Polyneuropathy associated with IgM monoclonal gammopathy (IgM-PNP) is a slowly progressive, senso... more Polyneuropathy associated with IgM monoclonal gammopathy (IgM-PNP) is a slowly progressive, sensorimotor neuropathy. It is assumed that complement activation contributes to IgM-PNP pathogenesis. We investigated whether innate differences in complement activity of the classical and mannose binding lectin (MBL) pathways are associated with IgM-PNP or its severity. We measured complement activity using ELISA and determined MBL serumc oncentrations and MBL gene polymorphisms in 83 patients and 83 healthy controls. We did not observe differences between IgM-PNP patients and healthy controls nor associations with different disease severities. Differences in innate complement activity are not likely to explain susceptibility to or severity of IgM-PNP.
The Editorial team of JDUHS greatfully acknowledges the contribution of the following honorable r... more The Editorial team of JDUHS greatfully acknowledges the contribution of the following honorable reviewers who helped and guided to improving the quality of the manuscripts published / evaluated in the year 2014.
Lipopolysaccharide (LPS) is the major mediator of gram-negative septic shock. Molecules that bind... more Lipopolysaccharide (LPS) is the major mediator of gram-negative septic shock. Molecules that bind LPS and neutralize its toxic effects could have important clinical applications. We showed that serum amyloid P component (SAP) neutralizes LPS. A SAP-derived peptide, consisting of amino acids 27 to 39, inhibited LPS-mediated effects in the presence of human blood. In this study, we used a pepscan of overlapping 15-mer peptides and distinguished two additional LPS-binding regions within the SAP molecule, identified in the regions spanning amino acids 61 to 75 and 186 to 200. The corresponding SAP-derived peptides, pep61-75 and pep186-200, inhibited the binding of fluorescein isothiocyanate-labeled LPS to monocytes as efficiently as a bactericidal/permeability-increasing protein (BPI)-derived 15-mer peptide comprising amino acids 85 to 99. The same SAP-derived peptides very potently inhibited LPS-induced priming of phagocytes in human blood. Also, SAP-derived pep186-200 caused a prolong...
The absolute number of membraneexpressed CD14, the most important endotoxin receptor, on human mo... more The absolute number of membraneexpressed CD14, the most important endotoxin receptor, on human monocytes and neutrophils shows remarkable variation in the literature. To quantify these numbers two fluorescence methods using fluorescein isothiocyanate (FUff)-labeled monoclonal antibodies (mAb) were applied. A commercially available set of standard beads was used in flow cytometry to quantitate CD14 with eight different mAbs. Independent from their isotype the various mAbs showed minor differences and indicated that peripheral blood monocytes expressed 99,500-134,600 (115,400 ± 10,600) and neutrophils 1,900-4,400 (3,300 ± 800) CD14 receptors. There was no significant difference in CD14 expression on leukocytes in unprocessed freshly obtained whole blood and after a Ficoll isolation procedure. However, a short temperature shift resulted in a 1.3-to 1.6-fold up-regulation of CD14. The results obtained with the reference beads were verified with fluorescence Scatchard analysis and spectrofluorometry using itiAb 26ic-FflC and showed 109,500 CD14 per monocyte and 6,700 CD14 per neutrophil. For comparison the number of CD14 on the monocytic THP-1 cells and Fc'y-receptors on human leukocytes were determined using the reference beads and flow cytometry and gave results comparable to published data. Our data indicate that resting human monocytes express roughly 110,000 CD14 molecules on their surface using a simple fluorometric assay. Correct determination ofthe number ofCDl4 and other cell surface receptors is of importance in the monitoring of septic patients.
Journal of immunology (Baltimore, Md. : 1950), 1998
LPS-binding proteins in plasma play an important role in modifying LPS toxicity. Significant prop... more LPS-binding proteins in plasma play an important role in modifying LPS toxicity. Significant properties have already been attributed to the LPS-binding protein (LBP). It accelerates LPS toxicity as well as incorporation into high-density lipoproteins, leading to neutralization of LPS in serum. A search for other LPS-binding components in serum, using LPS-coated magnetic beads, revealed a new LPS-binding protein. N-terminal microsequencing identified this protein as serum amyloid P component (SAP). Purified SAP bound to smooth and rough types of LPS via the lipid A part. SAP inhibited the binding of FITC-labeled ReLPS (LPS from Salmonella minnesota strain R595) to human monocytes and the ReLPS-induced priming of the oxidative burst of human neutrophils only in the presence of low concentrations of LBP. In search for the LPS binding site of SAP, we found that pep27-39, a 13-mer peptide consisting of amino acids 27-39 of SAP, competitively inhibited the binding of LPS to SAP. In additi...
The building blocks of bacterial flagella, flagellin monomers, are potent stimulators of host inn... more The building blocks of bacterial flagella, flagellin monomers, are potent stimulators of host innate immune systems. Recognition of flagellin monomers occurs by flagellin-specific pattern-recognition receptors, such as Toll-like receptor 5 (TLR5) in mammals and flagellin-sensitive 2 (FLS2) in plants. Activation of these immune systems via flagellin leads eventually to elimination of the bacterium from the host. In order to prevent immune activation and thus favor survival in the host, bacteria secrete many proteins that hamper such recognition. In our search for Toll like receptor (TLR) antagonists, we screened bacterial supernatants and identified alkaline protease (AprA) of Pseudomonas aeruginosa as a TLR5 signaling inhibitor as evidenced by a marked reduction in IL-8 production and NF-kB activation. AprA effectively degrades the TLR5 ligand monomeric flagellin, while polymeric flagellin (involved in bacterial motility) and TLR5 itself resist degradation. The natural occurring alkaline protease inhibitor AprI of P. aeruginosa blocked flagellin degradation by AprA. P. aeruginosa aprA mutants induced an over 100-fold enhanced activation of TLR5 signaling, because they fail to degrade excess monomeric flagellin in their environment. Interestingly, AprA also prevents flagellin-mediated immune responses (such as growth inhibition and callose deposition) in Arabidopsis thaliana plants. This was due to decreased activation of the receptor FLS2 and clearly demonstrated by delayed stomatal closure with live bacteria in plants. Thus, by degrading the ligand for TLR5 and FLS2, P. aeruginosa escapes recognition by the innate immune systems of both mammals and plants.
Bacterial flagellin molecules are strong inducers of innate immune responses in both mammals and ... more Bacterial flagellin molecules are strong inducers of innate immune responses in both mammals and plants. The opportunistic pathogen Pseudomonas aeruginosa secretes an alkaline protease called AprA that degrades flagellin monomers. Here, we show that AprA is widespread among a wide variety of bacterial species. In addition, we investigated the role of AprA in virulence of the bacterial plant pathogen P. syringae pv. tomato DC3000. The AprA-deficient DC3000 ΔaprA knockout mutant was significantly less virulent on both tomato and Arabidopsis thaliana. Moreover, infiltration of A. thaliana Col-0 leaves with DC3000 ΔaprA evoked a significantly higher level of expression of the defense-related genes FRK1 and PR-1 than did wild-type DC3000. In the flagellin receptor mutant fls2, pathogen virulence and defense-related gene activation did not differ between DC3000 and DC3000 ΔaprA. Together, these results suggest that AprA of DC3000 is important for evasion of recognition by the FLS2 recepto...
Phagocytes play a major role in host defense against staphylococci as well as in the pathophysiol... more Phagocytes play a major role in host defense against staphylococci as well as in the pathophysiology of Gram-positive septic shock. In Gram negative sepsis, the main mediator, LPS exerts its effects as easily suspendable mediator. In Gram positive sepsis the main mediator is still not found, therefore we studied the interaction of soluble staphylococcal products with phagocytes. Staphylococcus aureus supernates
Leukocyte migration is a key event both in host defense against invading pathogens as well as in ... more Leukocyte migration is a key event both in host defense against invading pathogens as well as in inflammation. Bacteria generate chemoattractants primarily by excretion (formylated peptides), complement activation (C5a), and subsequently through activation of leukocytes (e.g., leukotriene B4, platelet-activating factor, and interleukin 8). Here we describe a new protein secreted by Staphylococcus aureus that specifically impairs the response of neutrophils and monocytes to formylated peptides and C5a. This chemotaxis inhibitory protein of S. aureus (CHIPS) is a 14.1-kD protein encoded on a bacteriophage and is found in >60% of clinical isolates. CHIPS reduces the neutrophil recruitment toward C5a in a mouse peritonitis model, even though its activity is much more potent on human than on mouse cells. These findings suggest a new immune escape mechanism of S. aureus and put forward CHIPS as a potential new antiinflammatory therapeutic compound.
Polymorphonuclear leukocytes (PMN) can mediate cytotoxic reactions against virus infected targets... more Polymorphonuclear leukocytes (PMN) can mediate cytotoxic reactions against virus infected targets cells. We observed very efficient binding of PMN to HSV-infected fibroblasts when loaded with HSV-specific antibodies. Using electron microscopy, infected fibroblasts were found to be totally surrounded by PMN and the phagocytosis of virions and fragments of infected cells was demonstrated. To quantify and study this phenomenon, and to compare PMN with monocytes, we developed radiometric and fluorometric phagocytosis assays. Leukocytes were mixed with [3H]glucosamine-or FITC-labeled virus and incubated at 37 °C. PMN associated radioactivity or fluorescence per cell as measured by flow cytometry was determined. PMN phagocytosis was dependent on the presence of specific anti-HSV antibodies and could be enhanced by addition of complement. Monocytes were also able to phagocytize virions; however, the rate of uptake was less than that for PMN. Under optimal conditions the total amount of herpes simplex particles that could be associated with one PMN or monocyte was about 10,000. PMN and monocytes are capable of phagocytosis of HSV. This may be an important factor in preventing the spread of infection in vivo.
Proceedings of the National Academy of Sciences, 2015
Toll-like receptors (TLRs) are crucial in innate recognition of invading micro-organisms and thei... more Toll-like receptors (TLRs) are crucial in innate recognition of invading micro-organisms and their subsequent clearance. Bacteria are not passive bystanders and have evolved complex evasion mechanisms. Staphylococcus aureus secretes a potent TLR2 antagonist, staphylococcal superantigen-like protein 3 (SSL3), which prevents receptor stimulation by pathogen-associated lipopeptides. Here, we present crystal structures of SSL3 and its complex with TLR2. The structure reveals that formation of the specific inhibitory complex is predominantly mediated by hydrophobic contacts between SSL3 and TLR2 and does not involve interaction of TLR2–glycans with the conserved LewisX binding site of SSL3. In the complex, SSL3 partially covers the entrance to the lipopeptide binding pocket in TLR2, reducing its size by ∼50%. We show that this is sufficient to inhibit binding of agonist Pam2CSK4 effectively, yet allows SSL3 to bind to an already formed TLR2–Pam2CSK4 complex. The binding site of SSL3 over...
Human adherent cells, obtained by EDTA reversible adherence to plastic, are potent effectors in c... more Human adherent cells, obtained by EDTA reversible adherence to plastic, are potent effectors in cell-mediated cytotoxicity. Spontaneous cytotoxicity in a 2-hr assay against K562 target cells was shown to be largely mediated by contaminating natural killer (NK) cells. Treatment of adherent cells with NK-specific monoclonal antibody anti-Leu-11 plus complement abolished almost completely the spontaneous cytotoxicity. Spontaneous cytotoxicity by adherent cells was also reduced when the phorbol ester PMA was present in the assay. On the other hand, PMA induced a cytotoxic response in NK-cell depleted adherent cells after prolonged 18 hr incubation. The cell population responsible for this dichotomous effect of PMA on adherent cell-mediated cytotoxicity was shown to be monocytes, as revealed by monoclonal antibody treatment. Pure NK cell preparations were not affected by PMA in their cytolytic capacities. Reactive oxygen species are not involved in NK-cell mediated cytotoxicity, while PM...
The specific binding of human polymorphonuclear leukocytes (PMN) to antibody-coated target cells ... more The specific binding of human polymorphonuclear leukocytes (PMN) to antibody-coated target cells was characterized by flow cytometry. PMN were labeled with phycoerythrin-E (PE) via a granulocyte-specific monoclonal antibody (leu-M1) and mixed with fluorescein isothiocyanate-labeled K562 tumor cells sensitized with rabbit antiserum. Specific conjugates were formed as analyzed by two-color fluorescence in a flow cytometer. The formation of stable conjugates was dependent on initiation of contact, temperature, time, and antiserum concentration. Studies with inhibitors implicate that microfilaments, but not microtubules, Ca2+, Mg2+, or energy-dependent processes were a prerequisite for binding of PMN to the antibody-coated target cells. No conjugates were formed when uncoated target cells were used or when the experiment was performed in the presence of protein A, indicating that binding was specifically mediated through Fc receptors (FcR). Monoclonal antibodies against the FcRII and Fc...
In the present study we used flow cytometry to investigate the phagocytosis of fluorescein isothi... more In the present study we used flow cytometry to investigate the phagocytosis of fluorescein isothiocyanate-labeled herpes simplex virus type 1 (FITC-HSV-1) by rat alveolar macrophages and the effects of surfactant protein A (SP-A) on this process. The phagocytosis of FITC-HSV-1 by alveolar macrophages, which was studied as a model for virus phagocytosis in general, was strongly enhanced in the presence of SP-A. The SP-A-mediated phagocytosis was time and concentration dependent, reaching a maximal level after 15 min of incubation and at an SP-A concentration of 5 micrograms/ml. Using a fluorescence quenching technique, we could show that at least 65% of the viruses were indeed internalized by the macrophages. The addition of SP-A to the system was sufficient for the phagocytosis of FITC-HSV-1 by the alveolar macrophages, suggesting that SP-A acts as an opsonin. This hypothesis was further strengthened by the observation that F(ab')2 fragments of immunoglobulin G directed against ...
Journal of immunology (Baltimore, Md. : 1950), 1999
Interaction of LPS with monocytes and neutrophils is known to occur via CD14 and is strongly enha... more Interaction of LPS with monocytes and neutrophils is known to occur via CD14 and is strongly enhanced by LPS-binding protein (LBP). Integrins as well as CD14 play a role in the interaction of erythrocytes (E) coated with LPS or whole Gram-negative bacteria with phagocytes. We reasoned that the density of LPS on a particle is an important determinant in these interactions. Therefore, E were coated with different concentrations of LPS (ELPS). The binding of these ELPS to neutrophils was evaluated by flow cytometry. Simultaneously, we measured fMLP receptor expression to evaluate neutrophil activation. ELPS only bound to neutrophils in the presence of LBP. Blocking CD14 inhibited both activation and binding, whereas blocking complement (C) receptor 3 (CR3) inhibited binding but not activation. TNF activation restored ELPS binding in CD14-blocked cells but not in cells in which CR3 was blocked. Salmonella minnesota did bind to neutrophils independent of CR3 or CD14. The addition of LBP ...
Polyneuropathy associated with IgM monoclonal gammopathy (IgM-PNP) is a slowly progressive, senso... more Polyneuropathy associated with IgM monoclonal gammopathy (IgM-PNP) is a slowly progressive, sensorimotor neuropathy. It is assumed that complement activation contributes to IgM-PNP pathogenesis. We investigated whether innate differences in complement activity of the classical and mannose binding lectin (MBL) pathways are associated with IgM-PNP or its severity. We measured complement activity using ELISA and determined MBL serumc oncentrations and MBL gene polymorphisms in 83 patients and 83 healthy controls. We did not observe differences between IgM-PNP patients and healthy controls nor associations with different disease severities. Differences in innate complement activity are not likely to explain susceptibility to or severity of IgM-PNP.
The Editorial team of JDUHS greatfully acknowledges the contribution of the following honorable r... more The Editorial team of JDUHS greatfully acknowledges the contribution of the following honorable reviewers who helped and guided to improving the quality of the manuscripts published / evaluated in the year 2014.
Lipopolysaccharide (LPS) is the major mediator of gram-negative septic shock. Molecules that bind... more Lipopolysaccharide (LPS) is the major mediator of gram-negative septic shock. Molecules that bind LPS and neutralize its toxic effects could have important clinical applications. We showed that serum amyloid P component (SAP) neutralizes LPS. A SAP-derived peptide, consisting of amino acids 27 to 39, inhibited LPS-mediated effects in the presence of human blood. In this study, we used a pepscan of overlapping 15-mer peptides and distinguished two additional LPS-binding regions within the SAP molecule, identified in the regions spanning amino acids 61 to 75 and 186 to 200. The corresponding SAP-derived peptides, pep61-75 and pep186-200, inhibited the binding of fluorescein isothiocyanate-labeled LPS to monocytes as efficiently as a bactericidal/permeability-increasing protein (BPI)-derived 15-mer peptide comprising amino acids 85 to 99. The same SAP-derived peptides very potently inhibited LPS-induced priming of phagocytes in human blood. Also, SAP-derived pep186-200 caused a prolong...
The absolute number of membraneexpressed CD14, the most important endotoxin receptor, on human mo... more The absolute number of membraneexpressed CD14, the most important endotoxin receptor, on human monocytes and neutrophils shows remarkable variation in the literature. To quantify these numbers two fluorescence methods using fluorescein isothiocyanate (FUff)-labeled monoclonal antibodies (mAb) were applied. A commercially available set of standard beads was used in flow cytometry to quantitate CD14 with eight different mAbs. Independent from their isotype the various mAbs showed minor differences and indicated that peripheral blood monocytes expressed 99,500-134,600 (115,400 ± 10,600) and neutrophils 1,900-4,400 (3,300 ± 800) CD14 receptors. There was no significant difference in CD14 expression on leukocytes in unprocessed freshly obtained whole blood and after a Ficoll isolation procedure. However, a short temperature shift resulted in a 1.3-to 1.6-fold up-regulation of CD14. The results obtained with the reference beads were verified with fluorescence Scatchard analysis and spectrofluorometry using itiAb 26ic-FflC and showed 109,500 CD14 per monocyte and 6,700 CD14 per neutrophil. For comparison the number of CD14 on the monocytic THP-1 cells and Fc'y-receptors on human leukocytes were determined using the reference beads and flow cytometry and gave results comparable to published data. Our data indicate that resting human monocytes express roughly 110,000 CD14 molecules on their surface using a simple fluorometric assay. Correct determination ofthe number ofCDl4 and other cell surface receptors is of importance in the monitoring of septic patients.
Journal of immunology (Baltimore, Md. : 1950), 1998
LPS-binding proteins in plasma play an important role in modifying LPS toxicity. Significant prop... more LPS-binding proteins in plasma play an important role in modifying LPS toxicity. Significant properties have already been attributed to the LPS-binding protein (LBP). It accelerates LPS toxicity as well as incorporation into high-density lipoproteins, leading to neutralization of LPS in serum. A search for other LPS-binding components in serum, using LPS-coated magnetic beads, revealed a new LPS-binding protein. N-terminal microsequencing identified this protein as serum amyloid P component (SAP). Purified SAP bound to smooth and rough types of LPS via the lipid A part. SAP inhibited the binding of FITC-labeled ReLPS (LPS from Salmonella minnesota strain R595) to human monocytes and the ReLPS-induced priming of the oxidative burst of human neutrophils only in the presence of low concentrations of LBP. In search for the LPS binding site of SAP, we found that pep27-39, a 13-mer peptide consisting of amino acids 27-39 of SAP, competitively inhibited the binding of LPS to SAP. In additi...
The building blocks of bacterial flagella, flagellin monomers, are potent stimulators of host inn... more The building blocks of bacterial flagella, flagellin monomers, are potent stimulators of host innate immune systems. Recognition of flagellin monomers occurs by flagellin-specific pattern-recognition receptors, such as Toll-like receptor 5 (TLR5) in mammals and flagellin-sensitive 2 (FLS2) in plants. Activation of these immune systems via flagellin leads eventually to elimination of the bacterium from the host. In order to prevent immune activation and thus favor survival in the host, bacteria secrete many proteins that hamper such recognition. In our search for Toll like receptor (TLR) antagonists, we screened bacterial supernatants and identified alkaline protease (AprA) of Pseudomonas aeruginosa as a TLR5 signaling inhibitor as evidenced by a marked reduction in IL-8 production and NF-kB activation. AprA effectively degrades the TLR5 ligand monomeric flagellin, while polymeric flagellin (involved in bacterial motility) and TLR5 itself resist degradation. The natural occurring alkaline protease inhibitor AprI of P. aeruginosa blocked flagellin degradation by AprA. P. aeruginosa aprA mutants induced an over 100-fold enhanced activation of TLR5 signaling, because they fail to degrade excess monomeric flagellin in their environment. Interestingly, AprA also prevents flagellin-mediated immune responses (such as growth inhibition and callose deposition) in Arabidopsis thaliana plants. This was due to decreased activation of the receptor FLS2 and clearly demonstrated by delayed stomatal closure with live bacteria in plants. Thus, by degrading the ligand for TLR5 and FLS2, P. aeruginosa escapes recognition by the innate immune systems of both mammals and plants.
Bacterial flagellin molecules are strong inducers of innate immune responses in both mammals and ... more Bacterial flagellin molecules are strong inducers of innate immune responses in both mammals and plants. The opportunistic pathogen Pseudomonas aeruginosa secretes an alkaline protease called AprA that degrades flagellin monomers. Here, we show that AprA is widespread among a wide variety of bacterial species. In addition, we investigated the role of AprA in virulence of the bacterial plant pathogen P. syringae pv. tomato DC3000. The AprA-deficient DC3000 ΔaprA knockout mutant was significantly less virulent on both tomato and Arabidopsis thaliana. Moreover, infiltration of A. thaliana Col-0 leaves with DC3000 ΔaprA evoked a significantly higher level of expression of the defense-related genes FRK1 and PR-1 than did wild-type DC3000. In the flagellin receptor mutant fls2, pathogen virulence and defense-related gene activation did not differ between DC3000 and DC3000 ΔaprA. Together, these results suggest that AprA of DC3000 is important for evasion of recognition by the FLS2 recepto...
Phagocytes play a major role in host defense against staphylococci as well as in the pathophysiol... more Phagocytes play a major role in host defense against staphylococci as well as in the pathophysiology of Gram-positive septic shock. In Gram negative sepsis, the main mediator, LPS exerts its effects as easily suspendable mediator. In Gram positive sepsis the main mediator is still not found, therefore we studied the interaction of soluble staphylococcal products with phagocytes. Staphylococcus aureus supernates
Leukocyte migration is a key event both in host defense against invading pathogens as well as in ... more Leukocyte migration is a key event both in host defense against invading pathogens as well as in inflammation. Bacteria generate chemoattractants primarily by excretion (formylated peptides), complement activation (C5a), and subsequently through activation of leukocytes (e.g., leukotriene B4, platelet-activating factor, and interleukin 8). Here we describe a new protein secreted by Staphylococcus aureus that specifically impairs the response of neutrophils and monocytes to formylated peptides and C5a. This chemotaxis inhibitory protein of S. aureus (CHIPS) is a 14.1-kD protein encoded on a bacteriophage and is found in >60% of clinical isolates. CHIPS reduces the neutrophil recruitment toward C5a in a mouse peritonitis model, even though its activity is much more potent on human than on mouse cells. These findings suggest a new immune escape mechanism of S. aureus and put forward CHIPS as a potential new antiinflammatory therapeutic compound.
Polymorphonuclear leukocytes (PMN) can mediate cytotoxic reactions against virus infected targets... more Polymorphonuclear leukocytes (PMN) can mediate cytotoxic reactions against virus infected targets cells. We observed very efficient binding of PMN to HSV-infected fibroblasts when loaded with HSV-specific antibodies. Using electron microscopy, infected fibroblasts were found to be totally surrounded by PMN and the phagocytosis of virions and fragments of infected cells was demonstrated. To quantify and study this phenomenon, and to compare PMN with monocytes, we developed radiometric and fluorometric phagocytosis assays. Leukocytes were mixed with [3H]glucosamine-or FITC-labeled virus and incubated at 37 °C. PMN associated radioactivity or fluorescence per cell as measured by flow cytometry was determined. PMN phagocytosis was dependent on the presence of specific anti-HSV antibodies and could be enhanced by addition of complement. Monocytes were also able to phagocytize virions; however, the rate of uptake was less than that for PMN. Under optimal conditions the total amount of herpes simplex particles that could be associated with one PMN or monocyte was about 10,000. PMN and monocytes are capable of phagocytosis of HSV. This may be an important factor in preventing the spread of infection in vivo.
Proceedings of the National Academy of Sciences, 2015
Toll-like receptors (TLRs) are crucial in innate recognition of invading micro-organisms and thei... more Toll-like receptors (TLRs) are crucial in innate recognition of invading micro-organisms and their subsequent clearance. Bacteria are not passive bystanders and have evolved complex evasion mechanisms. Staphylococcus aureus secretes a potent TLR2 antagonist, staphylococcal superantigen-like protein 3 (SSL3), which prevents receptor stimulation by pathogen-associated lipopeptides. Here, we present crystal structures of SSL3 and its complex with TLR2. The structure reveals that formation of the specific inhibitory complex is predominantly mediated by hydrophobic contacts between SSL3 and TLR2 and does not involve interaction of TLR2–glycans with the conserved LewisX binding site of SSL3. In the complex, SSL3 partially covers the entrance to the lipopeptide binding pocket in TLR2, reducing its size by ∼50%. We show that this is sufficient to inhibit binding of agonist Pam2CSK4 effectively, yet allows SSL3 to bind to an already formed TLR2–Pam2CSK4 complex. The binding site of SSL3 over...
Human adherent cells, obtained by EDTA reversible adherence to plastic, are potent effectors in c... more Human adherent cells, obtained by EDTA reversible adherence to plastic, are potent effectors in cell-mediated cytotoxicity. Spontaneous cytotoxicity in a 2-hr assay against K562 target cells was shown to be largely mediated by contaminating natural killer (NK) cells. Treatment of adherent cells with NK-specific monoclonal antibody anti-Leu-11 plus complement abolished almost completely the spontaneous cytotoxicity. Spontaneous cytotoxicity by adherent cells was also reduced when the phorbol ester PMA was present in the assay. On the other hand, PMA induced a cytotoxic response in NK-cell depleted adherent cells after prolonged 18 hr incubation. The cell population responsible for this dichotomous effect of PMA on adherent cell-mediated cytotoxicity was shown to be monocytes, as revealed by monoclonal antibody treatment. Pure NK cell preparations were not affected by PMA in their cytolytic capacities. Reactive oxygen species are not involved in NK-cell mediated cytotoxicity, while PM...
The specific binding of human polymorphonuclear leukocytes (PMN) to antibody-coated target cells ... more The specific binding of human polymorphonuclear leukocytes (PMN) to antibody-coated target cells was characterized by flow cytometry. PMN were labeled with phycoerythrin-E (PE) via a granulocyte-specific monoclonal antibody (leu-M1) and mixed with fluorescein isothiocyanate-labeled K562 tumor cells sensitized with rabbit antiserum. Specific conjugates were formed as analyzed by two-color fluorescence in a flow cytometer. The formation of stable conjugates was dependent on initiation of contact, temperature, time, and antiserum concentration. Studies with inhibitors implicate that microfilaments, but not microtubules, Ca2+, Mg2+, or energy-dependent processes were a prerequisite for binding of PMN to the antibody-coated target cells. No conjugates were formed when uncoated target cells were used or when the experiment was performed in the presence of protein A, indicating that binding was specifically mediated through Fc receptors (FcR). Monoclonal antibodies against the FcRII and Fc...
In the present study we used flow cytometry to investigate the phagocytosis of fluorescein isothi... more In the present study we used flow cytometry to investigate the phagocytosis of fluorescein isothiocyanate-labeled herpes simplex virus type 1 (FITC-HSV-1) by rat alveolar macrophages and the effects of surfactant protein A (SP-A) on this process. The phagocytosis of FITC-HSV-1 by alveolar macrophages, which was studied as a model for virus phagocytosis in general, was strongly enhanced in the presence of SP-A. The SP-A-mediated phagocytosis was time and concentration dependent, reaching a maximal level after 15 min of incubation and at an SP-A concentration of 5 micrograms/ml. Using a fluorescence quenching technique, we could show that at least 65% of the viruses were indeed internalized by the macrophages. The addition of SP-A to the system was sufficient for the phagocytosis of FITC-HSV-1 by the alveolar macrophages, suggesting that SP-A acts as an opsonin. This hypothesis was further strengthened by the observation that F(ab')2 fragments of immunoglobulin G directed against ...
Journal of immunology (Baltimore, Md. : 1950), 1999
Interaction of LPS with monocytes and neutrophils is known to occur via CD14 and is strongly enha... more Interaction of LPS with monocytes and neutrophils is known to occur via CD14 and is strongly enhanced by LPS-binding protein (LBP). Integrins as well as CD14 play a role in the interaction of erythrocytes (E) coated with LPS or whole Gram-negative bacteria with phagocytes. We reasoned that the density of LPS on a particle is an important determinant in these interactions. Therefore, E were coated with different concentrations of LPS (ELPS). The binding of these ELPS to neutrophils was evaluated by flow cytometry. Simultaneously, we measured fMLP receptor expression to evaluate neutrophil activation. ELPS only bound to neutrophils in the presence of LBP. Blocking CD14 inhibited both activation and binding, whereas blocking complement (C) receptor 3 (CR3) inhibited binding but not activation. TNF activation restored ELPS binding in CD14-blocked cells but not in cells in which CR3 was blocked. Salmonella minnesota did bind to neutrophils independent of CR3 or CD14. The addition of LBP ...
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