release in response to M. bovis. Induction of apoptosis is closely linked to the emergence of macrophage resistance to M. bovis replication, which is dependent on endogenous TNF-alpha release.     

Suppression of macrophage responses to bacterial lipopolysaccharide (LPS) by secretory leukocyte protease inhibitor (SLPI) is independent of its anti-protease function

Secretory leukocyte protease inhibitor (SLPI), a potent serine protease inhibitor, has been shown to suppress macrophage responses to bacterial lipopolysaccharide (LPS). SLPI contains two topologically superimposable domains. Its C-terminal domain binds and inhibits target proteases. It is not clear whether SLPI's anti-protease function plays a role in the LPS-inhibitory action of SLPI. Four single amino acid substitution mutants of SLPI, M73G, M73F, M73E and M73K, were generated. Wild type SLPI is a potent inhibitor of chymotrypsin and elastase. Mutants M73G and M73F selectively lost inhibitory function towards chymotrypsin and elastase, respectively, whereas mutants M73K and M73E inhibited neither elastase nor chymotrypsin. Macrophage cell lines were established from RAW264.7 cells to stably express each SLPI mutant. Expression of the SLPI protease inhibition mutants suppressed NO and TNF production in response to LPS in a similar fashion as wild type SLPI. Expression of truncated forms of SLPI, containing only its N-terminus or its C-terminus, was similarly sufficient to confer inhibition of LPS responses. Thus, the LPS-inhibitory action of SLPI is independent of its anti-protease function.     

Current understanding of macrophage type 1 cytokine responses during intracellular infections

Macrophages are important effector cells in cell-mediated immunity against intracellular infection. Among cytokines that macrophages are able to release are IL-12 and TNF alpha. IL-12 is a critical linker between the innate and adaptive cell-mediated immunity, capable of Th1 differentiation and IFN gamma release by T and NK cells. IFN gamma is critically required for the activation of macrophage bactericidal activities. Recently emerging evidence suggests that macrophages are able to release not only IL-12 and TNF alpha but also IFN gamma. However, the mechanisms that control the release of each of these type 1 cytokines in macrophages appear different. While macrophages release TNF alpha in an indiscriminate and IL-12-independent way, the release of IL-12, particularly bioactive IL-12 p70, and IFN gamma is under tight control. We are just beginning to understand what controls the release of IL-12 p70, a question of fundamental importance to understanding the mechanisms underlying the initiation of cell-mediated immunity. Our recent findings have shed more insights into the regulatory mechanisms of macrophage IFN gamma responses. It has become evident that IL-12 is required not only for Th1 differentiation but also for IFN gamma responses by both T cells and macrophages during intracellular infection. In this overview, we have discussed about the current understanding of the regulation of macrophage type 1 cytokine responses during intracellular infection, based upon the recent findings from us and others.     

Studies on pyrogenic tolerance to bacterial lipopolysaccharide in rabbits.

The development of pyrogenic tolerance was studied in rabbits treated with varying doses of E. coli lipopolysaccharide (LPS). The following results were obtained. 1) Development of pyrogenic tolerance seemed to proceed in two steps: that is, the first in which tolerance appeared rapidly and the second in which tolerance proceeded more gradually or steadily in response to repeated injections of a constant dose. 2) Tolerance induced by the latter method was not absolute; the rabbits were still sensitive to increased doses of LPS. 3) Rabbits immunized with E. coli vaccine lost their pyrogenic sensitivity of parent LPS to some extent. 4) Following intracisternal injection of LPS into tolerant rabbit, pyrogenic response was not decreased but rather enhanced in comparison with control. 5) The contents of nor-epinephrine and serotonin in the brain did not differ between normal and tolerant rabbits. 6) The mechanisms of endotoxin tolerance remain to be further studied.     

Antibody responses of mice to alkaline detoxifield lipopolysaccharide.

The antibody responses of outbred normal mice and nude mice injected with alkaline detoxified lipopolysaccharide (Alk-LPS) were measured. In some cases the antibody against lipopolysaccharide (LPS) and native protoplasmic polysaccharide (NPP). The kinetics of the primary responses to Alk-LPS and NPP were similar, whereas LPS stimulated a more rapid appearance of antibodies in the primary responses. Alk-LPS stimulated only primary antibody responses in both types of mice and sensitized nude mice for secondary responses which could be triggered with LPS. However, secondary antibody responses could not be triggered in normal mice primed with Alk-LPS. These data suggested that, on a functional basis, Alk-LPS possessed the specific antigenic signal associated with LPS antigens but lack the second nonspecific mitogenic signal dependent on the lipid A portion of LPS.     

Physiologic and innate immunity responses to bacterial lipopolysaccharide administration in beef heifers supplemented with OmniGen-AF

Nutritional alternatives to strengthen animal immunocompetence are critical for welfare and productivity in livestock systems, such as beef cattle operations. This experiment evaluated physiological and innate immunity effects of supplementing an immunomodulatory feed ingredient (Omnigen-AF; Phibro Animal Health, Teaneck, NJ, USA) to beef heifers administered bacterial lipopolysaccharide (LPS). In total, 8 non-pregnant, non-lactating nulliparous Angus×Hereford heifers (676±4 days of age) were ranked by BW (473±8 kg), and assigned to crossover design containing two periods of 34 days each. Heifers were housed in individual pens and had ad libitum access to meadow foxtail (Alopecurus pratensis L.) hay, water and a granulated commercial vitamin+mineral mix. Within each period, heifers received (as-fed basis) 227 g/day of dried distillers grains including (OMN) or not (CON) 56 g of Omnigen-AF for 34 days. On day 28 of each period (0800 h), heifers received an intravenous bolus dose (0.5 μg/kg of BW, diluted in 5 ml of 0.9% sterile saline) of bacterial LPS (Escherichia coli 0111:B4). Hay DM intake was recorded daily from day 0 to 34. Blood was collected at −1, 0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 24, 48, 72, 96, 120 and 144 h relative to LPS administration. Heifer intravaginal temperature was recorded every 10 min from −0.5 to 10 h relative to LPS administration. No treatment effect was detected (P=0.35) for hay DM intake during the experiment. No treatment effects were detected (P⩾0.64) for intravaginal temperature and plasma concentrations of tumor necrosis-α, cortisol and haptoglobin, which increased (time effect, P<0.01) for OMN and CON heifers and peaked at 4.5, 2, 4 and 48 h, respectively, after LPS administration. No treatment effects were detected (P⩾0.35) for whole blood mRNA expression of chemokine ligand 5, tumor necrosis-α, cyclooxygenase 2 and interleukin 8, which also increased (time effect, P<0.01) for OMN and CON heifers and peaked at 0.5, 1.5, 2 and 2.5 h, respectively, after LPS administration. Whole blood mRNA expression of interleukin 8 receptor and L-selectin were also similar (P⩾0.61) between OMN and CON heifers, and decreased (time effect, P<0.01) for both treatments reaching nadir levels at 1 and 2.5 h, respectively, after LPS administration. Collectively, OMN supplementation did not modulate the physiological and innate immunity responses of beef heifers to bacterial LPS administration.     

Effects of bacterial lipopolysaccharide on protein synthesis in murine peritoneal macrophages: relationship to activation for macrophage tumoricidal function

Early biochemical events in the response of murine peritoneal macrophages to bacterial lipopolysaccharide (LPS) have been examined (i.e., 0-4 hr after initiation of treatment). At concentrations of 10 ng/ml or less, LPS stimulated the new or enhanced synthesis of a series of at least six polypeptides of 85, 80, 75, 65, 57, and 38 kD. This effect was dependent upon the lipid A moiety of LPS as lipid A itself could induce the changes and the effect of LPS could be blocked by inclusion of polymixin B sulfate in the culture medium. The effect was specific for LPS in that other endotoxin-free agents known to alter macrophage physiology could not produce the same changes. The time course of LPS stimulation of macrophage protein synthesis was remarkable in that the synthesis of all six proteins was transient even in the continued presence of LPS, being first detected approximately 1 hr after exposure and no longer apparent by 8-10 hr after treatment was initiated. Furthermore, both pulse-chase and cumulative radiolabeling studies indicated that at least two of the proteins (85 and 38 kD) were short-lived and did not accumulate in LPS-treated cells, suggesting the possibility that they participate in a regulatory rather than a functional role. Macrophage tumoricidal activation involves cooperation in response to two independent signals; interferon gamma and LPS. Pretreatment of macrophages with interferon gamma increased the sensitivity of macrophages to LPS-stimulated protein synthesis by one to two orders of magnitude documenting such cooperativity in molecular terms. The LPS-induced stimulation of specific protein synthesis could be reproduced by treatment of macrophages with heat killed Listeria monocytogenes, a gram-positive, endotoxin-negative bacterial stain which has been shown to substitute effectively for LPS in macrophage tumoricidal activation. Furthermore, reversible inhibition (i.e., treatment with cycloheximide) of protein synthesis during LPS treatment abrogated the acquisition of tumoricidal function. These results identify an early biochemical response to LPS which may be a necessary component of the intracellular transduction of signals which regulate macrophage functional development.     

Varying susceptibility of pulmonary cytokine production to lipopolysaccharide in mice

Objectives: The lipopolysaccharide (LPS)-induced acute lung injury (ALI) model has been widely applied for pathophysiological and pharmacological research. The aim of present study is to understand the variation of acute pulmonary inflammation between mouse strains. Methods: The present study investigated the susceptibility of acute production of inflammatory mediators, e.g. cytokines, chemokines and others, to LPS in C57BL/6J, Balb/cJ, DBA/1J, CD-1, NMRI, DBA/2J, A/J, and C3H/HeN mice. Results: The susceptibility to intra-tracheal challenge with LPS varied between measured variables, durations and strains. General lung hyper-reactive susceptibility to LPS-induced pulmonary production of 6–8 inflammatory mediators followed the order NMRI, Balb/cJ, C3H/HeN, A/J, C57BL/6J, DBA/1J, DBA/2J and CD-1 mice at 4 h, and A/J, C3H/HeN, CD-1, NMRI, C57BL/6J, Balb/cJ, DBA/2J and DBA/1J mice at 24 h. Conclusions: Our data provide information for scientists to consider the proper strain of mice for the measurement of specific inflammatory mediators and to select sensitive or resistant mouse strains for understanding genetic variation in the pathogenesis and for the screening of target-oriented drug development.     

Redox-active metals in commercial preparations of lipopolysaccharide: implications for studies of cellular responses to bacterial products

The mechanisms by which lipopolysaccharide (LPS) activates cells have been the subject of intense investigation for many years. Whereas much information on this process has been collected for mammalian species, little is known about the signalling path-ways operative in other animals. One general mode of cellular activation that has been recently pro-posed for pathways independent of the primary mammalian LPS receptor, CD14, involves reactive oxygen species (ROS) as intermediates in LPS-induced signalling pathways. Therefore, we used 2',7'-dichlorodihydrofluorescein, a fluorogenic probe of redox activity, to examine LPS-induced oxidative responses of a macrophage-like cell line from the rainbow trout, RTS11. Lipopolysaccharide dose-dependently increased oxidation of this probe by RTS11 cells, and a variety of other cell lines. This process was inhibited by catalase, superoxide dismutase and NG-methylarginine citrate, an inhibitor of nitric oxide synthases, suggesting the involvement of a diverse assortment of cellular ROS. More careful dissection of this phenomenon led us to conclude that the increase in oxidation was, in fact, due almost entirely to metals, particularly copper, in some LPS preparations, which is something to consider when experimenting with LPS.     

Molecular mechanism of lipopolysaccharide (LPS) in promoting biomineralization on bacterial surface

Biomineralization on bacterial surface is affected by biomolecules of bacterial cell surface. Lipopolysaccharide (LPS) is the main and outermost component on the extracellular membrane of Gram-negative bacteria. In the present study, the molecular mechanism of LPS in affecting biomineralization of Ag⁺/Cl^? colloids was investigated by taking advantages of two LPS structural deficient mutants of Escherichia coli. The two mutants were generated by impairing the expression of waaP or wbbH genes with CRISPR/Cas9 technology and it induced deficient polysaccharide chain of O-antigen (ΔwbbH) or phosphate groups of core oligosaccharide (ΔwaaP) in LPS structures. There were significant changes of the cell morphology and surface charge of the two mutants in comparing with that of wild type cells. LPS from ΔwaaP mutant showed increased ΔH_ITC upon interacting with free Ag⁺ ions than LPS from wild type cells or ΔwbbH mutant, implying the binding affinity of LPS to Ag⁺ ions is affected by the phosphate groups in core oligosaccharide. LPS from ΔwbbH mutant showed decreased endotherm (ΔQ) upon interacting with Ag⁺/Cl^? colloids than LPS from wild type or ΔwaaP mutant cells, implying LPS polysaccharide chain structure is critical for stabilizing Ag⁺/Cl^? colloids. Biomineralization of Ag⁺/Cl^? colloids on ΔwbbH mutant cell surface showed distinctive morphology in comparison with that of wild type or ΔwaaP mutant cells, which confirmed the critical role of O-antigen of LPS in biomineralization. The present work provided molecular evidence of the relationship between LPS structure, ions, and ionic colloids in biomineralization on bacterial cell surface.