LPS-responsive Cells Research Articles

Pentraxin 3 Regulates miR-21 Expression and Secretion in Brown Adipocytes During Lipopolysaccharide-Induced Inflammation.

This study investigates the role of pentraxin 3 (PTX3) in the regulation of inflammatory homeostasis involving anti-inflammatory miR-21 during lipopolysaccharide (LPS) stimulation in adipocytes. Using PTX3 knockout (PTX3 KO) mouse and primary stromal vascular cell models, this study determined the effect of PTX3 deficiency on the expression and secretion of miR-21 in brown adipose tissue (BAT) and brown adipocytes as well as the rescue effect of recombinant PTX3 on miR-21 during LPS-induced inflammation. Among three fat depots, BAT was the major tissue and fully differentiated brown adipocytes were the major cells that expressed miRNA-processing enzymes and produced miRNA. Moreover, brown adipocytes, but not stromal vascular cells, were the LPS-responsive cells in miR-21 production. PTX3 deficiency attenuated LPS-stimulated upregulation of miR-21 in sera and BAT. In wild-type brown adipocytes, LPS stimulation significantly upregulated cellular miR-21. Interestingly, this stimulatory effect of LPS was attenuated, and cellular and secreted miR-21 levels were reduced in PTX3 KO cells upon LPS stimulation. Treatment of recombinant PTX3 reversed the cellular and secreted levels of miR-21 and attenuated an LPS-stimulated increase in the expression of Tnf-α and Mcp1 genes in PTX3 KO adipocytes. PTX3 plays an anti-inflammatory role, in part through regulating miR-21 expression and secretion.

Impaired Inflammatory Responses in Murine Lrrk2-Knockdown Brain Microglia

LRRK2, a Parkinson's disease associated gene, is highly expressed in microglia in addition to neurons; however, its function in microglia has not been evaluated. Using Lrrk2 knockdown (Lrrk2-KD) murine microglia prepared by lentiviral-mediated transfer of Lrrk2-specific small inhibitory hairpin RNA (shRNA), we found that Lrrk2 deficiency attenuated lipopolysaccharide (LPS)-induced mRNA and/or protein expression of inducible nitric oxide synthase, TNF-α, IL-1β and IL-6. LPS-induced phosphorylation of p38 mitogen-activated protein kinase and stimulation of NF-κB-responsive luciferase reporter activity was also decreased in Lrrk2-KD cells. Interestingly, the decrease in NF-κB transcriptional activity measured by luciferase assays appeared to reflect increased binding of the inhibitory NF-κB homodimer, p50/p50, to DNA. In LPS-responsive HEK293T cells, overexpression of the human LRRK2 pathologic, kinase-active mutant G2019S increased basal and LPS-induced levels of phosphorylated p38 and JNK, whereas wild-type and other pathologic (R1441C and G2385R) or artificial kinase-dead (D1994A) LRRK2 mutants either enhanced or did not change basal and LPS-induced p38 and JNK phosphorylation levels. However, wild-type LRRK2 and all LRRK2 mutant variants equally enhanced NF-κB transcriptional activity. Taken together, these results suggest that LRRK2 is a positive regulator of inflammation in murine microglia, and LRRK2 mutations may alter the microenvironment of the brain to favor neuroinflammation.

Identification of lipopolysaccharide‐binding peptide regions within HMGB1 and their effects on subclinical endotoxemia in a mouse model

Lipopolysaccharide (LPS) triggers deleterious systemic inflammatory responses when released into the circulation. LPS-binding protein (LBP) in the serum plays an important role in modifying LPS toxicity by facilitating its interaction with LPS signaling receptors, which are expressed on the surface of LPS-responsive cells. We have previously demonstrated that high mobility group box 1 (HMGB1) can bind to and transfer LPS, consequently increasing LPS-induced TNF-α production in human peripheral blood mononuclear cells (PBMCs). We report here on the identification of two LPS-binding domains within HMGB1. Furthermore, using 12 synthetic HMGB1 peptides, we define the LPS-binding regions within each domain. Among them, synthetic peptides HPep1 and HPep6, which are located in the A and B box domains of HMGB1, bind to the polysaccharide and lipid A moieties of LPS respectively. Both HPep1 and HPep6 peptides inhibited binding of LPS to LBP and HMGB1, LBP-mediated LPS transfer to CD14, and cellular uptake of LPS in RAW264.7 cells. These peptides also inhibited LPS-induced TNF-α release in human PBMCs and induced lower levels of TNF-α in the serum in a subclinical endotoxemia mouse model. These results indicate that HMGB1 has two LPS-binding peptide regions that can be utilized to design anti-sepsis or LPS-neutralizing therapeutics.

Identification of lipopolysaccharide binding site on High Mobility Group Box 1 Protein (98.23)

Abstract Lipopolysaccharide (LPS), a bacterial endotoxin, triggers deleterious systemic inflammatory responses when released into blood circulation, causing severe sepsis, organ dysfunction and death. LPS binding components in serum play an important role in modifying LPS toxicity by facilitating its interaction with the LPS signaling receptor, which is expressed on LPS-responsive cells. We have previously shown that HMGB1 is able to bind LPS and this binding can increase LPS-induced TNF-α production in human peripheral blood monocytes. In this report, we identified several LPS binding regions within human HMGB1 sequence by pull-down experiments with synthetic biotinylated peptide derived from HMGB1. Using a conventional sequence-based comparison with several other LPS-binding proteins, we identified similar LPS-binding motifs in LPS-binding peptides from HMGB1, but inhibition assay demonstrated that the capacity of these peptides to interfere with LPS-HMGB1 interaction exhibits different characteristics We are now carrying out further experiments to evaluate which amino acid residues contribute to LPS binding and functional differences between different peptide in LPS-binding capacities. Our studies identified a defined region of HMGB1 involved in LPS binding, thereby implicating a physiological role of human HMGB1 as an LPS-binding protein. [This study was supported by a grant from the Korean Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (A080694) and Brain Korea 21 project to Medical Science.]

Effects of LPS-mediated bystander activation in the innate immune system

LPS induces dendritic cell (DC) activation, but the precise in vivo mechanism is unclear since DCs express low levels of TLR4. Here, it is shown that DCs can be activated in response to LPS through a bystander mechanism. This result was obtained using chimeric mice reconstituted with LPS-responsive and nonresponsive bone marrow cells. Thus, after indirect in vivo conditioning by LPS, bystander-activated DCs (LPS nonresponsive) up-regulated CD86. This up-regulation occurred even when LPS-responsive cells were MyD88 deficient. Functional analysis demonstrated that in vivo LPS conditioning endowed both the LPS-responsive and bystander cells with the ability to produce IFN-gamma in response to TLR9 stimulation in vitro. IFN-gamma production was also shown to be important for enhanced T-bet gene expression but not important for up-regulation of CD86. To investigate aspects of the mechanism, we used intracellular cytokine staining and found that NKDCs were responsible for at least some of the IFN-gamma production. Thus, our in vivo results demonstrated that bacterial LPS can bridge activation of various cellular populations of the innate immune system through a bystander mechanism.

Lipopolysaccharide Injection Induces Relapses of Experimental Autoimmune Encephalomyelitis in Nontransgenic Mice via Bystander Activation of Autoreactive CD4+ Cells

Infections sometimes associate with exacerbations of autoimmune diseases through pathways that are poorly understood. Ag-specific mechanisms such as cross-reactivity between a microbial Ag and a self-Ag have received no direct support. In this study, we show that injection of LPS induces experimental autoimmune encephalomyelitis in TCR-transgenic mice and relapse of encephalomyelitis in normal mice. This form of treatment induces proliferation and cytokine production in a fraction of effector/memory Th lymphocytes in vitro via physical contact of Th cells with CD4(-) LPS-responsive cells. TCR-mediated signals are not necessary; rather what is required is ligation of costimulatory receptors on Th cells by costimulatory molecules on the CD4(-) cells. This form of bystander activation provides an Ag-independent link between infection and autoimmunity that might fit the clinical and epidemiological data on the connection between infection and autoimmunity better than the Ag-specific models.

Endotoxin Tolerance Disrupts Chromatin Remodeling and NF-κB Transactivation at the IL-1β Promoter

The NF-kappaB family plays a crucial role in the pathogenesis of highly lethal septicemia by modulating transcription of many innate and adaptive immunity genes. Two phases of NF-kappaB activation occur: cytosolic activation and nuclear transactivation. Septicemia with multiorgan failure is associated with chronic activation of cytosolic NF-kappaB with translocation and accumulation of increased levels of nuclear p65 in blood leukocytes. Paradoxically, NF-kappaB-dependent transcription of many proinflammatory genes responding to bacterial LPS endotoxin (LPS) is persistently repressed during septicemia; this phenomenon of LPS tolerance is associated with immunosuppression and poor prognosis. This report suggests an explanation for this paradox. Using an in vitro human leukocyte model and chromatin immunoprecipitation assays, we find that both the cytosolic activation and nuclear transactivation phases of NF-kappaB occur in LPS responsive THP-1 promonocytes with recruitment and binding of NF-kappaB p65 at the IL-1beta promoter. However, transcriptionally repressed LPS-tolerant THP-1 cells do not bind NF-kappaB p65 at the IL-1beta promoter, despite cytosolic activation and accumulation of p65 in the nucleus. In contrast, NF-kappaB p50, which also accumulates in the nucleus, constitutively binds to the IL-1beta promoter NF-kappaB site in both LPS-responsive and LPS-tolerant cells. The level of p65 binding correlates with a binary shift in nucleosome remodeling between histone H3 phosphorylation at serine 10 and methylation of histone H3 at lysine 9. We conclude that LPS tolerance disrupts the transactivating stage of NF-kappaB p65 and altered nucleosome remodeling at the IL-1beta promoter in human leukocytes.

Accelerated internalization and detoxification of endotoxin by anti-lipopolysaccharide antibody is an Fc receptor–mediated process

Background. Interaction between lipopolysaccharide (LPS), LPS-binding protein, and the CD14 receptor at the surface of LPS-responsive cells results in inflammatory cytokine release and internalization and detoxification of LPS. Monoclonal antibodies (mAbs) raised against the deep-core lipid A or the O-linked polysaccharide moieties of LPS accelerate internalization and detoxification of LPS without stimulating cytokine release. This study was conducted to test the hypothesis that the antibody-mediated internalization of LPS is an Fc receptor (FcR)–mediated process. Methods. Fluoroisothiocyanate (FITC)–conjugated Escherichia coli O111:B4 LPS was incubated with RAW 264.7 cells and allowed to internalize for 2 hours in the presence and absence of anti-LPS, anti-CD14, and isotype control mAbs, and Fab fragments from the anti-CD14, anti–Fc receptor, and control mAbs. Tumor necrosis factor–α (TNF-α) release was measured by WEHI 164 cell bioassay. FITC-LPS uptake was measured by flow cytometry. Statistical analysis was by analysis of variance and Fisher exact test. Results. Addition of anti-LPS antibodies resulted in a 30- to 40-fold acceleration of LPS internalization (P <.01) in agreement with previous studies. This increase was blunted by anti-CD14 and also by isotype control holo-antibody (P <.01), but not by Fab fragments from anti-CD14 or isotype control antibody. Both anti-FcR antibodies and Fab fragments blocked anti-LPS antibody–stimulated uptake of FITC-LPS. Both intact anti-CD14 holo-antibody and Fab fragments blocked TNF-α release (P <.01). Conclusions. Clearance and detoxification of LPS are thought to be essential to the host response to endotoxin. It has been shown that antibodies to LPS accelerate its internalization by monocytic cell lines without increasing the elaboration of cytokines. We found that specific blockade of CD14 by Fab fragments could block TNF-α release but not alter the accelerated internalization of LPS produced by anti-LPS antibodies. In contrast, a nonspecific blockade of internalization was produced by competing antibody, which suggests a mechanistic role for the FcR. Specific blockade of FcR by either holo-antibody or Fab fragments blocked accelerated internalization, which confirms a FcR mechanism. We conclude that the accelerated internalization of LPS produced by anti-LPS antibody is an Fc receptor–mediated process. These results have significance for the development of adjuvant immunotherapy for gram-negative bacterial sepsis. (Surgery 2001;130:192-7.)

Cellular Events Mediated by Lipopolysaccharide-stimulated Toll-like Receptor 4: MD-2 IS REQUIRED FOR ACTIVATION OF MITOGEN-ACTIVATED PROTEIN KINASES AND Elk-1

Lipopolysaccharide (LPS) stimulates multiple signaling events, including nuclear factor-kappaB (NF-kappaB) activity and the mitogen-activated protein (MAP) kinases, ERK, JNK, and p38 in LPS-responsive cells, resulting in transcriptional activation and cytokine generation. LPS-induced signaling via toll-like receptor 4 (TLR4) results in the activation of the transcription factor NF-kappaB. Since LPS activates other signaling cascades in responsive cells, the objective of this study was to determine whether such events are mediated by TLR4 in response to LPS. We generated human embryonic kidney cells (HEK293) that stably express TLR4 (HEK-TLR4) and examined their responsiveness to LPS by measuring NF-kappaB activity and production of interleukin-8 (IL-8). A trans-reporting system was used to measure the activity of Elk-1, an ETS-domain transcription factor targeted by MAP kinase pathways. LPS stimulated NF-kappaB reporter activity and IL-8 production but not Elk-1 activity in HEK-TLR4 cells. When MD-2, a protein associated with the extracellular domain of TLR4, was expressed in these cells, there was a marked increase in Elk-1 activity as well as ERK, JNK, and p38 MAP kinase phosphorylation in response to LPS. TLR4-mediated NF-kappaB reporter activity and IL-8 production was enhanced by the expression of MD-2. This study demonstrates that expression of both TLR4 and MD-2 is required for LPS to activate or augment the MAP kinase pathways, Elk-1 stimulation, and IL-8 generation.

Lipophosphopeptidoglycan of Entamoeba histolytica Induces an Antiinflammatory Innate Immune Response and Downregulation of Toll-Like Receptor 2 (TLR-2) Gene Expression in Human Monocytes

Introduction Bacterial infection results in activation of the innate im-mune system. The recognition of a pathogen is mediated bya set of germline-encoded receptors that are referred to aspattern-recognition receptors (PRRs) that can directly rec-ognize invariant molecular structures known as pathogen-associated molecular patterns (PAMPs), which are sharedby large groups of microorganisms. One PAMP recognizedby PRRs is lipopolysaccharide (LPS), a mayor componentof the outer membrane of Gram-negative bacteria. LPSstimulates host cells and makes them produce various proin-flammatory cytokines, such as TNF- a , IL-1 a , and IL-6.LPS is captured by LPS-binding protein (LBP), which de-livers to CD14 on the cell surface. Because CD14 lacks anintracellular signaling domain, a transmembrane coreceptorhas been postulated in LPS-responsive cells that transducesthe LPS signaling into the cytoplasm. In Drosophila , theToll receptor participates in establishing the dorsoventralpatterns in the embryo and inducing antifungal immune re-sponse in the adult fly. Toll is a type I transmembrane pro-tein with a cytoplasmic domain with sequence homology tothe interleukin-1 receptor (IL-1R). Activation of Toll leadsto induction of genes through the transcription factor NF-kBpathway (1). Recently, several Toll-like receptors (TLRs)have been identified in macrophages and monocytes, basedon homology to Drosophila protein. Other studies havevariably suggested that TLR-2 and TLR-4 serve as the mainmediators of responses to LPS (2). In addition, TLR-2 mayalso act as a signaling receptor for other common bacterialstructures: peptidoglycan (PGN) and lipoteichoic acid(LTA) from Gram-positive bacteria and lipoarabinomannan(LAM) from Mycobacterium. Lipophosphopeptidoglycan (LPPG) antigens from Ent-amoeba histolytica were described for the first time by Isi-basi et al., and were later used to study its expression underdifferent culture conditions and in different E. histolytica strains using monoclonal antibodies. One antibody againstLPPG was able to inhibit adhesion of amebas and cytotoxic-ity to target cells. Recently, the expression of LPPG wascorrelated to amebic virulence. In addition, the protectionagainst invasive amebiasis by a single monoclonal antibodydirected against LPPG has been studied (3). We studiedLPPG, and found that, due to its polysaccharide nature, thisameba surface molecule behaves like a PAMP. LPPG isprobably recognized by TLR-2, but instead of inducing apro-inflammatory response, it induced production of inter-leukin-10 and downregulation of tlr -2 gene expression. Materials and Methods Isolation of LPPG. Trophozoites from E. histolytica strainHM-1 were cultivated under axenic conditions, and wereused to extract LPPG using the phenol–water method de-scribed by Westphal and Jann and modified by Isibasi et al.Contaminating LPS in LPPG preparations were determinedusing quantitative Limulus lysate assay (BioWhittaker). Inall preparations, 0.020 ng of LPS/ m g of carbohydrate wasdetected. Monocyte isolation and LPPG/LPS stimulation assay. Periph-eral blood mononuclear cells were isolated on Ficoll-paquegradients (Pharmacia LKB Biotecnology) and cultured (5 3 10

PU.1 and Interferon Consensus Sequence-binding Protein Regulate the Myeloid Expression of the Human Toll-like Receptor 4 Gene

The protein product of the Toll-like receptor (TLR) 4 gene has been implicated in the signal transduction events induced by lipopolysaccharide (LPS). In mice, destructive mutations of Tlr4 impede the normal response to LPS and cause a high susceptibility to Gram-negative infection. Expression of TLR4 mRNA in humans is restricted to a small number of cell types, including LPS-responsive myeloid cells, B-cells, and endothelial cells. To investigate the molecular basis for TLR4 expression in cells of myeloid origin, we cloned the human TLR4 gene and analyzed its putative 5'-proximal promoter. In transient transfections a region of only 75 base pairs upstream of the major transcription initiation site was sufficient to induce maximal luciferase activity in THP-1 cells. The sequence of this region is similar in human and mouse TLR4 genes and lacks a TATA box, typical Sp1-sites or CCAAT box sequences. Instead, it contains consensus-binding sites for Ets family transcription factors, octamer-binding factors, and a composite interferon response factor/Ets motif. The activity of the promoter in macrophages was strictly dependent on the integrity of both half sites of the composite interferon response factor/Ets motif, which was constitutively bound by the myeloid and B-cell-specific transcription factor PU.1 and interferon consensus sequence-binding protein. These results indicate that the two tissue-restricted transcription factors PU.1 and interferon consensus sequence-binding protein participate in the basal regulation of human TLR4 in myeloid cells. Cloning of the human TLR4 gene provides a basis for further investigation of the possible impact of genetic variations on the susceptibility to infection and sepsis.

Lipopolysaccharide-activated Kinase, an Essential Component for the Induction of the Antimicrobial Peptide Genes inDrosophila melanogaster Cells

Eukaryotic organisms use a similar Rel/NF-kappaB signaling cascade for the induction of innate immune genes. In Drosophila, lipopolysaccharide (LPS) signal-induced activation of the Rel/NF-kappaB family transcription factors is an essential step in the transcriptional activation of inducible antimicrobial peptide genes. However, the mechanism by which the LPS-induced signaling pathway proceeds remains largely unknown. Here we have cloned a novel Drosophila LPS-activated kinase (DLAK) that is structurally related to mammalian IkappaB kinases. DLAK is expressed and transiently activated in LPS-responsive Drosophila cells following LPS stimulation. Furthermore, DLAK can interact with Cactus, a Drosophila IkappaB and phosphorylate recombinant Cactus, in vitro. Overexpression of dominant-negative mutant DLAK (DLAK(K50A)) blocks LPS-induced Cactus degradation. DLAK-bound Cactus can be degraded in a LPS signal-dependent fashion, whereas the DLAK(K50A) mutant-bound Cactus is completely resistant to degradation in the presence of LPS. The DLAK(K50A) mutant also inhibits nuclear kappaB binding activity and kappaB-dependent diptericin reporter gene activity in a dose-dependent manner, but the kappaB-dependent diptericin reporter gene activity can be rescued by overexpression of wild type DLAK. Moreover, mRNA analysis of various kappaB-dependent antimicrobial peptide genes shows that LPS inducibility of these genes is greatly impaired in cells overexpressing DLAK(K50A). These results establish that DLAK is a novel LPS-activated kinase, which is an essential signaling component for the induction of antimicrobial peptide genes following LPS treatment in Drosophila cells.

Bacterial Lipopolysaccharide Activates Nuclear Factor-κB through Interleukin-1 Signaling Mediators in Cultured Human Dermal Endothelial Cells and Mononuclear Phagocytes

Bacterial lipopolysaccharide (LPS)-mediated immune responses, including activation of monocytes, macrophages, and endothelial cells, play an important role in the pathogenesis of Gram-negative bacteria-induced sepsis syndrome. Activation of NF-kappaB is thought to be required for cytokine release from LPS-responsive cells, a critical step for endotoxic effects. Here we investigated the role and involvement of interleukin-1 (IL-1) and tumor necrosis factor (TNF-alpha) signal transducer molecules in LPS signaling in human dermal microvessel endothelial cells (HDMEC) and THP-1 monocytic cells. LPS stimulation of HDMEC and THP-1 cells initiated an IL-1 receptor-like NF-kappaB signaling cascade. In transient cotransfection experiments, dominant negative mutants of the IL-1 signaling pathway, including MyD88, IRAK, IRAK2, and TRAF6 inhibited both IL-1- and LPS-induced NF-kappaB-luciferase activity. LPS-induced NF-kappaB activation was not inhibited by a dominant negative mutant of TRAF2 that is involved in TNF signaling. LPS-induced activation of NF-kappaB-responsive reporter gene was not inhibited by IL-1 receptor antagonist. TLR2 and TLR4 were expressed on the cell surface of HDMEC and THP-1 cells. These findings suggest that a signal transduction molecule in the LPS receptor complex may belong to the IL-1 receptor/toll-like receptor (TLR) super family, and the LPS signaling cascade uses an analogous molecular framework for signaling as IL-1 in mononuclear phagocytes and endothelial cells.

Role of Ceramide in Lipopolysaccharide (LPS)-induced Signaling: LPS INCREASES CERAMIDE RATHER THAN ACTING AS A STRUCTURAL HOMOLOG

Ceramide and ceramide-activated enzymes have been implicated in responses to bacterial lipopolysaccharide (LPS) and the proinflammatory cytokines tumor necrosis factor-alpha (TNF) and interleukin-1beta (IL-1). Although TNF and IL-1 cause elevation of cellular ceramide, which is thought to act as a second messenger, LPS has been proposed to signal by virtue of structural similarity to ceramide. We have investigated the relationship between ceramide and LPS by comparing the effects of a cell-permeable ceramide analog (C2-ceramide) and LPS on murine macrophage cell lines and by measuring ceramide levels in macrophages exposed to LPS. We found that while both C2-ceramide and LPS activated c-Jun N-terminal kinase (JNK), only LPS also activated extracellular signal-regulated kinases (ERKs). C2-ceramide was also unable to activate NF-kappaB, a transcription factor important for LPS-induced gene expression. Upon measurement of cellular ceramide in macrophage lines, we observed a small but rapid rise in ceramide, similar to that seen upon IL-1 or TNF treatment, suggesting LPS induces an increase in ceramide rather than interacting directly with ceramide-responsive enzymes. We found that C2-ceramide activated JNK and induced growth arrest in macrophages cell lines from both normal mice (Lpsn) and mice genetically unresponsive to LPS (Lpsd), whereas only Lpsn macrophages made these responses to LPS. Surprisingly, LPS treatment of Lpsd macrophages induced a rise in ceramide similar to that observed in LPS-responsive cells. These results indicate that the wild type Lps allele is not required for LPS-induced ceramide generation and suggest that ceramide elevation alone is insufficent stimulus for most responses to LPS.

Net inflammatory capacity of human septic shock plasma evaluated by a monocyte-based target cell assay: identification of interleukin-10 as a major functional deactivator of human monocytes.

We have developed a functional assay to study the inflammatory capacity of plasma collected from patients with severe gram-negative septic shock. In this assay, elutriation-purified, cryo-preserved human monocytes from one healthy donor are combined with plasma from patients with severe persistent septic shock for 5 h. Subsequently, the plasma is removed, medium added, and procoagulant activity (PCA) and secretion of tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) measured after 18-h incubation. Plasma from 10 patients (6 died) infected with Neisseria meningitidis previously shown to contain high levels of native lipopolysaccharide (LPS) (median 2,700 pg/ml), TNF-alpha, IL-6, IL-8, and complement activation products, had a low net spontaneous inflammatory capacity on the monocytes. The median levels of PCA, TNF-alpha, and IL-6 were 5, 0, and 4%, respectively, of the monocyte activities induced by normal plasma boosted with purified N. meningitidis (Nm)-LPS (2,500 pg/ml; net LPS-boosted capacity, 100%). The levels of PCA, TNF-alpha, and IL-6 obtained with plasma from shock patients were not different from those induced by plasma from 10 meningococcal patients without shock or with plasma from healthy persons. Boosting shock plasma with 2,500 pg/ml Nm-LPS had little effect on the monocyte activities since the median values of PCA, TNF-alpha, and IL-6 revealed a minimal increase from 5, 0, and 4% to 9, 2, and 6%, respectively. The shock plasmas revealed a strong LPS-inhibitory capacity that was largely absent in plasmas from 10 meningococcal patients without shock since the median levels of PCA, TNF-alpha, and IL-6 increased from 5, 0, and 0% to 135, 51, and 73%, respectively, after boosting with 2,500 pg/ml Nm-LPS. The LPS-inhibitory capacity was closely associated with the levels of IL-10. The median levels of IL-10 were 19,000 pg/ml in nine shock patients vs. 22 pg/ml in nine nonshock patients with systemic meningococcal disease. Removal of native IL-10 by immunoprecipitation restored the capacity of plasmas to induce monocyte activation either by native LPS or by boosting with Nm-LPS. IL-4 and TGF-beta were not detected in shock plasmas. In 24 patients with detectable meningococcal LPS ( > 10 pg/ml, 0.1 endotoxin units/ml), the levels of IL-10 were correlated to the levels of LPS (r = 0.79, P < 0.001). IL-10 declined from initiation of antibiotic therapy and paralleled the levels of native LPS. Decreasing levels of IL-10 in serially collected shock plasmas were directly related to increasing monocyte responsiveness after Nm-LPS boosting. These results suggest that IL-10 plays a major role in containing activation of monocytes and possibly other LPS-responsive cells during overwhelming meningococcemia.

Differences in mode of action of (β-lactam antibiotics influence morphology, LPS release and in vivo antibiotic efficacy

Antibiotic mediated release of endotoxin (lipopolysaccharide, LPS) from Gram-negative bacteria is implicated in septic shock. The β-lactam (cell wall active) class, in particular, has been deemed responsible for release of greater quantities of LPS than other classes of antibiotics. However, it is becoming increasingly recognized that variations in the ability to liberate free LPS exist within the β-lactam subclasses. Until recently, LPS-release studies have primarily included the cephalosporin, monobactam, and penicillin β-lactams, but not the carbapenem subclass. We document here that carbapenems significantly liberate less LPS than other β-lactam subclasses, and that disparity in LPS release also occurs within the carbapenems as well (i.e. imipenem vs meropenem). The propensity to release LPS correlates with the cumulative PBP binding affinity of each β-lactam antibiotic regardless of subclass. A direct correlation has been established between antibiotic concentration, frequency of antibiotic exposure, differential LPS release, PBP specificity (morphology, rate of bacterial lysis or cell mass) and antibiotic efficacy in two mouse models of infection. In the first, differences in the pathogenic (endotoxic) potential of potently biologically active smooth LPS (S-LPS) and poorly biologically active rough chemotype LPS (R-LPS) expressing bacteria underscores the in vivo importance of endotoxin in mediating lethality following antibiotic chemotherapy. Endotoxin is, however, not innately toxic and thus its lethal effects are mediated through LPS stimulation of host cells (i.e. macrophages) to produce deleterious levels of cytokines. In the second experimental model, therefore, the toxic effects of antibiotic-liberated endotoxin have been abrogated by pretreatment of mice with carrageenan (CGN) prior to challenge with endotoxin or Gram-negative bacteria. CGN treatment eliminates or markedly reduces the numbers of LPS-responsive mediator cells thus affecting mortality. In both animal models, an in vivo role for LPS is demonstrated through differential changes in antibiotic efficacy (ED 50) in response to differential effects of LPS via modulation of the endotoxic sensitivity/responsiveness of the host. We conclude that antibiotic class, concentration, dosing intervals (timing) and perhaps, method of treatment (i.e. bolus vs infusion) may each be important in the survival of experimental animals severely infected with Gram-negative bacteria.

LPS Responsiveness in Periodontal Ligament Cells is Regulated by Tumor Necrosis Factor-α

Gingival fibroblasts function as accessory immune cells and are capable of synthesizing cytokines in response to lipopolysaccharides (LPS) from Gram-negative microbes. Recently, we have isolated, cloned, and characterized two cell lines which exhibit characteristics of periodontal ligament (PDL) cells. In this report, we demonstrate that PDL cells showing osteoblast-like phenotype are not LPS-responsive cells. However, treatment of PDL cells with tumor necrosis factor-alpha (TNF-alpha) inhibits the expression of their osteoblast-like characteristics. As a consequence of this TNF-alpha-induced phenotypic change, PDL cells become LPS-responsive, i.e., synthesize several pro-inflammatory cytokines in response to LPS. These phenotypic changes occur at concentrations of TNF-alpha that are frequently observed in tissue exudates during periodontal inflammation, suggesting a physiological significance for these in vitro observations. It is of interest that TNF-alpha-induced phenotypic changes in PDL cells are transient, since removal of rhTNF-alpha from the supernatants of PDL cell cultures results in re-acquisition of the osteoblast-like characteristics and lack of LPS responsiveness of PDL cells. These results suggest that TNF-alpha, by regulating the PDL cell functions, may allow these cells to participate in the disease process as accessory immune cells at the expense of their structural properties.

Comparison of spleen cell proliferation in response to Brucella abortus 2308 lipopolysaccharide or proteins in mice vaccinated with strain 19 or RB51

Mice vaccinated with Brucella abortus 19 (S19) or RB51 (SRB51) had spleen cells which proliferated in response to proteins of 32, 27, 18, and < 18 kDa but not in response to proteins of 106, 80, and 49 kDa from B. abortus 2308 (S2308) following vaccination and challenge infection with S2308. Spleen cells from mice vaccinated with S19 but not with SRB51 had increased proliferation in response to S2308 lipopolysaccharide (LPS) following challenge infection with S2308. We previously reported that mice vaccinated with S19 or SRB51, which were analyzed in the current study, have increased resistance to infection with S2308 and that only mice vaccinated with S19 produce antibody to S2308 LPS (M. Stevens, S. Olsen, G. Pugh, Jr., and D. Brees, Infect. Immun. 63:264-270, 1995). The results from our current and previous studies support the contention that vaccination of mice with S19 or SRB51 induces protection from infection with S2308 by cell-mediated immune responses to the same immunodominant (32, 27, 18, and < 18 kDa) protein antigens of S2308. In addition, the absence of S2308 LPS-responsive spleen cells and antibody to S2308 LPS in mice vaccinated with SRB51 suggests that immune responses to LPS have no role in SRB51-induced protective immunity.

Transfection of CD14 into 70Z/3 cells dramatically enhances the sensitivity to complexes of lipopolysaccharide (LPS) and LPS binding protein.

Bacterial endotoxin (lipopolysaccharide [LPS]) causes fatal shock in humans and experimental animals. The shock is mediated by cytokines released by direct LPS stimulation of cells of monocytic origin (monocyte/macrophage [MO]). Recent studies have supported the concept that the plasma protein, LPS binding protein (LBP), plays an important role in controlling MO responses to LPS. Specifically, evidence has been presented to suggest that CD14, a membrane protein present in MO, serves as a receptor for complexes of LPS and the plasma protein LPS binding protein (LBP). In this function CD14 mediates attachment of LPS-bearing particles opsonized with LBP and appears to play an important role in regulating cytokine production induced by complexes of LPS and LBP. The CD14-, murine pre-B cell line 70Z/3 responds to LPS by synthesis of kappa light chains and consequent expression of surface IgM. To better understand the role of CD14 in controlling cellular responses to LPS, we investigated the effect of transfection of CD14 into 70Z/3 cells on LPS responsiveness. We report here that transfection of human or rabbit CD14 cDNA into 70Z/3 cells results in membrane expression of a glycosyl-phosphatidylinositol-anchored CD14. When LPS is complexed with LBP, CD14-bearing 70Z/3 cells bind more LPS than do the parental or 70Z/3 cells transfected with vector only. Remarkably, the expression of CD14 lowers the amount of LPS required to stimulate surface IgM expression by up to 10,000-fold when LPS dose-response curves in the CD14-, parental and CD14-bearing, transfected 70Z/3 cells are compared. In contrast, the response of CD14-bearing 70Z/3 cells and the parental 70Z/3 cell line (CD14-) to interferon gamma is indistinguishable. LPS stimulation of the parental and CD14-bearing 70Z/3 cells results in activation of NF-kB. These data provide evidence to support the concept that the LPS receptor in cells that constitutively express CD14 may be a multiprotein complex containing CD14 and membrane protein(s) common to a diverse group of LPS-responsive cells.

Purification and characterization of intraparenchymal lung lymphocytes.

We analyzed phenotypic and functional characteristics of intraparenchymal pulmonary lymphocytes in mice. As determined by flow cytometry, approximately 30% more T cells than B cells were found. Nearly all T cells bore the alpha beta TCR, most of which stained with either CD4+ or CD8+, although small numbers of double-negative T cells were present; the CD8+/CD4+ ratio was approximately 0.37. While practically all B cells bore surface IgM, and no IgA+ cells were found, and approximately more than 80% of the plasma cells produced IgA. Approximately half of the LPS-responsive cells produced IgA. Pulmonary B cell clonal precursors specific for the autoantigen, transferrin, were present in higher frequencies than those specific for the bacterial antigens, levan and Pseudomonas aeruginosa polysaccharide type I, or those producing antibodies against OVA. These results demonstrate that a distinct population of lymphocytes is present in the lungs.