PeproTech Inc.
Originally published online as doi:10.1189/jlb.0803360 on December 12, 2003

Published online before print December 12, 2003
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0803360v1
75/3/467    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lotz, S.
Right arrow Articles by Laskay, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lotz, S.
Right arrow Articles by Laskay, T.
(Journal of Leukocyte Biology. 2004;75:467-477.)
© 2004 by Society for Leukocyte Biology

Highly purified lipoteichoic acid activates neutrophil granulocytes and delays their spontaneous apoptosis via CD14 and TLR2

Sonja Lotz*, Eresso Aga*, Inga Wilde*, Ger van Zandbergen*, Thomas Hartung{dagger}, Werner Solbach* and Tamás Laskay*,1

* Institute for Medical Microbiology and Hygiene, University of Lübeck, Germany; and
{dagger} Department of Biochemical Pharmacology, University of Konstanz, Germany

1Correspondence: Institute for Medical Microbiology and Hygiene, University of Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, Germany. E-mail: Tamas.Laskay{at}hygiene.ukl.mu-luebeck.de


arrow
ABSTRACT
 
Lipoteichoic acid (LTA) is a major component of the cell membrane of gram-positive bacteria. Although LTA has become increasingly recognized as an immunomodulator, its effect on polymorphonuclear neutrophil granulocytes (PMN) is still not clear. The interaction between LTA and PMN, however, is of particular importance, as PMN are the first leukocytes that migrate to the site of infection and encounter bacterial pathogens. In the present study, the interaction of highly purified human PMN with endotoxin-free LTA from Staphylococcus aureus was investigated. After exposure to LTA, neutrophil granulocytes acquired typical activated cell morphology. LTA had a marked activating effect on the functions of PMN as well. Shedding of CD62L, degranulation, and priming for formyl-Met-Leu-Phe-mediated oxidative burst were induced in PMN upon exposure to LTA. Moreover, LTA treatment induced the release of proinflammatory cytokines such as interleukin-8, tumor necrosis factor {alpha}, and granulocyte-colony stimulating factor by PMN. The effects of LTA on PMN were found to be associated with nuclear factor-{kappa}B activation. Of particular interest was that LTA inhibited the spontaneous apoptosis and therefore, increased the lifespan of PMN. Experiments using blocking antibodies revealed that CD14 and Toll-like receptor 2 (TLR2) but not TLR4 play a major role in LTA-mediated effects on PMN. These data clearly show that LTA, a component of gram-positive bacteria, directly activates neutrophil granulocytes, the primary effector cells in the first line of defense against infectious challenge.

Key Words: PMN • LTA • gram-positive bacteria • cellular activation • inflammation


arrow
INTRODUCTION
 
Polymorphonuclear neutrophil granulocytes (PMN) are phagocytic cells that participate in inflammatory reactions as a first line of defense against microbial pathogens. Whereas lipopolysaccharide (LPS) is considered a major component of gram-negative bacteria, which stimulates PMN functions [1 ], the counterpart of LPS in gram-positive bacteria is not well characterized. As lipoteichoic acid (LTA) is one of the major membrane components of gram-positive bacteria [2 ], several previous investigations dealt with the response of PMN to exposure to LTA [3 4 5 ]. However, two major obstacles in these earlier studies were the low purity of the LTA and the loss of biological activity of LTA during purification. Commercially available LTA was often contaminated with LPS or other immunostimulatory substances [6 ], making repurification steps necessary. Repurified LTA from Staphylococcus aureus, however, was biologically inactive in the systems tested [7 ]. The results obtained with various commercial preparations of LTA and with highly purified LTA, therefore, were contradictory and left the question open whether LTA had a direct immunostimulatory effect on PMN. Recently, a novel purification technique provided a highly purified, biologically active LTA preparation. The novel technique [8 ] is based on a gentle extraction procedure using butanol. The purification methods applied in previous studies included a phenol extraction step, which resulted in the decomposition of LTA as a result of the loss of its alanine substituents [8 ]. The bioactive LTA preparation used in our study was also shown to be free of impurities such as LPS [8 ].

Although the activity of pure LTA on many cell types has been studied [9 10 11 ], it is still not clear whether LTA plays a role in the activation of neutrophil granulocytes. In the present study, the reaction of highly purified neutrophil granulocytes to endotoxin-free and bioactive LTA was investigated. Our results indicated that LTA directly activates granulocytes, resulting in morphological changes, shedding of CD62L, degranulation, cytokine release, and priming for formyl-Met-Leu-Phe (fMLP)-mediated oxidative burst. It is most interesting that through delaying the spontaneous apoptosis, LTA treatment led to an extended lifespan of PMN. Blocking experiments revealed that CD14 and Toll-like receptor 2 (TLR2) participate in the LTA-induced effect on neutrophil granulocytes.


arrow
MATERIALS AND METHODS
 
Purification of LTA
LTA was purified from S. aureus as described previously [8 ]. Briefly, S. aureus (DSM 20233) was cultured aerobically in a 42-L fermentor (MBR Rio Reactor) at 37°C and harvested at an OD578 of 15 (extrapolated) in a continuous flow centrifuge, resuspended in 0.1 M citrate buffer, pH 4.7, and disrupted with glass beads in a Braun disintegrator. A defrosted aliquot of bacteria was mixed with an equal volume ofn-butanol (Merck, Darmstadt, Germany) under stirring for 30 min at room temperature (RT). After centrifugation at 13,000 g for 20 min, the aquatic phase was lyophilized, resuspended with chromatography start buffer (15% n-propanol in 0.1 M ammonium acetate, pH 4.7), and centrifuged at 45,000 g for 15 min. The supernatant was subjected to hydrophobic interaction chromatography on octyl-Sepharose. The purity of the LTA was over 99%, according to nuclear magnetic resonance and mass spectrometry [8 ]. Endotoxin contamination was less than 30 pg LPS/mg LTA as determined by the Limulus amoebocyte lysate assay (QCL-1000; BioWhittaker, Apen, Germany).

Isolation of neutrophil granulocytes
Peripheral blood was collected by venipuncture from healthy adult volunteers using lithium-heparin. Granulocytes were isolated as described previously [12 , 13 ]. Briefly, blood was layered on a two-layer Histopaque gradient consisting of Histopaque 1077 (upper layer) and Histopaque 1119 (bottom layer; Sigma, Taufkirchen, Germany) and centrifuged for 5 min at 300 g followed by 20 min at 800 g. The plasma and the Histopaque 1077 layer consisting mainly of lymphocytes and monocytes were discarded. The granulocyte-rich layer in the Histopaque 1119 layer was collected, leaving the erythrocyte pellet at the bottom of the tube. Granulocytes were washed twice in phosphate-buffered saline (PBS), resuspended in complete medium (RPMI-1640 medium supplemented with 50 µM 2-mercaptoethanol, 2 mM L-glutamine, 10 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin, all from Biochrom, Berlin, Germany) and 10% low endotoxin fetal calf serum (FCS; Gibco, Karlsruhe, Germany; LPS content, 0.523 ng/ml), and further fractionated on a discontinuous Percoll (Amersham Biosciences, Uppsala, Sweden) gradient consisting of layers with densities of 1.105 g/ml (85%), 1.100 g/ml (80%), 1.093 g/ml (75%), 1.087 g/ml (70%), and 1.081 g/ml (65%). After centrifugation for 20 min at 800 g, the interface between the 80% and 85% Percoll layers was collected, washed twice in PBS, and resuspended in complete medium. All procedures were conducted at RT. Cell viability was >99%, as determined by trypan blue exclusion. The cell preparations contained >99% granulocytes, of which >95% were neutrophils, and 1–4% were eosinophils, as determined by morphological examination of Giemsa-stained cytocentrifuge (Shandon, Pittsburgh, PA) preparates.

As monocytes are known to respond to LTA with cytokine release [9 ], the number of contaminating monocytes in the granulocyte preparations was determined using an immunofluorescent staining. Freshly isolated neutrophil granulocytes (5x105 cells) were resuspended in fluorescein-activated cell sorter (FACS) buffer (PBS containing 1% human serum, 1% bovine serum albumin, and 0.01% sodium azide) in a V-bottom 96-well plate. After washing with FACS buffer, PMN were stained with a phycoerythrin (PE)-conjugated monoclonal antibody (mAb) to CD14 (clone TÜK4, Dako, Hamburg, Germany). Following two washing steps, the cells were fixed with paraformaldehyde (1% in PBS). Cytocentrifuge preparates were embedded in IMAGEN® mounting fluid (Dako), and the cells were observed under a Zeiss Axioskop2 fluorescent microscope. Monocytes that have a high expression of CD14 could be detected as brightly stained red cells. After counting cells in 50 high power fields (10,000 cells), an average of two monocytes was detected, which corresponds to a monocyte contamination of 0.02%.

Cell culture
PMN were incubated in complete medium (see above) at a concentration of 5 x 106/ml at 37°C in a humidified atmosphere containing 5% CO2 in tissue-culture plates with 96 flat-bottom wells (Greiner, Frickenhausen, Germany). All components of the cell-culture medium were of low endotoxin content. The complete cell-culture medium contained between 50 and 100 pg/ml endotoxin as determined by the Limulus amoebocyte lysate assay (Charles River Laboratories, Sulzfeld, Germany). LTA was used in a concentration range of 0.3–30 µg/ml. As the LTA preparation had an endotoxin content as low as 30 pg/mg LTA, LTA contributed solely 0.9 pg LPS/ml culture medium, even in the highest concentration (30 µg/ml) used. This endotoxin level is far below the endotoxin content of the culture medium and therefore, cannot account for the biological effects of the LTA preparation. LPS from Escherichia coli 0111:B4 (Sigma) was used at a concentration of 100 ng/ml. The morphology of PMN in the cell culture was visualized under an invert microscope (Zeiss Axiovert 25) and photographed with a Ricoh HR-10m camera (Ricoh, Tokyo, Japan).

Blocking experiments
Blocking experiments were performed to investigate the role of CD14, TLR2, and TLR4 in the LTA-mediated effects on PMN. Freshly isolated PMN were pretreated with 10 µg/ml anti-CD14 mAb [immunoglobulin G (IgG)1; clone 18D11, Diatec, Hamburg, Germany], 10 µg/ml anti-TLR2 mAb (IgG2a, clone TL2.1, Imgenex, San Diego, CA), and 10 µg/ml anti-TLR4 mAb (IgG2a, clone HAT125, Imgenex) for 30 min at RT and subsequently exposed to LTA. As isotype controls, 10 µg/ml IgG1 (Diatec) or 10 µg/ml IgG2a (eBioscience, San Diego, CA) was used.

Flow cytometry analysis
The cell-surface expression of CD66b as a specific marker for granulocytes [14 ] and of CD62L and CD11b as markers for PMN activation was analyzed [15 ]. PMN (5x105) were resuspended in FACS buffer (see above) in a V-bottom 96-well plate. After washing with FACS buffer, PMN were stained with fluorescein isothiocyanate (FITC)-conjugated mAb to human CD66b (clone 80H3, IgG1, Immunotech, Hamburg, Germany) and PE-conjugated mAb to CD62L (clone Dreg-56, IgG1, BD Biosciences, Heidelberg, Germany) or PE-conjugated mAb to CD11b (clone 2LPM19c, IgG1, Dako) in FACS buffer for 20 min on ice. Following two wash steps, the cells were fixed with paraformaldehyde (1% in PBS) and analyzed with a FACSCalibur® flow cytometer using CellQuest® software (Becton Dickinson, San Diego, CA). FITC- and PE-conjugated mouse IgG1 isotype-control antibodies were purchased from Dako.

Analysis of intracellular interleukin (IL)-8 by flow cytometry
PMN were cultured at a concentration of 5 x 106/ml in complete medium for 3 h before Brefeldin A (GolgiPlug®, BD Biosciences) was added, and the cultures were further incubated for 12 h. Subsequently, the cells were stained with FITC-conjugated anti-CD66b mAb (Immunotech) followed by permeabilization and intracellular staining of IL-8 using the Cytofix/Cytoperm Plus kit (BD Biosciences) and PE-labeled anti-IL-8 mAb (clone G 265-8, IgG2b, BD Biosciences), respectively. PE-labeled mouse IgG2b (BD Biosciences) was used as an isotype control. The cells were analyzed with a FACSCalibur® flow cytometer using CellQuest® software (Becton Dickinson).

Determination of cytokines in culture supernatants
Cell-free supernatants of PMN cultures were collected and stored at –20°C until cytokine determination. IL-8 was measured using an enzyme-linked immunosorbent assay (ELISA; CytoSets, Biosource, Camarillo, CA), according to the manufacturer’s instructions. The detection limit was 30 pg/ml. Granuloctye-colony stimulating factor (G-CSF) was determined by using the Quantikine HS immunoassay (R&D Systems, Wiesbaden-Nordenstadt, Germany) with a detection limit of 1 pg/ml. Tumor necrosis factor {alpha} (TNF-{alpha}) was analyzed by using the Opteia human TNF-{alpha} set (BD Biosciences) with a detection limit of 3 pg/ml.

Oxidative burst assay
The intracellular production of reactive oxygen radicals was assayed by using the substrate dihydrorhodamine 123 (DHR; Molecular Probes, Leiden, Netherlands), which is fluorescent upon interaction with reactive oxygen species (ROS; H2O2 and HO·) [16 ].

To investigate the priming effect of LTA on fMLP-induced oxidative burst, 1 x 106/200 µl PMN were pretreated with various concentrations of LTA for 90 min or without LTA in RPMI + 1% FCS. Then, DHR was added for 5 min followed by stimulation with 1 µM fMLP (Sigma) for 5 min. The reaction was stopped on ice, and the fluorescent intensity of the cells was analyzed immediately by flow cytometry.

Analysis of PMN apoptosis
Morphological assessment of PMN apoptosis
In apoptotic neutrophils, morphological changes are striking and include separation of nuclear lobes and darkly stained pyknotic nuclei [17 , 18 ]. Nuclear morphology was assessed on Giemsa-stained cytocentrifuge slides. Cell morphology was examined under oil immersion light microscopy. A minimum of 200 cells/slide was examined and graded as apoptotic/nonapoptotic.

Annexin-V binding
Annexin-V exhibits calcium-dependent binding to phosphatidylserine (PS), expressed in the outer membrane leaflet of apoptotic cells [19 ]. Labeling of apoptotic PMN with Annexin-V–FITC (Roche Molecular Biologicals, Mannheim, Germany) was performed as recommended by the manufacturer. Labeled cells were analyzed by flow cytometry using a FACSCalibur® with CellQuest® software (Becton Dickinson).

Deoxyuridine triphosphate (dUTP) nick end-labeling (TUNEL) assay of chromatin fragmentation
The TUNEL assay with fluorescein-dUTP (in situ cell death detection kit, Roche Molecular Biologicals) was used to detect DNA strand breaks in apoptotic cells [20 ]. Briefly, freshly isolated PMN (106 cells per probe) were incubated for 24 h in the presence or absence of LTA. The cells were fixed with 4% paraformaldeyde in PBS and incubated for 1 h on a shaker at RT followed by a washing step. The cells were then permeabilized with 100 µl 0.1% Triton X-100 in 0.1% sodium citrate for 2 min on ice. After washing twice, the cells were resuspended in 50 µl TUNEL reaction mixture or in LABEL solution as negative control for 60 min at 37°C in a humidified atmosphere in the dark. The cells were washed again and resuspended in 400 µl PBS and analyzed by flow cytometry (see above).

Western blot analysis of phosphorylated inhibitor of {kappa}B (P-I{kappa}B)
PMN were cultured in the absence or presence of 10 or 30 µg/ml LTA for 30, 90, or 150 min. After adding ice-cold RPMI-1640 medium, the cells were pelleted at 800 g for 5 min at 4°C. Cell pellets were washed and dissolved in 2x sample buffer [50 mM Tris, pH 6.80, 4% sodium dodecyl sulfate (wt/vol), 10% 2-mercaptoethanol (vol/vol), 20% glycerol (vol/vol)], containing a protease and phosphatase-inhibitor cocktail [1 mM EDTA (Sigma), 0.5 mM EGTA (Sigma), 1 mM phenylmethylsulfonyl fluoride (Sigma), and 10 µg/ml each of aprotinin (Sigma), leupeptin (Calbiochem, Schwalbach, Germany), pepstatin-A (Sigma), 0.5 mM dithiothreitol (Sigma), 2 mM levamisol (Sigma), 0.5 mM benzamidin (Sigma), 1 mM sodium orthovanadate (Sigma), 10 mM ß-glycerophosphate (Sigma), and complete miniprotease inhibitor (Roche Molecular Biologicals; one tablet dissolved in 1 ml distilled water and used in a final dilution of 1:5]. Samples were then heated for 7 min at 95°C, and equivalents of 1 x 106 cells/lane were loaded in 15% denaturing polyacrylamide gels.

Proteins were blotted onto a nitrocellulose membrane at 21 V constant voltage for 60 min in a Transblot® semidry transfer cell (BioRad, Hercules, CA). The membrane was blocked in PBS containing 0.05% Tween-20 and 3% low-fat skimmed milk for 60 min at RT and was subsequently incubated with a 1:500 dilution of mouse antiphospho-I{kappa}B (IgG1) antibody (clone IMG-156, Biocarta, Hamburg, Germany) overnight at 4°C. After extensive washing, the membranes were incubated with a horseradish peroxidase-coupled polyclonal goat anti-mouse-IgG secondary antibody (Dako) for 90 min at RT, and the bands were visualized using the chemiluminescent Western blotting detection system (SuperSignal® West Dura extended duration substrate, Pierce, Bonn, Germany). To control equal protein load, the membranes were stripped and incubated with a 1:5000 dilution of anti-ß-actin antibody (clone AC-74, Sigma) for 90 min at RT followed by chemiluminescence detection as described above.

Statistical analysis
Data are expressed as mean ± SD. Statistical significance was assessed by the paired or unpaired Student’s t-test. Throughout the figures, P values of <0.05 are indicated by one asterisk; P values of <0.01 by double asterisks.


arrow
RESULTS
 
Exposure of neutrophils to LTA results in changes in cell morphology
Neutrophil activation is associated with marked changes in the cell morphology. Typically, shortly after activation, PMN appear as elongated, motile cells [21 , 22 ]. In addition, enhanced expression of adhesion molecules on activated PMN leads then to formation of aggregates [23 ]. PMN incubated for 2 h in the presence of 10 µg/ml LTA had the morphology of activated cells, i.e., elongated shape with a rough surface (Fig. 1B ). In contrast, PMN incubated in medium alone showed a round form and a smooth surface (Fig. 1A) . No aggregate formation could be observed in cultures of PMN incubated overnight in medium alone (Fig. 1C) . In the presence of LTA, the cells formed aggregates (Fig. 1D) . The morphological changes were identical to those observed after stimulation of PMN with 100 ng/ml LPS (data not shown) [24 ]. These morphological observations clearly show that LTA induces the activation of highly purified PMN.



View larger version (139K):
[in this window]
[in a new window]
  		Figure 1. PMN acquire activated cell shape after exposure to LTA. PMN (5x106/ml) were incubated for 2 h (A, B) or 24 h (C, D) in medium alone (A, C) or in the presence of 10 µg/ml LTA (B, D; original magnification, 400x).

Treatment with LTA leads to CD62L shedding on neutrophils
To support the morphological data, CD62L expression was monitored on PMN after exposure to LTA. As activation of PMN leads to shedding of L-selectin (CD62L) from the cell surface [15 ], analysis of CD62L expression is a widely used method to investigate PMN activation [25 ]. Freshly isolated granulocytes had a high level of CD62L expression on the cell surface (Fig. 2 ). The majority of PMN remained CD62L-positive after in vitro culture in medium alone. In the presence of LTA, however, PMN lost CD62L surface expression progressively in a concentration-dependent manner after 3 and 6 h (Fig. 2) .



View larger version (49K):
[in this window]
[in a new window]
  		Figure 2. Coincubation with LTA leads to the down-regulation of CD62L expression. PMN were stained with anti-CD62L-PE (FL2) antibody and analyzed by flow cytometry. Dot blots of freshly isolated PMN (0 h) and PMN incubated for 3 or 6 h in medium alone or with 3 or 10 µg/ml LTA are shown. The dot plots show results of a representative experiment. The percentages of CD62L-negative cells of the representative experiment and in brackets, the mean ± SD of three experiments are given. Significant differences in induction of CD62L shedding are indicated. SSC, Side-scatter.

LTA up-regulates the expression of CD11b and CD66b on PMN
Granule exocytosis is an important function of activated neutrophils [26 ]. During this process, the granule membrane incorporates into the cell membrane. Consequently, increasing expression of granule-membrane markers such as CD11b [27 ] and CD66b [14 ] can be observed on the cell surface of activated PMN. Freshly isolated PMN were found to express CD11b on their surface (Fig. 3A ). The presence of LTA (10 µg/ml) in the culture medium led to a marked up-regulation of this surface marker (Fig. 3A) .



View larger version (32K):
[in this window]
[in a new window]
  		Figure 3. LTA treatment leads to enhanced CD11b and CD66b expression on PMN, which were stained with anti-CD11b-PE (A) or anti-CD66b-FITC (B) antibody or with an isotype-control antibody (gray-filled histograms) and analyzed by flow cytometry. Left panel, Freshly isolated PMN; right panel, PMN incubated for 6 h at a cell concentration of 5 x 106/ml in medium alone (broken line) or with 10 µg/ml LTA (solid line). The histograms and mean fluorescence intensities (MFI) are from one representative experiment. In brackets, mean ± SD of the MFI of three to four experiments is given. Significant up-regulations of the expression after LTA treatment are indicated.

CD66b is a specific marker for granulocytes [14 ]. As this molecule is also stored in the granules of PMN, granule exocytosis leads to increased cell-surface expression of CD66b [14 ]. After coincubation with LTA, the CD66b expression was up-regulated on PMN (Fig. 3B) . The up-regulation of CD11b and CD66b demonstrates that LTA induced the degranulation of PMN and therefore indicates that LTA potently activates neutrophil granulocytes.

LTA alone does not induce oxidative burst in PMN but primes for fMLP-induced production of reactive oxygen intermediates (ROI)
The ability to induce the production of ROI is characteristic for many neutrophil-activating agents [26 ]. Hence,we analyzed the ability of LTA to induce an oxidative burst in human PMN. Intracellular production of ROI was assayed by using DHR, which is fluorescent on interaction with ROS. PMN were incubated in medium alone or with LTA, which did not induce intracellular production of ROI (not shown).

A characteristic feature of low-level PMN activation is the priming, i.e., the amplification of the neutrophil’s functional response to other stimuli [28 ]. LPS, for example, has a priming effect on the fMLP-mediated oxidative burst of PMN [29 ]. fMLP alone induced a relatively weak oxidative response, where only 34% of PMN contained ROI. Preincubation with 10 µg/ml LTA markedly increased the number of positive cells (Fig. 4 ). These data show that although LTA alone does not induce an oxidative response in PMN, treatment with LTA results in priming of PMN to fMLP-induced oxidative burst.



View larger version (18K):
[in this window]
[in a new window]
  		Figure 4. LTA primes PMN for fMLP-induced oxidative burst. (A) PMN were incubated at 5 x 106/ml for 90 min in culture medium followed by a 5-min incubation in the presence (solid line) or absence (dotted line) of 1 µM fMLP. (B) PMN were incubated at 5 x 106/ml for 90 min in culture medium containing 10 µg/ml LTA followed by a 5-min incubation in the presence (solid line) or absence (dotted line) of 1 µM fMLP. Intracellular production of ROI was assessed by using the fluorogenic substrate DHR and measured by flow cytometry. The histograms show a representative experiment. The percentages of PMN containing ROI from a representative experiment and in brackets, the mean ± SD of four experiments are given. LTA-mediated significant difference in the ratio of burst-positive cells is given.

LTA induces cytokine secretion by PMN
Upon activation, PMN acquire the capacity to produce and release several cytokines including IL-8, TNF-{alpha}, and G-CSF [30 ]. Although IL-8 is produced by a variety of cell types, neutrophil granulocytes are the major source of this proinflammatory cytokine [30 ]. The release of IL-8 by neutrophils has been proven as a very sensitive method to detect low-level activation of neutrophils, where the stimulation is not strong enough to activate the oxidative burst [31 ].

PMN were incubated for 24 h with various concentrations of LTA. PMN in medium alone released low levels of IL-8. In the presence of LTA, a dose-dependent induction of IL-8 release was observed (Fig. 5 ). The amounts of IL-8 released after exposure to 10 µg/ml LTA were comparable with those measured in PMN cultures after coincubation with 100 ng/ml LPS (not shown).



View larger version (10K):
[in this window]
[in a new window]
  		Figure 5. LTA treatment induces the secretion of IL-8, TNF-{alpha}, and G-CSF by PMN. Neutrophils were incubated at 5 x 106/ml for 24 h with different concentrations of LTA. The IL-8, TNF-{alpha} and G-CSF content in the supernatants were measured by ELISA. The results are mean ± SD of three to four experiments. Significant increases in the amount of cytokine after stimulation are indicated.

The above data could also be verified by intracellular staining of IL-8 in PMN. In line with previous reports [32 ], we observed that freshly isolated PMN contain intracellular IL-8 (not shown). As compared with PMN incubated in medium alone, a dose-dependent increase of intracellular IL-8 was observed after exposure to LTA for 15 h (Fig. 6 ). These data indicate that treatment with LTA leads to the synthesis of IL-8 rather than simply inducing the release of preformed IL-8 by PMN.



View larger version (28K):
[in this window]
[in a new window]
  		Figure 6. LTA induces synthesis of IL-8 in PMN. Neutrophil granulocytes were cultured at 5 x 106/ml for 3 h in the absence (broken line) or presence of 3 µg/ml LTA (gray line) or 10 µg/ml LTA (black line). Brefeldin A was then added to the cultures, and the cells were further incubated for an additional 12 h followed by intracellular staining with anti-IL-8 mAb or isotype-control mAb (gray-filled histogram) and analyzed by flow cytometry as described in Materials and Methods. The MFI values of IL-8 staining are given. The results are representative of two experiments.

In addition to IL-8, LTA treatment of PMN resulted in the release of TNF-{alpha} and G-CSF as well (Fig. 5) .

CD14 and TLR2 play a major role in the LTA-mediated activation of PMN
Having established a direct, stimulatory effect of LTA on PMN, we addressed the question of LTA-binding receptors on PMN. In a previous study, CD14 was found to be involved in the LTA-induced effects on monocytes [9 , 10 ]. As PMN express CD14, we tested whether blockage of the CD14 receptor affects the response of PMN to LTA. PMN were pretreated for 30 min with the anti-CD14 mAb and subsequently exposed to 10 µg/ml LTA. CD14 blockage prevented the LTA-induced shedding of CD62L (Fig. 7A ) and markedly inhibited the LTA-induced IL-8 release (Fig. 7B) by human PMN. These data indicate that CD14 plays a major role in the LTA-mediated activation of PMN.



View larger version (27K):
[in this window]
[in a new window]
  		Figure 7. LTA-mediated effects on PMN depend on CD14 and TLR2. (A) Anti-CD14 prevents the shedding of CD62L. Freshly isolated neutrophils were stained with anti-CD62L-PE (FL2) antibody and analyzed by flow cytometry. Neutrophils were incubated at 5 x 106/ml for 30 min at RT in medium or with anti-CD14 mAb (10 µg/ml) or isotype control. Subsequently, the cells were cultured for 3 h in medium alone or in the presence of 10 µg/ml LTA. The dot plots show results from a representative experiment. The percentages of CD62L-negative cells are given for the representative experiment and in brackets, the mean ± SD of three experiments. (B) Anti-CD14 and anti-TLR2 attenuate the LTA-induced IL-8 release by PMN. Neutrophils (5x106/ml) were pretreated with 10 µg/ml anti-CD14 mAb, anti-TLR2 mAb, anti-TLR4 mAb, and IgG1 and IgG2a isotype-control mAb or without antibody for 30 min at RT before exposure to 10 µg/ml LTA for 24 h. ELISA measured the IL-8 content in the supernatant. Results are mean ± SD of three experiments. Significant reductions of IL-8 release between treatments with control versus specific antibodies are indicated. (C) Blocking CD14 and TLR2 reduces the antiapoptotic effect of LTA. Neutrophils (5x106/ml) were preincubated with anti-CD14 mAb, anti-TLR2 mAb, and anti-TLR4 mAb with IgG1 and IgG2a isotype-control mAb or in the absence of antibody for 30 min at RT before exposure to 10 µg/ml LTA for 24 h. The ratio of apoptotic PMN was determined by Annexin-V staining as described in Materials and Methods. The data show the LTA-mediated inhibition of spontaneous PMN apoptosis after the given pretreatments. Results are mean ± SD of three to four experiments. Significant reductions of apoptosis inhibition between treatments with control versus specific antibodies are indicated.

In addition to CD14, TLR2 was reported to be required for LTA-mediated cytokine production in monocytes and macrophages [33 ]. Therefore, blocking experiments were performed to investigate the role of TLR2 and TLR4 on the LTA-induced IL-8 release by neutrophil granulocytes. PMN were pretreated for 30 min with the anti-TLR2 and anti-TLR4 mAb and subsequently exposed to 10 µg/ml LTA. Blocking TLR2 but not TLR4 had a marked inhibitory effect on the LTA-induced IL-8 release (Fig. 7B) by human PMN, indicating that in addition to CD14, TLR2 is also involved in the LTA-mediated effects on PMN.

Coincubation with LTA inhibits the apoptosis of PMN in a CD14- and TLR2-dependent manner
Neutrophils undergo constitutive apoptosis when aged in vitro. Aging granulocytes exhibit classical features of apoptosis such as cell shrinkage, cytoplasmic condensation, and condensation of nuclear heterochromatin [17 , 18 ]. Accordingly, neutrophil apoptosis can be assessed by various parameters including changes in cellular morphology. Using these criteria, the percentage of apoptotic cells was determined in highly purified granulocytes cultured in vitro in the absence or presence of LTA. The ratio of apoptotic cells was strongly reduced when neutrophils were coincubated with LTA (Fig. 8A ). LTA protected neutrophils from apoptosis without evidence of significant necrotic death as assessed by trypan blue exclusion (not shown).



View larger version (24K):
[in this window]
[in a new window]
  		Figure 8. Exposure to LTA inhibits the spontaneous apoptosis of PMN. (A) PMN (5x106/ml) were cultured for 24 h, 48 h, and 72 h without or with 10 µg/ml LTA. The percentage of apoptotic PMN was determined by microscopial evaluation of >200 cells on cytocentrifuge preparations stained with Giemsa. Results are mean ± SD of three experiments. Significant differences between LTA-treated and nontreated cultures at each time point are indicated. (B) PMN apoptosis was detected by TUNEL assay in freshly isolated PMN (left panel), in PMN incubated for 24 h in medium alone (middle panel), or in PMN incubated for 24 h with 10 µg/ml LTA (right panel). The DNA strand breaks were labeled with a fluorescent marker using TUNEL technique and analyzed by flow cytometry. A representative experiment is shown. The percentages of TUNEL-positive apoptotic cells are given for a representative experiment and in brackets, the mean ± SD of three experiments. Significant differences between LTA-treated and nontreated cells are indicated. (C) Annexin-V binding of freshly isolated PMN (left panel), of PMN incubated for 24 h without (middle panel), or of PMN with 10 µg/ml LTA (right panel). The cells were stained with Annexin-V–FITC and analyzed by flow cytometry. Histograms from a representative experiment are shown. The percentages of Annexin-V-positive apoptotic cells are given for the representative experiment and in brackets, the mean ± SD of five experiments. Significant differences between LTA-treated and nontreated cells are indicated.

The delay of PMN apoptosis by LTA was further substantiated by the use of the TUNEL assay, which discloses the apoptotic fragmentation of nuclear DNA. After incubation for 24 h in medium alone, 54% of the neutrophils were apoptotic (TUNEL-positive) in comparison with only 27% in the presence of 10 µg/ml LTA (Fig. 8B) .

An early marker of apoptosis is the appearance of PS on the outer membrane, a process that is called membrane flip-flop [19 ]. PS can be detected by staining with Annexin-V. As shown in Figure 7B , freshly isolated neutrophil granulocytes are PS-negative. Sixty-two percent of the cells became Annexin-V-positive after 24 h of incubation in medium alone (Fig. 8C) . Coincubation with LTA (10 µg/ml) resulted in a marked decrease in the ratio of Annexin-V-positive cells (Fig. 8C) .

Taken together, our data, obtained with morphological analysis, Annexin-V staining, and TUNEL assay, show that LTA protects neutrophils from spontaneous apoptosis. In an independent set of experiments, we could demonstrate that the antiapoptotic effect of LTA lasted for several days. After incubation with 10 µg/ml LTA for 2 days, less than half of the cells were apoptotic, whereas nearly all PMN in the control culture had apoptotic nuclear morphology (Fig. 8A) . Even after 3 days, when all PMN in the control cultures had an apoptotic nuclear morphology, 34% of the LTA-treated cells were still nonapoptotic (Fig. 8A) .

Stimulation of PMN with granulocyte macrophage (GM)-CSF and interferon-{gamma} (IFN-{gamma}) has been reported to delay the PMN apoptosis [34 ]. It is more important that after treatment with these cytokines for 3 days, PMN were reported to express major histocompatibility complex class II (MHC-II) molecules [35 ], which could be confirmed in our laboratory. We tested whether LTA-mediated activation leads to MHC-II expression on PMN. However, LTA in a concentration of 10 µg/ml, neither alone nor in combination with IFN-{gamma}, induced MHC-II expression by PMN (data not shown).

As CD14 and TLR2 were found to be involved in the LTA-induced IL-8 release by PMN, blocking experiments were performed to investigate whether the same receptors participate in the antiapoptotic effect of LTA. PMN were pretreated for 30 min with anti-CD14, anti-TLR2, and anti-TLR4 mAb and were subsequently exposed to 10 µg/ml LTA. The rate of Annexin-V-positive apoptotic PMN was determined after 24 h. The antiapoptotic effect of LTA was diminished after blocking CD14 and TLR2 but not TLR4 (Fig. 7C) . These data show that CD14 and TLR2 are involved in the LTA-mediated, antiapoptotic effect on human PMN.

LTA treatment leads to nuclear factor (NF)-{kappa}B activation in neutrophil granulocytes
The above data clearly demonstrated activation of PMN by LTA. Although the NF-{kappa}B pathway is a major way of PMN activation [36 ], NF-{kappa}B-independent mechanisms of PMN activation have been also described [37 ]. Therefore, we tested whether NF-{kappa}B was involved in the LTA-induced effects on PMN functions. As a marker for NF-{kappa}B activation, the amount of P-I{kappa}B was assessed by Western blotting. Dose-dependent I{kappa}B phosphorylation was observed in LTA-treated PMN within the time period of 30–150 min (Fig. 9 ). Therefore, it is evident that the NF-{kappa}B pathway is involved in the activation of PMN through LTA.



View larger version (58K):
[in this window]
[in a new window]
  		Figure 9. LTA treatment results in I{kappa}B phosphorylation in PMN, which were incubated at a concentration of 5 x 106/ml for 30, 90, and 150 min with or without LTA (10 µg/ml; A) or 30 µg/ml (B). P-I{kappa}B in the cell lysates was analyzed by Western blot. Subsequently, the membranes were stripped and reprobed for ß-actin.


arrow
DISCUSSION
 
In this article, we report the direct activation of human PMN by LTA. We present data that LTA is able to induce in PMN morphological changes, shedding of CD62L, degranulation, and cytokine release. Moreover, LTA treatment leads to priming of PMN to fMLP-induced oxidative burst, but LTA alone does not induce oxidative burst in PMN. Exposure to LTA delays the spontaneous apoptosis, resulting in an extended lifespan of PMN. We present evidence that CD14 and TLR2 play major roles in LTA-induced effects on PMN, which involves the activation of NF-{kappa}B.

The highly purified, biologically active LTA preparation used in our present study was previously shown to stimulate the cytokine production of monocytes [9 ]. These cytokines include the proinflammatory cytokines TNF-{alpha}, IL-1ß, IL-6, and IL-8, which are known to possess the capacity to stimulate PMN. The goal of our study was to investigate the direct effects of LTA on PMN. Therefore, indirect effects of LTA mediated by monocytes had to be ruled out; e.g., special care had to be taken to avoid monocyte contamination. Our technique for PMN isolation resulted in a highly pure granulocyte population containing solely two or less monocytes per 10,000 granulocytes. The marked biological effect of LTA on this highly pure PMN population argues strongly against a possible indirect effect mediated by soluble factors released by contaminating leukocytes other then PMN.

In our in vitro studies, LTA concentrations of 0.3–10 µg/ml had detectable, stimulatory activity on PMN functions. This concentration is relatively high as compared with the 200 ng/ml, which activates the release of TNF-{alpha} in peripheral blood mononuclear cell cultures [10 ]. However, similar differences were reported between monocytes and granulocytes concerning their responsiveness to LPS. Whereas 10–100 ng/ml LPS can stimulate monocytes [38 ], a concentration as high as 1 µg/ml is the generally used LPS concentration for the direct activation of PMN [39 , 40 ]. This is thought to be associated with the low-level expression of the LPS-binding receptors on PMN. For example, only 3300 CD14 molecules were found on a single PMN, whereas over 100,000 were on a monocyte [41 ]. As CD14 is also a receptor for LTA [10 ], this difference may explain the relatively low response of PMN to LTA.

LTA concentrations can reach high levels at infectious sites with high bacterial load. LTA concentrations up to 10 µg/ml were detected in cultures of S. aureusin vitro [42 ]. Moreover, a previous study showed that the local concentration of LTA and TA can reach a level as high as 26 µg/ml in infected tissues in vivo [43 ]. This is presumably associated with the fact that 107 gram-positive bacteria contain as much as 1 µg LTA, whereas 107 gram-negative contain solely 20 ng LPS [44 ].

In infected tissues, however, LTA represents only one of several stimuli that act on PMN. Additional bacterial products such as fMLP, CpG DNA, and flagellin can all be recognized by pattern-recognition receptors. These various factors often act in a synergistic way, as demonstrated for muramyldipeptide with LTA [45 ]. Moreover, in infected tissues, various cell types, which can act in a synergistic way when acting together or in concert with microbial components such as LPS or LTA, release cytokines [46 ]. Such interactions, however, were not analyzed in our present work. We used a highly purified population (>99.9%) of PMN to investigate the direct effect of LTA on PMN and to rule out that the observed activating effect was a result of indirect activation mediated by LTA-induced products of other cells such as monocytes.

Neutrophil granulocytes contain a large number of granules (hence, granulocytes), which based on their content, can be divided into azurophil, specific, gelatinase granules as well as secretory vesicles. The membrane of specific granules serves as a reservoir of receptors and membrane-bound proteins involved in cell adhesion, signal transduction, and activation of microbicidal pathways [47 ]. The fusion of granules with the cell membrane during degranulation changes the surface expression of these membrane receptors [26 ], and this principle has been used in this study to measure and quantify neutrophil degranulation using flow cytometry. We demonstrated the enhanced cell-surface expression of CD11b and CD66b on PMN upon exposure to LTA. Using this approach, we could clearly show that treatment with LTA results in PMN granule exocytosis, which is a clear sign of PMN activation.

After being recruited to the site of infection, PMN recognize and phagocytose gram-negative [48 ] and -positive bacteria [49 ], resulting in the activation of these antimicrobial effector cells. PMN activation includes a broad range of phenotypic and functional changes, which occur in several stages [26 , 50 ]. A low level of activation leads to events such as the modulation of receptor expression on the cell surface and release of cytokines. Such low-level activation results also in the "priming" of PMN [25 ]; i.e., the cells become highly responsive to subsequent stimuli. Functions that require higher levels of activating stimuli include the oxidative burst. Some activating agents cannot evoke all functions [31 ]. LPS, for example, as a single stimulus does not have the capacity to induce oxidative burst, albeit it can induce activation-associated changes in the surface-receptor expression, prime neutrophils for enhanced induction of ROS by another activating stimulus, and mediate cytokine release [25 ]. LTA treatment alone did not activate a full-blown oxidative burst in PMN but resulted in the priming of PMN for fMLP-induced production of ROI.

We presented data that LTA treatment leads to release of cytokines from PMN. After pathogen challenge, IL-8 is one of the most important chemotactic factors that mediate local neutrophil recruitment [30 ]. The autocrine production of IL-8 by activated PMN is regarded as an amplifying loop to attract more neutrophils to the site of infection [31 ]. Intracellular staining in PMN revealed that treatment with LTA results not only in the release of preformed IL-8 but stimulates the new synthesis of this neutrophil-derived chemokine as well. IL-8 is a prototype of proinflammatory cytokines under NF-{kappa}B regulation [51 ]. Measuring I{kappa}B phosphorylation as a read-out system, rapid activation of NF-{kappa}B was observed in PMN upon exposure to LTA. In addition to IL-8, LTA treatment also induced the release of G-CSF and TNF-{alpha}, which is a further, additional sign of the activated state of PMN.

PMN are inherently short-lived cells with a half-life of only 6–10 h, after which they undergo spontaneous apoptosis [17 ]. Although apoptosis is an intrinsic cell process, it is modulated by signals from the local micromilieu. The lifespan of mature neutrophils can be extended in vitro by incubation with proinflammatory cytokines including GM-CSF and G-CSF, IL-8, and IL-1ß and bacterial products such as LPS and fMLP [34 , 52 ]. This implies that PMN die rapidly via apoptosis if not engaged in function. After activation, however, inflammatory PMN have an extended lifespan that enables these important cells of the innate immune system to execute their antimicrobial effector functions. In this study, we demonstrated for the first time that LTA, a component of gram-positive bacteria, can directly delay the spontaneous apoptosis of PMN, resulting in an extended lifespan of these cells. In case of cytokine-induced delay of PMN apoptosis, NF-{kappa}B activation was shown to be involved [53 ]. As LTA activates NF-{kappa}B in PMN very rapidly, this way is also likely to play a role in the LTA-induced modulation of programmed cell death in PMN.

IL-8 and G-CSF, which are released after exposure to LTA, could mediate the observed delay of spontaneous PMN apoptosis. However, the levels of these cytokines in the PMN cultures were much lower as compared with concentrations that were reported to inhibit PMN apoptosis [54 , 55 ]. Therefore, the delay in spontaneous PMN apoptosis is likely a direct effect of LTA rather than being mediated by LTA-induced cytokines.

Using blocking experiments with specific mAb, here, we showed for the first time the CD14- and TLR2-dependent, direct effect of LTA on PMN. Blocking CD14 in primary human monocytes was previously shown to diminish LTA-mediated effects [56 ]. In contrast to monocytes, CD14 is expressed at a low level on PMN [41 ]. The strongly attenuated effect of LTA on PMN after CD14 blockade shows, however, that this molecule plays a major role in the LTA-mediated effects on PMN.

Here, we showed that in primary human neutrophils, in addition to blocking CD14, treatment with anti-TLR2 antibody also reduced a LTA-induced cellular response. Transfection studies with human embryonic kidney and Chinese hamster ovary cells revealed that CD14 and TLR2 participate in the response to LTA [56 , 57 ]. However, few previous reports dealt with the effect of different TLR-2 ligands on PMN. In these works, TLR2 agonists such as peptidoglycan, zymosan, and tripalmitoyl-cyteinyl-seryl-(lysyl)3-lysine (Pam3CSK4) were studied [46 , 58 ], but the effect of LTA on PMN was not investigated. In one study, the stimulation of PMN with the TLR2 agonists zymosan, peptidoglycan, and nonmannose-capped lipoarabinomannan resulted in a release of low amounts of IL-8 by PMN but did not trigger the generation of ROI [46 ]. Hayashi et al. [58 ] showed a stimulatory effect of TLR ligands zymosan and Pam3CSK4 on PMN. However, the low purity of granulocytes in the mentioned study makes it difficult to say whether the observed effect was a result of the direct interaction with PMN. Studies with TLR2 ligands such as Pam3CSK4, however, may be of limited value regarding the role of LTA, as LTA and Pam3CSK4 were recently shown to have distinct binding domains on the TLR2 molecule [59 ]. The importance of the response of PMN to LTA is highlighted by the fact that in addition to peptidoglycan, LTA is the main immunostimulatory cell-wall component of gram-positive bacteria [60 ], which are responsible for an increasing number of systemic infections [61 ].

Ligand specificity for a number of TLR2 activators requires heterodimerization with additional TLR molecules, TLR1 and TLR6 [62 , 63 ]. Studies suggest that TLR2/TLR1 heterodimers mediate responses to Pam3CSK4. It is still to be clarified whether LTA binds to TLR2/TLR1 or TLR2/TLR6 heterodimers. TLR2/TLR1 and TLR2/TLR6 heterodimers are capable of inducing differential cellular responses [64 , 65 ]. In line with these data, different TLR2 agonists were shown to induce distinct effects on human mast cells [66 ].

Using blocking experiments with specific mAb, here, we showed for the first time that CD14 and TLR2 are involved in the LTA-mediated, antiapoptotic effect on human PMN. Interaction with distinct binding domains on TLR2 may explain the apparent discrepancy of our present finding with a previous report, in which TLR2 activation via Pam3CSK4 did not prevent neutrophil apoptosis [67 ]. Moreover, no direct comparison of the LTA effect with that of other TLR2 ligands can be made, as for example, the biological activity of Pam3-Cys-Ser-Ser-Asn-Ala was shown to be independent of CD14 [68 ].

Taken together, LTA has a direct stimulatory activity on the functions of human PMN, which when exposed to LTA, show all typical features of activation, i.e., elongated cell shape, granule degranulation, shedding of CD62L, release of IL-8, TNF-{alpha}, and G-CSF, a primed state for fMLP-induced oxidative burst, and an extended lifespan. The LTA-induced stimulation of PMN involves CD14, TLR2, and the activation of NF-{kappa}B. These data suggest that LTA is a potent component of gram-positive bacteria in terms of recognition by and stimulation of PMN.


arrow
ACKNOWLEDGEMENTS
 
This work was supported by grants from the Deutsche Forschungsgesellschaft (La1267/1-1, GRK288/B8, SFB367/B10). The authors thank Ms. Birgit Hansen for invaluable, methodical help.

Received August 1, 2003; revised October 28, 2003; accepted November 12, 2003.


arrow
REFERENCES
 
    1
  1. Wilson, M. E. (1985) Effects of bacterial endotoxins on neutrophil function Rev. Infect. Dis. 7,404-418[Medline]
    2. 2
  2. Fischer, W. (1988) Physiology of lipoteichoic acids in bacteria Adv. Microb. Physiol. 29,233-302[Medline]
    3. 3
  3. Saetre, T., Kahler, H., Foster, S. J., Lyberg, T. (2000) Peptidoglycan and lipoteichoic acid, components of the streptococcal cell wall, have marked and differential effects on adhesion molecule expression and the production of reactive oxygen species in human whole blood leukocytes Scand. J. Clin. Lab. Invest. 60,311-321[CrossRef][Medline]
    4. 4
  4. Lavkan, A. H., Astiz, M. E., Rackow, E. C. (1998) Effects of proinflammatory cytokines and bacterial toxins on neutrophil rheologic properties Crit. Care Med. 26,1677-1682[CrossRef][Medline]
    5. 5
  5. Ginsburg, I., Fligiel, S. E., Ward, P. A., Varani, J. (1988) Lipoteichoic acid-antilipoteichoic acid complexes induce superoxide generation by human neutrophils Inflammation 12,525-548[CrossRef][Medline]
    6. 6
  6. Morath, S., Geyer, A., Spreitzer, I., Hermann, C., Hartung, T. (2002) Structural decomposition and heterogeneity of commercial lipoteichoic acid preparations Infect. Immun. 70,938-944[Abstract/Free Full Text]
    7. 7
  7. Kusunoki, T., Hailman, E., Juan, T. S., Lichenstein, H. S., Wright, S. D. (1995) Molecules from Staphylococcus aureus that bind CD14 and stimulate innate immune responses J. Exp. Med. 182,1673-1682[Abstract/Free Full Text]
    8. 8
  8. Morath, S., Geyer, A., Hartung, T. (2001) Structure-function relationship of cytokine induction by lipoteichoic acid from Staphylococcus aureus J. Exp. Med. 193,393-397[Abstract/Free Full Text]
    9. 9
  9. Ellingsen, E., Morath, S., Flo, T., Schromm, A., Hartung, T., Thiemermann, C., Espevik, T., Golenbock, D., Foster, D., Solberg, R., Aasen, A., Wang, J. (2002) Induction of cytokine production in human T cells and monocytes by highly purified lipoteichoic acid: involvement of Toll-like receptors and CD14 Med. Sci. Monit. 8,BR149-BR156[Medline]
    10. 10
  10. Hermann, C., Spreitzer, I., Schroder, N. W., Morath, S., Lehner, M. D., Fischer, W., Schutt, C., Schumann, R. R., Hartung, T. (2002) Cytokine induction by purified lipoteichoic acids from various bacterial species-role of LBP, sCD14, CD14 and failure to induce IL-12 and subsequent IFN-{gamma} release Eur. J. Immunol. 32,541-551[CrossRef][Medline]
    11. 11
  11. Jacinto, R., Hartung, T., McCall, C., Li, L. (2002) Lipopolysaccharide- and lipoteichoic acid-induced tolerance and cross-tolerance: distinct alterations in IL-1 receptor-associated kinase J. Immunol. 168,6136-6141[Abstract/Free Full Text]
    12. 12
  12. Aga, E., Katschinski, D. M., van Zandbergen, G., Laufs, H., Hansen, B., Muller, K., Solbach, W., Laskay, T. (2002) Inhibition of the spontaneous apoptosis of neutrophil granulocytes by the intracellular parasite Leishmania major J. Immunol. 169,898-905[Abstract/Free Full Text]
    13. 13
  13. Laufs, H., Muller, K., Fleischer, J., Reiling, N., Jahnke, N., Jensenius, J. C., Solbach, W., Laskay, T. (2002) Intracellular survival of Leishmania major in neutrophil granulocytes after uptake in the absence of heat-labile serum factors Infect. Immun. 70,826-835[Abstract/Free Full Text]
    14. 14
  14. Ducker, T. P., Skubitz, K. M. (1992) Subcellular localization of CD66, CD67, and NCA in human neutrophils J. Leukoc. Biol. 52,11-16[Abstract]
    15. 15
  15. Kishimoto, T. K., Jutila, M. A., Berg, E. L., Butcher, E. C. (1989) Neutrophil Mac-1 and MEL-14 adhesion proteins inversely regulated by chemotactic factors Science 245,1238-1241[Abstract/Free Full Text]
    16. 16
  16. Smith, J. A., Weidemann, M. J. (1993) Further characterization of the neutrophil oxidative burst by flow cytometry J. Immunol. Methods 162,261-268[CrossRef][Medline]
    17. 17
  17. Payne, C. M., Glasser, L., Tischler, M. E., Wyckoff, D., Cromey, D., Fiederlein, R., Bohnert, O. (1994) Programmed cell death of the normal human neutrophil: an in vitro model of senescence Microsc. Res. Tech. 28,327-344[CrossRef][Medline]
    18. 18
  18. Squier, M. K., Sehnert, A. J., Cohen, J. J. (1995) Apoptosis in leukocytes J. Leukoc. Biol. 57,2-10[Abstract]
    19. 19
  19. Homburg, C. H., de Haas, M., dem Borne, A. E., Verhoeven, A. J., Reutelingsperger, C. P., Roos, D. (1995) Human neutrophils lose their surface Fc{gamma}RIII and acquire Annexin V binding sites during apoptosis in vitro Blood 85,532-540[Abstract/Free Full Text]
    20. 20
  20. Gorczyca, W., Gong, J., Darzynkiewicz, Z. (1993) Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays Cancer Res. 53,1945-1951[Abstract/Free Full Text]
    21. 21
  21. Klut, M. E., Whalen, B. A., Hogg, J. C. (1997) Activation-associated changes in blood and bone marrow neutrophils J. Leukoc. Biol. 62,186-194[Abstract]
    22. 22
  22. Stickle, J. E. (1996) The neutrophil. Function, disorders, and testing Vet. Clin. North Am. Small Anim. Pract. 26,1013-1021[Medline]
    23. 23
  23. Niwa, M., Kanamori, Y., Kohno, K., Matsuno, H., Kozawa, O., Kanamura, M., Uematsu, T. (2000) Usefulness of grading of neutrophil aggregate size by laser-light scattering technique for characterizing stimulatory and inhibitory effects of agents on aggregation Life Sci. 67,1525-1534[CrossRef][Medline]
    24. 24
  24. Fennell, K. O., Creamer, H. R., Gabler, W. L., Brown, A. C., Bullock, W. W. (1984) Activated rat neutrophils. A sequential quantitative assay for aggregation and NBT reduction Inflammation 8,287-300[CrossRef][Medline]
    25. 25
  25. Neufert, C., Pai, R. K., Noss, E. H., Berger, M., Boom, W. H., Harding, C. V. (2001) Mycobacterium tuberculosis 19-kDa lipoprotein promotes neutrophil activation J. Immunol. 167,1542-1549[Abstract/Free Full Text]
    26. 26
  26. van Eeden, S. F., Klut, M. E., Walker, B. A., Hogg, J. C. (1999) The use of flow cytometry to measure neutrophil function J. Immunol. Methods 232,23-43[CrossRef][Medline]
    27. 27
  27. Sengelov, H., Kjeldsen, L., Diamond, M. S., Springer, T. A., Borregaard, N. (1993) Subcellular localization and dynamics of Mac-1 ({alpha}m ß2) in human neutrophils J. Clin. Invest. 92,1467-1476
    28. 28
  28. Swain, S. D., Rohn, T. T., Quinn, M. T. (2002) Neutrophil priming in host defense: role of oxidants as priming agents Antioxid. Redox Signal. 4,69-83[CrossRef][Medline]
    29. 29
  29. Guthrie, L. A., McPhail, L. C., Henson, P. M., Johnston, R. B., Jr (1984) Priming of neutrophils for enhanced release of oxygen metabolites by bacterial lipopolysaccharide. Evidence for increased activity of the superoxide-producing enzyme J. Exp. Med. 160,1656-1671[Abstract/Free Full Text]
    30. 30
  30. Cassatella, M. A. (1999) Neutrophil-derived proteins: selling cytokines by the pound Adv. Immunol. 73,369-509[Medline]
    31. 31
  31. Albanyan, E. A., Vallejo, J. G., Smith, C. W., Edwards, M. S. (2000) Nonopsonic binding of type III group B Streptococci to human neutrophils induces interleukin-8 release mediated by the p38 mitogen-activated protein kinase pathway Infect. Immun. 68,2053-2060[Abstract/Free Full Text]
    32. 32
  32. Altstaedt, J., Kirchner, H., Rink, L. (1996) Cytokine production of neutrophils is limited to interleukin-8 Immunology 89,563-568[CrossRef][Medline]
    33. 33
  33. Morath, S., Stadelmaier, A., Geyer, A., Schmidt, R. R., Hartung, T. (2002) Synthetic lipoteichoic acid from Staphylococcus aureus is a potent stimulus of cytokine release J. Exp. Med. 195,1635-1640[Abstract/Free Full Text]
    34. 34
  34. Colotta, F., Re, F., Polentarutti, N., Sozzani, S., Mantovani, A. (1992) Modulation of granulocyte survival and programmed cell death by cytokines and bacterial products Blood 80,2012-2020[Abstract/Free Full Text]
    35. 35
  35. Gosselin, E. J., Wardwell, K., Rigby, W. F., Guyre, P. M. (1993) Induction of MHC class II on human polymorphonuclear neutrophils by granulocyte/macrophage colony-stimulating factor, IFN-{gamma}, and IL-3 J. Immunol. 151,1482-1490[Abstract]
    36. 36
  36. McDonald, P. P., Bald, A., Cassatella, M. A. (1997) Activation of the NF-{kappa}B pathway by inflammatory stimuli in human neutrophils Blood 89,3421-3433[Abstract/Free Full Text]
    37. 37
  37. Jozsef, L., Zouki, C., Petasis, N. A., Serhan, C. N., Filep, J. G. (2002) Lipoxin A4 and aspirin-triggered 15-epi-lipoxin A4 inhibit peroxynitrite formation, NF-{kappa}B and AP-1 activation, and IL-8 gene expression in human leukocytes Proc. Natl. Acad. Sci. USA 99,13266-13271[Abstract/Free Full Text]
    38. 38
  38. Dickensheets, H. L., Freeman, S. L., Smith, M. F., Donnelly, R. P. (1997) Interleukin-10 upregulates tumor necrosis factor receptor type-II (p75) gene expression in endotoxin-stimulated human monocytes Blood 90,4162-4171[Abstract/Free Full Text]
    39. 39
  39. Cassatella, M. A., Bazzoni, F., Ceska, M., Ferro, I., Baggiolini, M., Berton, G. (1992) IL-8 production by human polymorphonuclear leukocytes. The chemoattractant formyl-methionyl-leucyl-phenylalanine induces the gene expression and release of IL-8 through a pertussis toxin-sensitive pathway J. Immunol. 148,3216-3220[Abstract]
    40. 40
  40. Marie, C., Roman-Roman, S., Rawadi, G. (1999) Involvement of mitogen-activated protein kinase pathways in interleukin-8 production by human monocytes and polymorphonuclear cells stimulated with lipopolysaccharide or Mycoplasma fermentans membrane lipoproteins Infect. Immun. 67,688-693[Abstract/Free Full Text]
    41. 41
  41. Antal-Szalmas, P., Strijp, J. A., Weersink, A. J., Verhoef, J., Van Kessel, K. P. (1997) Quantitation of surface CD14 on human monocytes and neutrophils J. Leukoc. Biol. 61,721-728[Abstract]
    42. 42
  42. van Langevelde, P., van Dissel, J. T., Ravensbergen, E., Appelmelk, B. J., Schrijver, I. A., Groeneveld, P. H. (1998) Antibiotic-induced release of lipoteichoic acid and peptidoglycan from Staphylococcus aureus: quantitative measurements and biological reactivities Antimicrob. Agents Chemother. 42,3073-3078[Abstract/Free Full Text]
    43. 43
  43. Schneider, O., Michel, U., Zysk, G., Dubuis, O., Nau, R. (1999) Clinical outcome in pneumococcal meningitis correlates with CSF lipoteichoic acid concentrations Neurology 53,1584-1587[Abstract/Free Full Text]
    44. 44
  44. Ingraham, J. M., Maaloe, O., Neidhart, F. C. (1983) Growth of the Bacterial Cell ,3 Sinauer Associates Sunderland, MA.
    45. 45
  45. Yang, S., Tamai, R., Akashi, S., Takeuchi, O., Akira, S., Sugawara, S., Takada, H. (2001) Synergistic effect of muramyldipeptide with lipopolysaccharide or lipoteichoic acid to induce inflammatory cytokines in human monocytic cells in culture Infect. Immun. 69,2045-2053[Abstract/Free Full Text]
    46. 46
  46. Kurt-Jones, E. A., Mandell, L., Whitney, C., Padgett, A., Gosselin, K., Newburger, P. E., Finberg, R. W. (2002) Role of Toll-like receptor 2 (TLR2) in neutrophil activation: GM-CSF enhances TLR2 expression and TLR2-mediated interleukin 8 responses in neutrophils Blood 100,1860-1868[Abstract/Free Full Text]
    47. 47
  47. Borregaard, N., Lollike, K., Kjeldsen, L., Sengelov, H., Bastholm, L., Nielsen, M. H., Bainton, D. F. (1993) Human neutrophil granules and secretory vesicles Eur. J. Haematol. 51,187-198[Medline]
    48. 48
  48. Rozenberg-Arska, M., Salters, M. E., van Strijp, J. A., Geuze, J. J., Verhoef, J. (1985) Electron microscopic study of phagocytosis of Escherichia coli by human polymorphonuclear leukocytes Infect. Immun. 50,852-859[Abstract/Free Full Text]
    49. 49
  49. Vandenbroucke-Grauls, C. M., Thijssen, H. M., Verhoef, J. (1985) Phagocytosis of staphylococci by human polymorphonuclear leukocytes is enhanced in the presence of endothelial cells Infect. Immun. 50,250-254[Abstract/Free Full Text]
    50. 50
  50. Springer, T. A. (1994) Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm Cell 76,301-314[CrossRef][Medline]
    51. 51
  51. Roebuck, K. A. (1999) Regulation of interleukin-8 gene expression J. Interferon Cytokine Res. 19,429-438[CrossRef][Medline]
    52. 52
  52. Lee, A., Whyte, M. K., Haslett, C. (1993) Inhibition of apoptosis and prolongation of neutrophil functional longevity by inflammatory mediators J. Leukoc. Biol. 54,283-288[Abstract]
    53. 53
  53. Nolan, B., Kim, R., Duffy, A., Sheth, K., De, M., Miller, C., Chari, R., Bankey, P. (2000) Inhibited neutrophil apoptosis: proteasome dependent NF-{kappa}B translocation is required for TRAF-1 synthesis Shock 14,290-294[Medline]
    54. 54
  54. Kettritz, R., Gaido, M. L., Haller, H., Luft, F. C., Jennette, C. J., Falk, R. J. (1998) Interleukin-8 delays spontaneous and tumor necrosis factor-{alpha}-mediated apoptosis of human neutrophils Kidney Int. 53,84-91[CrossRef][Medline]
    55. 55
  55. Cox, G., Gauldie, J., Jordana, M. (1992) Bronchial epithelial cell-derived cytokines (G-CSF and GM-CSF) promote the survival of peripheral blood neutrophils in vitro Am. J. Respir. Cell Mol. Biol. 7,507-513
    56. 56
  56. Schroder, N. W., Morath, S., Alexander, C., Hamann, L., Hartung, T., Zahringer, U., Gobel, U. B., Weber, J. R., Schumann, R. R. (2003) Lipoteichoic acid (LTA) of Streptococcus pneumoniae and Staphylococcus aureusactivates immune cells via Toll-like receptor (TLR)-2, lipopolysaccharide-binding protein (LBP), and CD14, whereas TLR-4 and MD-2 are not involved J. Biol. Chem. 278,15587-15594[Abstract/Free Full Text]
    57. 57
  57. Schwandner, R., Dziarski, R., Wesche, H., Rothe, M., Kirschning, C. J. (1999) Peptidoglycan- and lipoteichoic acid-induced cell activation is mediated by Toll-like receptor 2 J. Biol. Chem. 274,17406-17409[Abstract/Free Full Text]
    58. 58
  58. Hayashi, F., Means, T. K., Luster, A. D. (2003) Toll-like receptors stimulate human neutrophil function Blood 102,2660-2669[Abstract/Free Full Text]
    59. 59
  59. Meng, G., Grabiec, A., Vallon, M., Ebe, B., Hampel, S., Bessler, W., Wagner, H., Kirschning, C. J. (2003) Cellular recognition of tri-/di-palmitoylated peptides is independent from a domain encompassing the N-terminal seven leucine-rich repeat (LRR)/LRR-like motifs of TLR2 J. Biol. Chem. 278,39822-39829[Abstract/Free Full Text]
    60. 60
  60. De Kimpe, S. J., Kengatharan, M., Thiemermann, C., Vane, J. R. (1995) The cell wall components peptidoglycan and lipoteichoic acid from Staphylococcus aureus act in synergy to cause shock and multiple organ failure Proc. Natl. Acad. Sci. USA 92,10359-10363[Abstract/Free Full Text]
    61. 61
  61. Fluit, A. C., Schmitz, F. J., Verhoef, J. (2001) Frequency of isolation of pathogens from bloodstream, nosocomial pneumonia, skin and soft tissue, and urinary tract infections occurring in European patients Eur. J. Clin. Microbiol. Infect. Dis. 20,188-191[CrossRef][Medline]
    62. 62
  62. Ozinsky, A., Underhill, D. M., Fontenot, J. D., Hajjar, A. M., Smith, K. D., Wilson, C. B., Schroeder, L., Aderem, A. (2000) The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between Toll-like receptors Proc. Natl. Acad. Sci. USA 97,13766-13771[Abstract/Free Full Text]
    63. 63
  63. Takeuchi, O., Sato, S., Horiuchi, T., Hoshino, K., Takeda, K., Dong, Z., Modlin, R. L., Akira, S. (2002) Cutting edge: role of Toll-like receptor 1 in mediating immune response to microbial lipoproteins J. Immunol. 169,10-14[Abstract/Free Full Text]
    64. 64
  64. Hirschfeld, M., Weis, J. J., Toshchakov, V., Salkowski, C. A., Cody, M. J., Ward, D. C., Qureshi, N., Michalek, S. M., Vogel, S. N. (2001) Signaling by Toll-like receptor 2 and 4 agonists results in differential gene expression in murine macrophages Infect. Immun. 69,1477-1482[Abstract/Free Full Text]
    65. 65
  65. Jones, B. W., Means, T. K., Heldwein, K. A., Keen, M. A., Hill, P. J., Belisle, J. T., Fenton, M. J. (2001) Different Toll-like receptor agonists induce distinct macrophage responses J. Leukoc. Biol. 69,1036-1044[Abstract/Free Full Text]
    66. 66
  66. McCurdy, J. D., Olynych, T. J., Maher, L. H., Marshall, J. S. (2003) Cutting edge: distinct Toll-like receptor 2 activators selectively induce different classes of mediator production from human mast cells J. Immunol. 170,1625-1629[Abstract/Free Full Text]
    67. 67
  67. Sabroe, I., Prince, L. R., Jones, E. C., Horsburgh, M. J., Foster, S. J., Vogel, S. N., Dower, S. K., Whyte, M. K. (2003) Selective roles for Toll-like receptor (TLR)2 and TLR4 in the regulation of neutrophil activation and life span J. Immunol. 170,5268-5275[Abstract/Free Full Text]
    68. 68
  68. Muroi, M., Ohnishi, T., Azumi-Mayuzumi, S., Tanamoto, K. (2003) Lipopolysaccharide-mimetic activities of a Toll-like receptor 2-stimulatory substance(s) in enterobacterial lipopolysaccharide preparations Infect. Immun. 71,3221-3226[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
R. Bucki, F. J. Byfield, A. Kulakowska, M. E. McCormick, W. Drozdowski, Z. Namiot, T. Hartung, and P. A. Janmey
Extracellular Gelsolin Binds Lipoteichoic Acid and Modulates Cellular Response to Proinflammatory Bacterial Wall Components
J. Immunol., October 1, 2008; 181(7): 4936 - 4944.
[Abstract] [Full Text] [PDF]

Home page
 CVIHome page
S. Meron-Sudai, A. Matityahou, Y. Keisari, K. H. Cox, D. L. Hasty, and I. Ofek
Lipoteichoic Acid Synergizes with Glycosphingolipids To Potently Stimulate Secretion of Interleukin-6 from Human Blood Cells
Clin. Vaccine Immunol., September 1, 2008; 15(9): 1309 - 1315.
[Abstract] [Full Text] [PDF]

Home page
 Nephrol Dial TransplantHome page
C.-S. Lin, J. G. Wann, C.-W. Hsiao, D. W. Tai, J.-S. Chen, Y.-H. Hsu, H.-C. Huang, and C.-F. Chao
Intracellular acidification enhances neutrophil phagocytosis in chronic haemodialysis patients: possible role of CD11b/CD18
Nephrol. Dial. Transplant., May 1, 2008; 23(5): 1642 - 1649.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. Knapp, S. von Aulock, M. Leendertse, I. Haslinger, C. Draing, D. T. Golenbock, and T. van der Poll
Lipoteichoic Acid-Induced Lung Inflammation Depends on TLR2 and the Concerted Action of TLR4 and the Platelet-Activating Factor Receptor
J. Immunol., March 1, 2008; 180(5): 3478 - 3484.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
H. G. Kim, N.-R. Kim, M. G. Gim, J. M. Lee, S. Y. Lee, M. Y. Ko, J. Y. Kim, S. H. Han, and D. K. Chung
Lipoteichoic Acid Isolated from Lactobacillus plantarum Inhibits Lipopolysaccharide-Induced TNF-{alpha} Production in THP-1 Cells and Endotoxin Shock in Mice
J. Immunol., February 15, 2008; 180(4): 2553 - 2561.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
W. Stenzel, S. Soltek, M. Sanchez-Ruiz, S. Akira, H. Miletic, D. Schluter, and M. Deckert
Both TLR2 and TLR4 Are Required for the Effective Immune Response in Staphylococcus aureus-Induced Experimental Murine Brain Abscess
Am. J. Pathol., January 1, 2008; 172(1): 132 - 145.
[Abstract] [Full Text] [PDF]

Home page
 Innate ImmunityHome page
C. Hermann
Review: Variability of host pathogen interaction
Innate Immunity, August 1, 2007; 13(4): 199 - 218.
[Abstract] [PDF]

Home page
 BloodHome page
G. Elson, I. Dunn-Siegrist, B. Daubeuf, and J. Pugin
Contribution of Toll-like receptors to the innate immune response to Gram-negative and Gram-positive bacteria
Blood, February 15, 2007; 109(4): 1574 - 1583.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Mueller, C. Stamme, C. Draing, T. Hartung, U. Seydel, and A. B. Schromm
Cell Activation of Human Macrophages by Lipoteichoic Acid Is Strongly Attenuated by Lipopolysaccharide-binding Protein
J. Biol. Chem., October 20, 2006; 281(42): 31448 - 31456.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
L. C. Parker, M. K. B. Whyte, S. K. Dower, and I. Sabroe
The expression and roles of Toll-like receptors in the biology of the human neutrophil
J. Leukoc. Biol., June 1, 2005; 77(6): 886 - 892.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0803360v1
75/3/467    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lotz, S.
Right arrow Articles by Laskay, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lotz, S.
Right arrow Articles by Laskay, T.