The CD14 region spanning amino acids 57-64 is critical for interaction with the extracellular Toll-like receptor 2 domain

CD14 has been shown to enhance Toll-like receptor 2 (TLR2)-mediated signaling in response to peptidoglycan. Anti-CD14 monoclonal antibody MEM-18, whose epitope was located at the amino acid residues 57-64, blocked the binding of sCD14 to the recombinant soluble form of the extracellular TLR2 domain (sTLR2). The deletion mutant sCD14Delta57-64 lacking the amino acid residues 57-64 failed to bind to sTLR2. Cotransfection of wild type mCD14 but not mCD14Delta57-64 with TLR2 enhanced NF-kappaB activation in response to peptidoglycan. These results indicate that the CD14 region spanning amino acids 57-64 is critical for interacting with TLR2 and enhancing TLR2-mediated peptidoglycan signaling.     

Interaction of soluble form of recombinant extracellular TLR4 domain with MD-2 enables lipopolysaccharide binding and attenuates TLR4-mediated signaling

TLRs have been implicated in recognition of pathogen-associated molecular patterns. TLR4 is a signaling receptor for LPS, but requires MD-2 to respond efficiently to LPS. The purposes of this study were to examine the interactions of the extracellular TLR4 domain with MD-2 and LPS. We generated soluble forms of rTLR4 (sTLR4) and TLR2 (sTLR2) lacking the putative intracellular and transmembrane domains. sTLR4 consisted of Glu(24)-Lys(631). MD-2 bound to sTLR4, but not to sTLR2 or soluble CD14. BIAcore analysis demonstrated the direct binding of sTLR4 to MD-2 with a dissociation constant of K(D) = 6.29 x 10(-8) M. LPS-conjugated beads precipitated MD-2, but not sTLR4. However, LPS beads coprecipitated sTLR4 and MD-2 when both proteins were coincubated. The addition of sTLR4 to the medium containing the MD-2 protein significantly attenuated LPS-induced NF-kappaB activation and IL-8 secretion in wild-type TLR4-expressing cells. These results indicate that the extracellular TLR4 domain-MD-2 complex is capable of binding LPS, and that the extracellular TLR4 domain consisting of Glu(24)-Lys(631) enables MD-2 binding and LPS recognition to TLR4. In addition, the use of sTLR4 may lead to a new therapeutic strategy for dampening endotoxin-induced inflammation.     

Recombinant soluble forms of extracellular TLR4 domain and MD-2 inhibit lipopolysaccharide binding on cell surface and dampen lipopolysaccharide-induced pulmonary inflammation in mice

In this study, we sought the possibility of a new therapeutic strategy for dampening endotoxin-induced inflammation using soluble form of extracellular rTLR4 domain (sTLR4) and soluble form of rMD-2 (sMD-2). Addition of sTLR4 plus sMD-2 was significantly effective in inhibiting LPS-elicited IL-8 release from U937 cells and NF-kappaB activation in the cells transfected with TLR4 and MD-2 when compared with a single treatment with sTLR4 or sMD-2. Thus, we investigated the role of the extracellular TLR4 domain in interaction of lipid A with MD-2. Biotinylated sTLR4 failed to coprecipitate [(3)H]lipid A when it was sedimented with streptavidin-agarose, demonstrating that the extracellular TLR4 domain does not directly bind lipid A by itself. The amounts of lipid A coprecipitated with sMD-2 significantly increased when coincubated with sTLR4, and sTLR4 increased the affinity of lipid A for the binding to sMD-2. Soluble CD14 is required for the sTLR4-stimulated increase of lipid A binding to sMD-2. We also found that addition of sTLR4 plus sMD-2 inhibited the binding of Alexa-conjugated LPS to the cells expressing TLR4 and MD-2. Murine lungs that had received sTLR4 plus sMD-2 with LPS did not show any findings indicative of interstitial edema, neutrophil flux, and hemorrhage. Co-instillation of sTLR4 plus sMD-2, but not sTLR4 or sMD-2 alone, significantly decreased neutrophil infiltration and TNF-alpha levels in bronchoalveolar lavage fluids from LPS-treated mice. This study provides novel usage of sTLR4 and sMD-2 as an antagonist against endotoxin-induced pulmonary inflammation.     

TLR2 recognizes a bacterial lipopeptide through direct binding

The TLRs play an important role in the initiation of cellular innate immune responses to a wide range of bacterial products, including LPS and lipoproteins. Although rapid progress has been made on signaling functions of activated TLRs, the molecular mechanisms that lead to TLR activation are still poorly understood. We report in this study that the extracellular domain of TLR2 interacts directly with synthetic bacterial lipopeptide (sBLP), a potent analog of bacterial lipoproteins. Using fluorescently labeled sBLP complexed to soluble recombinant CD14 (rsCD14), we observed specific binding of sBLP to the surface of cells expressing TLR2 transgenes and to a recombinant soluble form of the TLR2 ectodomain. TLR2-mediated binding of sBLP at the cell surface did not require prior induction of intracellular signals. In addition, using a chimeric TLR2/TLR4 construct, we showed that the leucine-rich region of TLR2 carries the specificity for binding of the agonist and for initiating signaling. Specific binding of fluorescent sBLP to purified sTLR2 required sCD14. However, sCD14 was not part of the complex formed by soluble TLR2 and sBLP. Together, these data provide evidence that TLR2 recognizes sBLP through direct binding.     

Differential interactions of fimbriae and lipopolysaccharide from Porphyromonas gingivalis with the Toll-like receptor 2-centred pattern recognition apparatus

The lipopolysaccharide (LPS) and fimbriae of Porphyromonas gingivalis play important roles in periodontal inflammation and pathogenesis. We investigated fimbriae and LPS from several P. gingivalis strains in terms of relative dependence on Toll-like receptor (TLR) signalling partners or accessory pattern-recognition molecules mediating ligand transfer to TLRs, and determined induced assembly of receptor complexes in lipid rafts. Fimbriae could utilize TLR1 or TLR6 for cooperative TLR2-dependent activation of transfected cell lines, in contrast to LPS and a mutant version of fimbriae which displayed preference for TLR1. Whether used to activate human cell lines or mouse macrophages, fimbriae exhibited strong dependence on membrane-expressed CD14 (mCD14), which could not be substituted for by soluble CD14 (sCD14). In contrast, sCD14 efficiently substituted for mCD14 in LPS-induced cellular activation. LPS-binding protein was more important for LPS- than for fimbria-induced cell activation, whereas the converse was true for CD11b/CD18. Cell activation by LPS or fimbriae required lipid raft function and formation of heterotypic receptor complexes (TLR1-2/CD14/CD11b/CD18), although wild-type fimbriae additionally recruited TLR6. In summary, TLR2 activation by P. gingivalis LPS or fimbriae involves differential dependence on accessory signalling or ligand-binding receptors, which may differentially influence innate immune responses.     

Engagement of CD14 mediates the inflammatory potential of monosodium urate crystals

Phagocyte ingestion of monosodium urate (MSU) crystals can induce proinflammatory responses and trigger acute gouty inflammation. Alternatively, the uptake of MSU crystals by mature macrophages can be noninflammatory and promote resolution of gouty inflammation. Macrophage activation by extracellular MSU crystals involves apparent recognition and ingestion mediated by TLR2 and TLR4, with subsequent intracellular recognition linked to caspase-1 activation and IL-1beta processing driven by the NACHT-LRR-PYD-containing protein-3 inflammasome. In this study, we examined the potential role in gouty inflammation of CD14, a phagocyte-expressed pattern recognition receptor that functionally interacts with both TLR2 and TLR4. MSU crystals, but not latex beads, directly bound recombinant soluble (s) CD14 in vitro. CD14(-/-) bone marrow-derived macrophages (BMDMs) demonstrated unimpaired phagocytosis of MSU crystals but reduced p38 phosphorylation and approximately 90% less IL-1beta and CXCL1 release. Attenuated MSU crystal-induced IL-1beta release in CD14(-/-) BMDMs was mediated by decreased pro-IL-1beta protein expression and additionally by decreased caspase-1 activation and IL-1beta processing consistent with diminished NACHT-LRR-PYD-containing protein-3 inflammasome activation. Coating of MSU crystals with sCD14, but not sTLR2 or sTLR4, restored IL-1beta and CXCL1 production in CD14(-/-) BMDMs in vitro. Gain of function of CD14 directly enhanced TLR4-mediated signaling in response to MSU crystals in transfected Chinese hamster ovary cells in vitro. Last, MSU crystal-induced leukocyte influx at 6 h was reduced by approximately 75%, and local induction of IL-1beta decreased by >80% in CD14(-/-) mouse s.c. air pouches in vivo. We conclude that engagement of CD14 is a central determinant of the inflammatory potential of MSU crystals.     

The extracellular toll-like receptor 2 domain directly binds peptidoglycan derived from Staphylococcus aureus

Toll-like receptor 2 (TLR2) has been recognized to mediate cell signaling in response to peptidoglycan (PGN), a major cell wall component of Gram-positive bacteria. The mechanism by which TLR2 recognizes PGN is unknown. It is not even clear whether TLR2 directly binds to PGN. In this study, we generated a soluble form of recombinant TLR2 (sTLR2) possessing only its putative extracellular domain by using the baculovirus expression system to examine the direct interaction between sTLR2 and PGN. sTLR2 bound avidly to insoluble PGN (iPGN) from Staphylococcus aureus coated onto microtiter wells in a concentration-dependent manner. In contrast, sTLR2 exhibited a very weak binding to lipopolysaccharide. iPGN cosedimented sTLR2 after the mixture of iPGN and sTLR2 had been incubated and centrifuged. sTLR2 partially attenuated the iPGN-induced NF-kappaB activation in TLR2-transfected HEK 293 cells and the iPGN-induced IL-8 secretion in U937 cells. One of anti-human TLR2 monoclonal antibodies, which blocked iPGN-induced NF-kappaB activation in TLR2-transfected cells, inhibited the binding of sTLR2 to iPGN. In addition, we found that sCD14 interacted with sTLR2 and increased the binding of sTLR2 to iPGN. From these results, we conclude that the extracellular TLR2 domain directly binds to PGN.     

Soluble forms of Toll-like receptor (TLR)2 capable of modulating TLR2 signaling are present in human plasma and breast milk

Dysregulation of the initial, innate immune response to bacterial infection may lead to septic shock and death. Toll-like receptors (TLRs) play a crucial role in this innate immune response, and yet the regulatory mechanisms controlling microbial-induced TLR triggering are still to be fully understood. We have therefore sought specific regulatory mechanisms that may modulate TLR signaling. In this study, we tested for the possible existence of a functionally active soluble form of TLR2. We demonstrated the existence of natural soluble forms of TLR2 (sTLR2), which we show to be capable of modulating cell activation. We found that blood monocytes released sTLR2 constitutively and that the kinetics of sTLR2 release increased upon cell activation. Analysis of cells expressing the human TLR2 cDNA or its c-myc-tagged version indicated that sTLR2 resulted from the posttranslational modification of the TLR2 protein in an intracellular compartment. Moreover, an intracellular pool of sTLR2 is maintained. sTLR2 was found naturally expressed in breast milk and plasma. Milk sTLR2 levels mirrored those of the TLR coreceptor soluble CD14. Depletion of sTLR2 from serum resulted in an increased cellular response to bacterial lipopeptide. Notably, serum sTLR2 was lower in tuberculosis patients. Coimmunoprecipitation experiments and computational molecular docking studies showed an interaction between sTLR2 and soluble CD14 in plasma and milk. These findings suggest the existence of a novel and specific innate immune mechanism regulating microbial-induced TLR triggering, and may lead to new therapeutics for the prevention and/or treatment of severe infectious diseases.     

Innate immune responses are increased in chronic obstructive pulmonary disease

Background

Chronic obstructive pulmonary disease (COPD) is characterised by irreversible airflow obstruction, neutrophilic airway inflammation and chronic bacterial colonisation, however the role of the innate immune response in the pathogenesis of COPD remains unclear.

Methods

Induced sputum was obtained from adults with COPD (n = 22), and healthy controls (n = 29) and was processed for differential cell counts. The sputum supernatant was assayed for innate immune mediators using ELISA, whilst sputum gene expression was measured using real-time PCR. Peripheral blood neutrophils were isolated and their response to lipopolysaccaride (LPS) stimulation was assessed in a subgroup of participants with COPD (n = 13) and healthy controls (n = 21).

Results

Participants with COPD had significantly higher protein levels of interleukin (IL)-8, and neutrophil elastase (NE) and detection of oncostatin M (OSM) compared to healthy controls. Gene expression for toll-like receptor (TLR) 2, IL-8 and OSM were also significantly higher in COPD participants. The level of IL-1β, surfactant protein (SP)-A, matrix metalloproteinase (MMP)-9 and TLR4 mRNA was not significantly different between groups. The level of innate immune response markers were highly associated with the presence of sputum neutrophils, each other and the degree of airflow limitation (FEV₁/FVC). Peripheral blood neutrophils from participants with COPD had an increased response to stimulation by LPS; with a greater fold increase in the production of IL-8 and MMP-9 protein, and gene expression of IL-8, TLR2 and TLR4.

Conclusions

The innate immune response is increased in the airways and circulating neutrophils in COPD, and may be an important mechanism involved in disease pathogenesis.     

Toll-like receptor (TLR)2 and TLR4 in human peripheral blood granulocytes: a critical role for monocytes in leukocyte lipopolysaccharide responses

Leukocyte responsiveness to LPS is dependent upon CD14 and receptors of the Toll-like receptor (TLR) family. Neutrophils respond to LPS, but conflicting data exist regarding LPS responses of eosinophils and basophils, and expression of TLRs at the protein level in these granulocyte lineages has not been fully described. We examined the expression of TLR2, TLR4, and CD14 and found that monocytes expressed relatively high levels of cell surface TLR2, TLR4, and CD14, while neutrophils also expressed all three molecules, but at low levels. In contrast, basophils expressed TLR2 and TLR4 but not CD14, while eosinophils expressed none of these proteins. Tested in a range of functional assays including L-selectin shedding, CD11b up-regulation, IL-8 mRNA generation, and cell survival, neutrophils responded to LPS, but eosinophils and basophils did not. In contrast to previous data, we found, using monocyte depletion by negative magnetic selection, that neutrophil responses to LPS were heavily dependent upon the presence of a very low level of monocytes, and neutrophil survival induced by LPS at 22 h was monocyte dependent. We conclude that LPS has little role in the regulation of peripheral blood eosinophil and basophil function, and that, even in neutrophils, monocytes orchestrate many previously observed leukocyte LPS response patterns.     

MD-2 mediates the ability of tetra-acylated and penta-acylated lipopolysaccharides to antagonize Escherichia coli lipopolysaccharide at the TLR4 signaling complex

We have demonstrated previously that tetra-acylated LPS derived from the oral bacterium, Porphyromonas gingivalis, and penta-acylated msbB LPS derived from a mutant strain of Escherichia coli can antagonize the ability of canonical hexa-acylated E. coli LPS to signal through the TLR4 signaling complex in human endothelial cells. Activation of the TLR4 signaling complex requires the coordinated function of LPS binding protein (LBP), CD14, MD-2, and TLR4. To elucidate the specific molecular components that mediate antagonism, we developed a recombinant human TLR4 signaling complex that displayed efficient LPS-dependent antagonism of E. coli LPS in HEK293 cells. Notably, changes in the expression levels of TLR4 in HEK293 cells modulated the efficiency of antagonism by P. gingivalis LPS. Both soluble (s) CD14 and membrane (m) CD14 supported efficient P. gingivalis LPS-dependent and msbB LPS-dependent antagonism of E. coli LPS in the recombinant TLR4 system. When cells expressing TLR4, MD-2, and mCD14 were exposed to LPS in the absence of serum-derived LBP, efficient LPS-dependent antagonism of E. coli LPS was still observed indicating that LPS-dependent antagonism occurs downstream of LBP. Experiments using immunoprecipitates of sCD14 or sMD-2 that had been pre-exposed to agonist and antagonist indicated that LPS-dependent antagonism occurs partially at sCD14 and potently at sMD-2. This study provides novel evidence that expression levels of TLR4 can modulate the efficiency of LPS-dependent antagonism. However, MD-2 represents the principal molecular component that tetra-acylated P. gingivalis LPS and penta-acylated msbB LPS use to antagonize hexa-acylated E. coli LPS at the TLR4 signaling complex.     

Sex Differences in Monocyte Activation in Systemic Lupus Erythematosus (SLE)

Introduction

TLR7/8 and TLR9 signaling pathways have been extensively studied in systemic lupus erythematosus (SLE) as possible mediators of disease. Monocytes are a major source of pro-inflammatory cytokines and are understudied in SLE. In the current project, we investigated sex differences in monocyte activation and its implications in SLE disease pathogenesis.

Methods

Human blood samples from 27 healthy male controls, 32 healthy female controls, and 25 female patients with SLE matched for age and race were studied. Monocyte activation was tested by flow cytometry and ELISA, including subset proportions, CD14, CD80 and CD86 expression, the percentage of IL-6-producing monocytes, plasma levels of sCD14 and IL-6, and urine levels of creatinine.

Results

Monocytes were significantly more activated in women compared to men and in patients with SLE compared to controls in vivo. We observed increased proportions of non-classic monocytes, decreased proportions of classic monocytes, elevated levels of plasma sCD14 as well as reduced surface expression of CD14 on monocytes comparing women to men and lupus patients to controls. Plasma levels of IL-6 were positively related to sCD14 and serum creatinine.

Conclusion

Monocyte activation and TLR4 responsiveness are altered in women compared to men and in patients with SLE compared to controls. These sex differences may allow persistent systemic inflammation and resultant enhanced SLE susceptibility.     

Electron tomography of negatively stained complex viruses: application in their diagnosis

Pattern recognition receptors are a key component of the first line host defense against infection, recognizing specific microbial products. We hypothesize that monocyte hyporesponsiveness in human sepsis is associated with a downregulation of the pattern recognition receptors Toll-like receptor (TLR)-2 and TLR4. Protein expression of CD14, TLR2 and TLR4 on blood monocytes was examined using flow cytometry from 29 patients with sepsis and 14 healthy controls. In addition LPS stimulated TNF-α and IL-10 production was studied in a 24 hour whole blood assay. We found an increased expression of CD14, TLR2 and TLR4 in patients with sepsis compared to controls (p < 0.01). In patients with sepsis, death was associated with significant lower CD14 and TLR2 expression at admission (CD14: 25.7 +- 19.1 vs 39.1 +- 17.3 mean fluorescence intensity [MFI], p = 0.02; TLR2: 21.8 +- 9.4 vs. 30.9 +- 9.6, p = 0.01). At 72 hours the TLR2 expression on monocytes was associated with the IL-10 inducibility after LPS stimulation (r = 0.52, p = 0.02) and the CD14 expression with the IL-6, IL-10 and TNF inducibility. We conclude that septic patients are characterized by an increased expression of CD14, TLR2 and TLR4 on monocytes compared to controls. Death is associated with downregulation of TLR2 and CD14 expression on monocytes correlating with reduced cytokine inducibility. We suggest that CD14 and TLR2 are a key factor in monocyte hyporesponsibility during severe sepsis.     

Varying importance of soluble and membrane CD14 in endothelial detection of lipopolysaccharide

The endothelial response to LPS is critical in the recruitment of leukocytes, thereby allowing the host to survive Gram-negative infection. Herein, we investigated the roles of soluble CD14 (sCD14) and membrane CD14 (mCD14) in the endothelial response to low level LPS (0.1 ng/ml), intermediate level LPS (10 ng/ml), and high level LPS (1000 ng/ml). Removal of sCD14 from serum and sCD14-negative serum prevented low level LPS detection and subsequent response. Addition of recombinant sCD14 back into the endothelial system rescued the endothelial response. GPI-linked mCD14 removal from endothelium or endothelial treatment with a CD14 mAb prevented responses to low-level LPS even in the presence of sCD14. This demonstrates essential nonoverlapping roles for both mCD14 and sCD14 in the detection of low-level LPS. At intermediate levels of LPS, sCD14 was not required, but blocking mCD14 still prevented endothelial LPS detection and E-selectin expression, even in the presence of sCD14, suggesting that sCD14 cannot substitute for mCD14. At very high levels of LPS, the absence of mCD14 and sCD14 did not abrogate TLR4-dependent, E-selectin synthesis in response to LPS. The MyD88 independent pathway was detected in endothelium (presence of TRIF-related adaptor molecule TRAM). The MyD88-independent response (IFN-beta) in endothelium required mCD14 even at the highest LPS dose tested. Our results demonstrate an essential role for endothelial mCD14 that cannot be replaced by sCD14. Furthermore, we have provided evidence for a TRAM pathway in endothelium that is dependent on mCD14 even when other responses are no longer mCD14 dependent.     

Decreased levels of soluble Toll-like Receptor 2 in patients with asthma

Background:

Recently, reports have indicated a role for the membrane form of Toll-like Receptor 2 (TLR2) in asthma pathogenesis. In this study we examined soluble TLR2 levels in serum and sputum of asthmatic and healthy subjects.

Methods:

Serum and sputum samples were obtained from 33 asthmatic and 19 healthy subjects. The asthmatics were classified into four groups according to the Global Initiative for Asthma. A sandwich ELISA was developed to measure soluble TLR2 (sTLR2) in serum and sputum. TLR2 mRNA expression was determined by semi-quantitative RT-PCR of all sputum samples.

Results:

The mean sTLR2 levels from serum and sputum of asthmatics were significantly lower than those from healthy subjects. Moreover, sTLR2 concentration decreased concomitantly with asthma severity. The differences observed, however, were not statistically significant. TLR2/GAPDH mRNA of sputum leukocytes was also significantly lower in asthmatics than in healthy subjects.

Conclusion:

This study demonstrated for the first time thatsTLR2 levels are lower in serum and sputum samples from asthmatic than from healthy subjects, and this could be an indicator of TLR2 expression. We also found that sTLR2 concentration in serum decreased concomitantly with an increase of asthma severity clinical score. Key Words: Asthma, Expression, TLR2 mRNA, Soluble Toll-like receptor     

Endotoxin Exposure during Sensitization to Blomia tropicalis Allergens Shifts TH2 Immunity Towards a TH17-Mediated Airway Neutrophilic Inflammation: Role of TLR4 and TLR2

Experimental evidence and epidemiological studies indicate that exposure to endotoxin lipopolysaccharide (eLPS) or other TLR agonists prevent asthma. We have previously shown in the OVA-model of asthma that eLPS administration during alum-based allergen sensitization blocked the development of lung TH2 immune responses via MyD88 pathway and IL-12/IFN-γ axis. In the present work we determined the effect of eLPS exposure during sensitization to a natural airborne allergen extract derived from the house dust mite Blomia tropicalis (Bt). Mice were subcutaneously sensitized with Bt allergens co-adsorbed onto alum with or without eLPS and challenged twice intranasally with Bt. Cellular and molecular parameters of allergic lung inflammation were evaluated 24 h after the last Bt challenge. Exposure to eLPS but not to ultrapure LPS (upLPS) preparation during sensitization to Bt allergens decreased the influx of eosinophils and increased the influx of neutrophils to the airways. Inhibition of airway eosinophilia was not observed in IFN-γdeficient mice while airway neutrophilia was not observed in IL-17RA-deficient mice as well in mice lacking MyD88, CD14, TLR4 and, surprisingly, TLR2 molecules. Notably, exposure to a synthetic TLR2 agonist (PamCSK4) also induced airway neutrophilia that was dependent on TLR2 and TLR4 molecules. In the OVA model, exposure to eLPS or PamCSK4 suppressed OVA-induced airway inflammation. Our results suggest that B. tropicalis allergens engage TLR4 that potentiates TLR2 signaling. This dual TLR activation during sensitization results in airway neutrophilic inflammation associated with increased frequency of lung TH17 cells. Our work highlight the complex interplay between bacterial products, house dust mite allergens and TLR signaling in the induction of different phenotypes of airway inflammation.     

The role of CD14 in neutrophil recruitment within the liver microcirculation during endotoxemia

During Gram-negative sepsis and endotoxemia, CD14 is essential for the recognition of LPS by the TLR4 complex and subsequent generation of systemic inflammation. However, CD14-independent responses to LPS have been reported in vitro and in vivo in selected tissues including the skin. As the liver is a key target organ for neutrophil sequestration and inflammatory pathology during sepsis and endotoxemia, we investigated the role of CD14 in the recruitment of neutrophils into the liver in a mouse model of endotoxemia. Using dynamic in vivo imaging of the liver, we observed that neutrophil recruitment within the sinusoids and post-sinusoidal venules occurred equivalently between LPS-treated wild-type and CD14-knockout mice. Neutrophil recruitment within the liver was completely independent of CD14 regardless of whether it was expressed on cells of hematopoietic or nonhematopoietic origin or in serum as soluble CD14. Whereas CD14 expression was essential for activation of circulating neutrophils and for the development of LPS-induced systemic inflammation (pulmonary neutrophil sequestration, leukopenia, and increased serum proinflammatory cytokine levels), deficiency of CD14 did not limit the adhesion strength of neutrophils in vitro. Furthermore, wild-type and CD14-knockout mice displayed identical deposition of serum-derived hyaluronan-associated protein within liver sinusoids in response to LPS, indicating that the sinusoid-specific CD44/hyaluronan/serum-derived hyaluronan-associated protein-dependent pathway of neutrophil adhesion is activated independently of CD14. Therefore, the liver microcirculation possesses a unique CD14-independent mechanism of LPS detection and activation of neutrophil recruitment.     

Metalloproteinase-Dependent TLR2 Ectodomain Shedding is Involved in Soluble Toll-Like Receptor 2 (sTLR2) Production

Toll-like receptor (TLR) 2, a type I membrane receptor that plays a key role in innate immunity, recognizes conserved molecules in pathogens, and triggering an inflammatory response. It has been associated with inflammatory and autoimmune diseases. Soluble TLR2 (sTLR2) variants have been identified in human body fluids, and the TLR2 ectodomain can negatively regulate TLR2 activation by behaving as a decoy receptor. sTLR2 generation does not involve alternative splicing mechanisms, indicating that this process might involve a post-translational modification of the full-length receptor; however, the specific mechanism has not been studied. Using CD14⁺ peripheral human monocytes and the THP-1 monocytic leukemia-derived cell line, we confirm that sTLR2 generation increases upon treatment with pro-inflammatory agents and requires a post-translational mechanism. We also find that the constitutive and ligand-induced release of sTLR2 is sensitive to pharmacological metalloproteinase activator and inhibitors leading us to conclude that metalloproteinase TLR2 shedding contributes to soluble receptor production. By expressing human TLR2 in ADAM10- or ADAM17-deficient MEF cells, we find both enzymes to be implicated in TLR2 ectodomain shedding. Moreover, using a deletion mutant of the TLR2 juxtamembrane region, we demonstrate that this domain is required for sTLR2 generation. Functional analysis suggests that sTLR2 generated by metalloproteinase activation inhibitsTLR2-induced cytokine production by this monocytic leukemia-derived cell line. The identification of the mechanisms involved in regulating the availability of soluble TLR2 ectodomain and cell surface receptors may contribute further research on TLR2-mediated processes in innate immunity and inflammatory disorders.     

NTPDase1 controls IL-8 production by human neutrophils

The ectonucleotidase NTPDase1 (CD39) terminates P2 receptor activation by the hydrolysis of extracellular nucleotides (i.e., the P2 receptor ligands). In agreement with that role, exacerbated inflammation has been observed in NTPDase1-deficient mice. In this study, we extend these observations by showing that inhibition of NTPDase1 markedly increases IL-8 production by TLR-stimulated human neutrophils. First, immunolabeling of human blood neutrophils and neutrophil-like HL60 cells displayed the expression of NTPDase1 protein, which correlated with the hydrolysis of ATP at their surface. NTPDase1 inhibitors (e.g., NF279 and ARL 67156) as well as NTPDase1-specific small interfering RNAs markedly increased IL-8 production in neutrophils stimulated with LPS and Pam(3)CSK(4) (agonists of TLR4 and TLR1/2, respectively) but not with flagellin (TLR5) and gardiquimod (TLR7 and 8). This increase in IL-8 release was due to the synergy between TLRs and P2 receptors. Indeed, ATP was released from neutrophils constitutively and accumulated in the medium upon NTPDase1 inhibition by NF279. Likewise, both human blood neutrophils and neutrophil-like HL60 cells produced IL-8 in response to exogenous nucleotides, ATP being the most potent inducer. In agreement, P2Y(2) receptor knockdown in neutrophil-like HL60 cells markedly decreased LPS- and Pam(3)CSK(4)-induced IL-8 production. In line with these in vitro results, injection of LPS in the air pouches of NTPDase1-deficient mice triggered an increased production of the chemokines MIP-2 and keratinocyte-derived chemokine (i.e., the rodent counterparts of human IL-8) compared with that in wild-type mice. In summary, NTPDase1 controls IL-8 production by human neutrophils via the regulation of P2Y(2) activation.     

Direct binding of Toll-like receptor 2 to zymosan,and zymosan-induced NF-kappa B activation and TNF-alpha secretion are down-regulated by lung collectin surfactant protein A

The lung collectin surfactant protein A (SP-A) has been implicated in the regulation of pulmonary host defense and inflammation. Zymosan induces proinflammatory cytokines in immune cells. Toll-like receptor (TLR)2 has been shown to be involved in zymosan-induced signaling. We first investigated the interaction of TLR2 with zymosan. Zymosan cosedimented the soluble form of rTLR2 possessing the putative extracellular domain (sTLR2). sTLR2 directly bound to zymosan with an apparent binding constant of 48 nM. We next examined whether SP-A modulated zymosan-induced cellular responses. SP-A significantly attenuated zymosan-induced TNF-alpha secretion in RAW264.7 cells and alveolar macrophages in a concentration-dependent manner. Although zymosan failed to cosediment SP-A, SP-A significantly reduced zymosan-elicited NF-kappaB activation in TLR2-transfected human embryonic kidney 293 cells. Because we have shown that SP-A binds to sTLR2, we also examined whether SP-A affected the binding of sTLR2 to zymosan. SP-A significantly attenuated the direct binding of sTLR2 to zymosan in a concentration-dependent fashion. From these results, we conclude that 1) TLR2 directly binds zymosan, 2) SP-A can alter zymosan-TLR2 interaction, and 3) SP-A down-regulates TLR2-mediated signaling and TNF-alpha secretion stimulated by zymosan. This study supports an important role of SP-A in controlling pulmonary inflammation caused by microbial pathogens.