Journal of Leukocyte Biology eBioscience full spectrum cell analysis
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.0902442 on July 8, 2005

Published online before print July 8, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0902442v1
78/4/992    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 Hattar, K.
Right arrow Articles by Sibelius, U.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hattar, K.
Right arrow Articles by Sibelius, U.
(Journal of Leukocyte Biology. 2005;78:992-1000.)
© 2005 by Society for Leukocyte Biology

Anti-proteinase 3 antibodies (c-ANCA) prime CD14-dependent leukocyte activation

Katja Hattar*, Sandra van Bürck*, Annette Bickenbach*, Ulrich Grandel*, Ulrich Maus*,{dagger}, Jürgen Lohmeyer*, Elena Csernok{ddagger}, Thomas Hartung§, Werner Seeger*, Friedrich Grimminger* and Ulf Sibelius*,1

* Department of Internal Medicine, Justus-Liebig-University, Giessen, Germany;
{dagger} Hannover Medical School, Department of Internal Medicine, Germany;
{ddagger} Department of Rheumatology, Medical University of Lübeck, Germany; and
§ Department of Biochemical Pharmacology, University of Konstanz, Germany

1Correspondence: Department of Internal Medicine, Justus-Liebig-University Giessen, D-35385 Giessen, FRG. E-mail: ulf.sibelius{at}innere.med.uni-giessen.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In Wegener’s granulomatosis (WG), a pathogenetic role has been proposed for circulating anti-neutrophil-cytoplasmic antibodies (ANCA) targeting proteinase 3 (PR3). Disease activation in WG appears to be triggered by bacterial infections. In the present study, we characterized the effect of anti-PR3 antibodies on in vitro activation of isolated monocytes and neutrophils by the bacterial cell-wall components lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Although sole incubation of monocytes and neutrophils with monoclonal anti-PR3 antibodies induced the release of minor quantities of the chemokine interleukin-8 (IL-8), preincubation with anti-PR3 antibodies, but not with isotype-matched control immunogloblin G (IgG), resulted in a markedly enhanced IL-8 liberation upon LPS challenge. The priming response was evident after 2 h of preincubation with anti-PR3 and peaked after 6 h. The anti-PR3-related priming was also observed for tumor necrosis factor {alpha} (TNF-{alpha}) and IL-6 synthesis. Comparable priming occurred when leukocytes were preincubated with ANCA-IgG derived from WG serum but not with normal IgG. The priming effect of the anti-PR3 antibody pretreatment was reproduced for LTA challenge of monocytes and neutrophils but not for leukocyte stimulation with TNF-{alpha}. Flow cytometric analysis revealed an increase in monocyte and neutrophil membrane CD14 expression during the anti-PR3 priming. We conclude that cytoplasmic ANCA specifically prime CD14-dependent monocytes and neutrophils for activation. The resulting enhanced responsiveness to bacterial pathogens may contribute to the development and maintenance of inflammatory lesions during active WG.

Key Words: monocytes/macrophages • neutrophils • autoantibodies • cell surface molecules • cytokines


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Wegener’s granulomatosis (WG) is characterized by granulomatous inflammation of the upper and lower respiratory tract and necrotizing vasculitis, primarily involving the kidneys. The vasculitic lesions are characterized by an early phase of neutrophil accumulation, followed by a monocytic infiltrate [1 , 2 ]. As the degree of leukocyte activation was found to correlate with the extent of the disease [3 4 5 ], activated neutrophils and monocytes appear to be the major effector cells in the development of inflammatory lesions in WG [6 ].

Is is proposed that disease activation is triggered by bacterial infections, as the onset of symptoms peaks in the winter [7 ], and antibiotics have a beneficial effect in the treatment of refractory WG [8 , 9 ]. Moreover, chronic nasal carriage of Staphylococcus aureus is associated with higher relapse rates of the disease [10 ]. The immunostimulatory cell-wall components of S. aureus include lipoteichoic acid (LTA), a macroamphiphile found in the cell membranes of virtually all Gram-positive bacteria [11 ], whereas lipopolysaccharides (LPS) are well-characterized, pathogenic factors of Gram-negative bacteria. LPS and LTA are capable of activating a wide variety of inflammatory functions in neutrophils and monocytes, including the induction of adhesion, the activation of the respiratory burst, and cytokine secretion [12 13 14 15 ]. Cellular activation by LPS and LTA is largely mediated by ligation of the CD14 molecule [16 , 17 ], a glycosylphosphatidylinositol (GPI)-linked membrane protein, and involves the activation of Toll-like receptors (TLRs) [18 , 19 ]. Whether an inadequate or overwhelming inflammatory response to invading microorganisms is responsible for the association between infection and disease exacerbations in WG or whether these bacterial cell-wall components directly stimulate activation of autoreactive B- and T-lymphocytes, thus further triggering the autoimmune process, remains unclear.

An established association does, however, exist between the occurrence of anti-neutrophil-cytoplasmic antibodies (ANCA), especially those targeting proteinase 3 (PR3), and the development of active WG [20 ]. The PR3 ANCA are reported to be causally involved in the pathogenesis of the disease, as the autoantibody titer correlates with the disease activity in vivo [21 ], and ANCA directly activate a wide variety of inflammatory functions in neutrophils in vitro, such as secretion of oxygen radicals, proteases, and lipid mediators, once the autoantigen PR3 is expressed on the leukocyte cell surface [22 23 24 25 ] under inflammatory conditions. Additionally, in isolated monocytes, anti-PR3 antibodies stimulate the release of proinflammatory cytokines [26 , 27 ].

In the present study, we focused on the interaction between anti-PR3 antibodies and bacterial cell-wall components in leukocyte activation. We used LPS as the major immunostimulatory component of Gram-negative and LTA as a prototype of Gram-positive cell-wall components. Both agents are activators of leukocyte interleukin-8 (IL-8) generation [13 , 15 ], which is pertinent, as a pathogenetic role has recently been attributed to this CXC chemokine in ANCA-associated glomerulonephritis [28 ]. Dual stimulation with anti-PR3 and LPS as well as LTA was performed. It is most interesting that the WG autoantibody caused a strong priming of the leukocyte cytokine response triggered upon subsequent stimulation with both prototype bacterial cell-wall components, and an up-regulation of membrane CD14 was proposed as one underlying mechanism.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Ficoll-Paque and Protein G Sepharose were purchased from Pharmacia (Uppsala, Sweden), fetal calf serum (FCS) was from Greiner (Frickenhausen, Germany), and all media and supplements were from Gibco (Eggenstein, Germany), unless otherwise indicated. LPS (Escherichia coli, 0111:B4), fluorescein isothiocyanate (FITC)-conjugated LPS, polymyxin B (PMB) immobilized on agarose, and isotype control mouse immunoglobulin G1(IgG1c; MOPC-21) were from Sigma (Deisenhofen, Germany). Antibodies used for tumor necrosis factor {alpha} (TNF-{alpha}), IL-6, and IL-8 enzyme-linked immunosorbent assay (ELISA; MAB 208; BAF 208) as well as recombinant human cytokines were purchased from R&D Systems (Wiesbaden, Germany). Peroxidase-conjugated streptavidin [horseradish peroxidase (HRP)] and 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulphonate; ABTS) were from Zymed Laboratories (San Francisco, CA). The murine monoclonal anti-CD-14 antibody (MY4) was from Coulter Immunotech (Hamburg, Germany), and the phycoerythrin (PE)-labeled anti-CD14 antibody LeuM3 and the PE/FITC-labeled irrelevant control antibodies (Simultest IgG2ac/IgG1) were from Becton Dickinson (Heidelberg, Germany). PE-labeled anti-human TLR2 (clone TL2.1) and anti-TLR4 (clone HTA25) were purchased from eBioscience (San Diego, CA). Pooled human IgG (Octagam) was obtained from Octapharma (Langenfeld, Germany), and the Limulus amebocyte cell lysate (LAL) test for the detection of endotoxin was from Chromogenix (Mölndal, Sweden). Cell culture plasticware was purchased from Falcon (Mannheim, Germany). All other chemicals were from Merck (Darmstadt, Germany).

Purification of LTA from S. aureus
LTA was purified by a structure-preserving isolation procedure as described [29 ]. Briefly, the classic phenol-water extraction at 68°C and subsequent dialysis were replaced by butanol extraction at room temperature and lyophilization. S. aureus (DSM 20233) was grown in a 42 L fermenter and harvested by centrifugation at 4°C. The pelleted bacteria were sonicated (Branson, Danbury, CT) on ice or disrupted with a cell mill (Büchi, Uster, Switzerland). LTA was negative for endotoxins in the chromogenic LAL assay; i.e., the LAL reactivity was lower than that for 1 pg LPS/ml.

Anti-PR3 antibodies
Murine monoclonal antibodies targeting PR3 were prepared by hybridoma technology, as described previously [30 ]. The clone WGM2 (IgG1) was chosen for further experiments. Cytoplasmic (c)-ANCA were purified from the sera of patients with monospecific, anti-PR3-positive WG by adsorption on a protein G column, as described [31 ], and human IgGc was prepared similarly using sera from five healthy volunteers. The IgG concentration of the eluates was determined according to standard procedures. PR3 specificity of the monoclonal and serum-derived antibodies was assessed by antigen-specific ELISA. Using the kinetic-OLC LAL test, endotoxin contamination of the murine antibody WGM2 and human anti-PR3 antibodies was below 15 pg/ml. The monoclonal anti-PR3 antibody 12.8 was purchased from Research Diagnostics (Flanders, NY) and was applied to a polymyxin-agarose column to remove contaminating endotoxin. After PMB treatment, endotoxin contamination of antibody preparation was below 15 pg/ml.

Isolation of neutrophils
Neutrophils were isolated from venous blood of healthy donors by centrifugation over a Ficoll-Paque gradient as described previously [23 ]. In brief, EDTA-anticoagulated blood was layered over Ficoll-Paque and centrifuged at 400 g for 35 min. After removal of mononuclear cells, erythrocytes were sedimented in 10% polyvinyl alcohol. Residual erythrocytes were removed by hypotonic lysis, and cells were washed twice in Ca++/Mg++-free phosphate-buffered saline (PBS) and finally, resuspended in RPMI containing 1% FCS at 10 x 106 polymorphonuclear neutrophil/ml. Cell purity was >97%, as quantified by flow cytometry, and cell viability was >96%, as assessed by trypan blue dye exclusion.

Isolation of monocytes
Monocytes were isolated by countercurrent centrifugal elutriation from leukocyte-enriched buffy coats, kindly provided by the local blood bank. Initially, peripheral blood mononuclear cells were separated by density gradient centrifugation on Ficoll-Paque gradients (400 g, 35 min). Then, monocytes were purified by elutriation in a Beckmann centrifugal elutriator (JE-5.0 elutriation system). Purity was determined by fluorescence-associated cell sorting, and only fractions containing ≥90% monocytes, <1% granulocytes, and <10% lymphocytes were used for experiments. Cell viability was always > 97%, as assessed by the trypan blue dye exclusion test.

Cell culture and stimulation
Monocytes and neutrophils were resuspended in RPMI supplemented with 1% FCS, plated in 24-well tissue-culture plates at 106/ml (monocytes) or 107/ml (neutrophils), each at 500 µl/well, and incubated at 37°C in a 5% CO2-humidified atmosphere. To induce surface expression of PR3 on neutrophils, cells were stimulated with TNF-{alpha} (2 ng/ml) for 30 min, whereas isolated monocytes were constitutively expressing surface PR3. Prior to stimulation with LPS (10 ng/ml), LTA (1 µg/ml), or TNF-{alpha} (10 ng/ml), cells were preincubated with media alone, with murine monoclonal anti-PR3 antibodies (1 µg/ml), or with purified c-ANCA IgG (100 µg/ml), originating from WG serum for various time periods (2–12 h). Mouse IgG1 isotype control (1 µg/ml) and normal human IgG (100 µg/ml) from healthy donors were used as control antibodies. In experiments designed to investigate the effects of anti-CD14 antibodies on agonist-induced IL-8 generation, the antibody MY-4 (5 µg/ml) was added simultaneously with LPS, LTA, or TNF-{alpha}. After 18 h of incubation, cell supernatants were harvested, debris was removed by centrifugation, and samples were stored at –20°C until further processing.

Cytokine ELISAs
Release of TNF-{alpha}, IL-6, and IL-8 was determined in a direct sandwich ELISA. In brief, immunoassay plates were coated with mouse monoclonal anti-human TNF-{alpha}, IL-6, or IL-8 antibodies at a concentration of 4 µg/ml. After a blocking period, samples were added. Recombinant human TNF-{alpha}, IL-6, and IL-8 were used for standard titration curves. To sandwich the antigen, biotinylated antibodies were applied at the following concentrations: 400 ng/ml polyclonal anti-TNF-{alpha}, 50 ng/ml anti-IL-6, or 40 ng/ml anti-IL-8. Plates were subsequently incubated with HRP-conjugated streptavidin followed by addition of substrate (H2O2 and ABTS). Absorbance was measured at 450 nm in a microplate reader using SLT LabInstruments software (Creilsheim, Germany) to analyze the generated data. IL-8 and IL-6 ELISA were sensitive to 15 pg/ml and TNF-{alpha} to 7 pg/ml.

Flow cytometry
For the determination of CD14, TLR2, and TLR4 surface expression, flow cytometry was performed. In brief, after incubation with anti-PR3 antibodies or IgGc, leukocytes were washed twice in ice-cold PBS containing 0.1% bovine serum albumin and 0.02% sodium acid. Then, 2 x 105 cells were distributed to each well of flexible round-bottom microtiter plates. Prior to the addition of a PE-labeled monoclonal anti-CD-14 antibody (LeuM3, 10 µg/ml), PE-labeled antibodies targeting TLR2 (TL2.1; 20 µg/ml) or TLR4 (HTA 25; 20 µg/ml), FITC-labeled LPS, or equal concentrations of the respective isotype-matched control antibodies, 20 µl pooled human Ig (100 µg/ml), were added to block leukocyte FcIgG receptors. As a negative control, incubation with a PE/FITC-labeled irrelevant antibody was performed. After 30 min of incubation with the specific antibodies or LPS, three washes were performed, and cells were resuspended in PBS and kept on ice until flow cytometric analysis, which was performed on a FACScan (Becton Dickinson, Mountain View, CA) using forward and orthogonal light-scatter to select viable cells. CellQuest® research software (Becton Dickinson, Mountain View, CA) was used to analyze the generated data.

Statistics
For statistical comparison, one-way ANOVA was performed, followed by Tukey’s honestly significant difference test when appropriate. A level of P< 0.05 was considered to be significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Anti-PR3 antibodies amplify LPS-induced, proinflammatory cytokine secretion in monocytes and neutrophils
Incubation of isolated monocytes and neutrophils with various concentrations of murine monoclonal anti-PR3 antibodies (WGM2; 0.01–10 µg/ml) for 24 h resulted in a dose-dependent release of IL-8 into the cell supernatants of monocytes and neutrophils, and equal concentrations of an isotype-matched mouse IgGc were ineffective (Fig. 1 ). However, when compared with leukocyte activation by 10 ng/ml LPS (111.62±23.13 ng/ml IL-8 for monocytes and 7.11±1.89 ng/ml IL-8 for neutrophils), the quantities of IL-8 formation elicited, even by treatment with high concentrations of anti-PR3 antibodies (10 µg/ml), were comparatively low (25.3±4.6 ng/ml IL-8 for monocytes and 1.1±0.22 ng/ml IL-8 for neutrophils).



View larger version (13K):
[in this window]
[in a new window]
  		Figure 1. Dose-dependent release of IL-8 from leukocytes in response to anti-PR3 antibodies. Isolated monocytes (0.5x106/ml) or neutrophils (5x106/ml) were exposed to various concentrations of monoclonal anti-PR3 antibodies (WGM2, anti-PR3; 0.01–10 µg/ml) or equal concentrations of isotype-matched IgGc (MOPC-21) for 24 h, or sham incubation was performed. IL-8 released into the supernatant is given in ng/ml. Data reflect mean ± SEM of at least three independent experiments. Values marked * differ significantly from IgGc-incubated leukocytes.

 
When leukocytes were preincubated with 1 µg/ml WGM2 for 6 h and subsequently challenged with LPS (10 ng/ml) for a further 18 h, a massive amplification of LPS-induced IL-8 formation was noted in monocytes and neutrophils (Fig. 2 ). The anti-PR3-elicited priming could be reproduced by a second monoclonal anti-PR3 antibody (12.8) targeting a different epitope of the autoantigen but not by an isotype-matched mouse IgGc.



View larger version (11K):
[in this window]
[in a new window]
  		Figure 2. Amplification of LPS-induced IL-8 release after preincubation with anti-PR3 antibodies. Isolated monocytes (0.5x106/ml) or neutrophils (5x106/ml) were preincubated for 6 h with two different monoclonal anti-PR3 antibodies (WGM2 or 12.8; 1 µg/ml) with equal amounts of an isotype-matched IgGc, or sham incubation was performed (control). Subsequently, leukocytes were challenged with LPS (10 ng/ml) for 18 h. IL-8 released into the supernatant is given in ng/ml. Data reflect mean ± SEM of at least five independent experiments. Values marked differ significantly from sham-incubated leukocytes.

 
Apart from amplification of leukocyte IL-8 secretion, a comparable priming effect for monocyte TNF-{alpha} and IL-6 release occurred in anti-PR3-pretreated cells (Fig. 3 ). In fact, release of TNF-{alpha} increased from 4.2 ± 0.95 ng/ml to 9.0 ± 3.6 ng/ml in WGM2- and to 12.3 ± 1.4 ng/ml in 12.8-pretreated cells in response to LPS stimulation (n=4). In contrast, both anti-PR3 antibodies elicited the release of only low quantities of this proinflammatory cytokine in the absence of LPS (0.25±0.03 ng/ml for WGM2 and 0.43±0.09 ng/ml for 12.8, n=4). Comparable results were obtained for IL-6: The LPS-induced synthesis of this cytokine was increased from 34 ± 0.7 ng/ml to 82.9 ± 1.3 ng/ml upon priming with WGM2 and to 95.4 ± 3.7 ng/ml upon priming with 12.8 (n=4), and IL-6 release induced by the anti-PR3 antibodies per se was low (1.25±0.34 ng/ml for WGM2 and 1.86±0.24 ng/ml for 12.8, n=4). These data again support the concept of primed leukocyte behavior upon anti-PR3 challenge.



View larger version (10K):
[in this window]
[in a new window]
  		Figure 3. Reproduction of the anti-PR3-related priming for monocyte TNF-{alpha} and IL-6 synthesis. Isolated monocytes (0.5x106/ml) were preincubated for 6 h with two different monoclonal anti-PR3 antibodies (WGM2 or 12.8; 1 µg/ml) with equal amounts of an isotype-matched IgGc, or sham incubation was performed (control). Subsequently, leukocytes were challenged with LPS (10 ng/ml) for 18 h. TNF-{alpha} (upper figure) and IL-6 (lower figure) released into the supernatant are given in ng/ml. Data reflect the means ± SEM of at least four independent experiments given. Values marked differ significantly from sham-incubated leukocytes.

 
When leukocytes were preincubated with three different human ANCA-IgG (100 µg/ml) preparations, purified from the sera of patients with monospecific anti-PR3-positive WG, amplification of LPS-induced IL-8 generation was reproduced (Fig. 4 ), and normal human IgG was ineffective. Similar to monoclonal anti-PR3 antibodies, treatment with ANCA IgG per se elicited the release of some minor quantities of IL-8 from monocytes (20.2±6.8 ng/ml) and neutrophils (1.1±0.33 ng/ml), thus confirming that the increase in LPS-induced IL-8 generation upon ANCA preincubation was not simply a result of an additive effect but a result of a priming phenomenon.



View larger version (14K):
[in this window]
[in a new window]
  		Figure 4. Amplification of LPS-induced IL-8 release after preincubation with c-ANCA IgG. Isolated monocytes (0.5x106/ml) or neutrophils (5x106/ml) were preincubated for 6 h with three different c-ANCA-IgG preparations (100 µg/ml) or with equal amounts of normal human IgG (IgGc), or sham incubation was performed (control). Subsequently, leukocytes were challenged with LPS (10 ng/ml) for 18 h. IL-8 released into the supernatant is given in ng/ml. Data reflect the mean ± SEM of at least three independent experiments. Values marked differ significantly from sham-incubated leukocytes.

 
Time course of the anti-PR3-elicited priming response
Next, analysis of the kinetics of the anti-PR3-elicited priming response was performed. Therefore, monocytes and neutrophils were incubated with monoclonal anti-PR3 antibodies (WGM2) for various time periods prior to LPS challenge. Although similar with respect to the magnitude of the priming reaction, the kinetics of the leukocyte response to anti-PR3 pretreatment differed between monocytes and neutrophils (Fig. 5 ): Although monocytes responded only with a slight elevation of IL-8 release after a 2-h priming period with anti-PR3, LPS-induced IL-8 generation was already significantly elevated at this time-point in neutrophils. In monocytes and neutrophils, priming was maximally enhanced after 6 h of anti-PR3 treatment. Longer priming periods (12 h) were still effective in monocytes but not in neutrophils.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 5. Time course of the anti-PR3-related priming for LPS-induced IL-8 generation. Isolated monocytes (0.5x106/ml) or neutrophils (5x106/ml) were preincubated for various time periods with 1 µg/ml monoclonal anti-PR3 antibodies (WGM2) with equal amounts of an isotype-matched IgGc, or sham incubation was performed. Subsequently, leukocytes were challenged with LPS (10 ng/ml) for 18 h. IL-8 released into the supernatant is given as percentage of LPS-induced IL-8 generation in sham-primed cells. Data reflect the mean ± SEM of at least four independent experiments. Values marked differ significantly from sham-incubated leukocytes.

 
Reproduction of the anti-PR3-related priming for LTA but not for TNF-{alpha} stimulation
When investigating whether the anti-PR3-related priming was also true for other agonists, an impressive amplification of monocyte and neutrophil IL-8 generation upon stimulation with S. aureus LTA was obtained (Fig. 6 ). After a 6-h pretreatment period with monoclonal anti-PR3 antibodies but not with isotype-matched IgGc, the LTA-induced IL-8 liberation from monocytes and neutrophils was approximately doubled in anti-PR3-pretreated cells, corresponding well with the priming response previously observed for LPS-induced leukocyte activation. In contrast, when the proinflammatory cytokine TNF-{alpha} was used for leukocyte activation, no priming for IL-8 generation was observed after anti-PR3 pretreatment (Fig. 6) .



View larger version (12K):
[in this window]
[in a new window]
  		Figure 6. Anti-PR3 priming of LTA-induced IL-8 release—comparison with TNF-{alpha} challenge. Isolated monocytes (0.5x106/ml) or neutrophils (5x106/ml) were preincubated for 6 h with monoclonal anti-PR3 antibodies (WGM2; 1 µg/ml) with equal amounts of an isotype-matched IgGc, or sham incubation was performed. Subsequently, leukocytes were challenged with LTA (1 µg/ml) or TNF-{alpha} (10 ng/ml) for 18 h. IL-8 released into the supernatant is given in ng/ml. Data reflect the means ± SEM of at least four independent experiments. Values marked differ significantly from sham-incubated leukocytes.

 
Anti-PR3 antibodies prime leukocytes by up-regulation of CD14 expression
As it is well known that LPS- and LTA- but not TNF-{alpha}-induced leukocyte activation proceeds via CD14, we speculated that the autoantibodies modified the leukocyte response to the bacterial cell-wall components via regulation at the receptor level. Therefore, monocytes and neutrophils were assessed for CD14 surface expression by flow cytometry after anti-PR3 or IgGc challenge. As observed for CD14 expression after a priming period for 6 h, anti-PR3 antibodies markedly increased the membrane expression of this CD14 in both leukocyte types as compared with isotype-matched IgGc (Fig. 7 ).



View larger version (25K):
[in this window]
[in a new window]
  		Figure 7. Up-regulation of leukocyte membrane CD14 in response to anti-PR3. Isolated monocytes (A; 0.5x106/ml) or neutrophils (B; 5x106/ml) were incubated with IgGc (1 µg/ml; gray line) or anti-PR3 antibodies (WGM2, 1 µg/ml; black line). After 6 h of incubation, CD14 membrane expression was assessed by flow cytometry. Filled graphs represent isotype control-labeled cells. Representative data of three independent flow cytometric studies are given.

 
The dependence on CD14 of LPS- or LTA-induced IL-8 generation was evident from the inhibitory capacity of the function-blocking anti-CD14 antibody MY-4 (5 µg/ml) on agonist-induced IL-8 generation. When MY-4 was admixed prior to LPS challenge, IL-8 generation was inhibited by 63 ± 16.3% in monocytes and by 75.1 ± 5.9% in neutrophils (Table 1) . Comparable effects were obtained for LTA-induced IL-8 generation: 64.9 ± 22.3% inhibition in monocytes and 53.08 ± 6.1% inhibition in neutrophils was observed in MY-4-treated cells. In contrast, leukocyte preincubation with MY-4 did not affect the TNF-{alpha}-related generation of IL-8 in both leukocyte types (data not shown).


View this table:
[in this window]
[in a new window]
  		Table 1. Effect of Anti-CD14 Antibodies on the Anti-RP3-Induced Priming of Leukocyte IL-8 Generation in Response to LPS

 
Moreover, when the anti-CD14 antibody MY-4 was used in experiments with monocytes and neutrophils undergoing preceding anti-PR3 priming for 6 h, strong inhibition of the LPS-induced IL-8 generation was again noted (Table 1) .

It is interesting that the kinetics of CD14 up-regulation, as assessed in monocytes and neutrophils after 2, 6, and 12 h of anti-PR3 treatment by flow cytometry, correlated well with the previously assessed time dependence of the priming of the cytokine response: Corresponding to priming of LPS-induced IL-8 generation in monocytes, we found a maximal up-regulation of CD14 after 6 h of preincubation, which was still evident after 12 h in this leukocyte type, versus a faster and more transient response in neutrophils, with up-regulation of CD14 already evident after 2 h of anti-PR3 treatment, peaking at 6 h and rapidly declining thereafter (Fig. 8 ).



View larger version (11K):
[in this window]
[in a new window]
  		Figure 8. Time course of up-regulation of CD14 expression in response to anti-PR3. Isolated monocytes (0.5x106/ml) or neutrophils (5x106/ml) were incubated with IgGc (1 µg/ml) or anti-PR3 antibodies (WGM2, 1 µg/ml). After 2, 6, or 12 h of incubation, CD14 membrane expression was assessed by flow cytometry. Data are expressed as CD14 expression as a percentage over controls, and controls represent IgGc-incubated cells. Data reflect the mean ± SEM of at least three independent experiments.

 
Anti-PR3 antibodies do not affect surface expression of TLR2 or TLR4 on monocytes
As the subtypes of TLRs TLR2 and TLR4 have recently been identified as key molecules in mediating LPS (TLR4)- and LTA (TLR2)-induced cellular activation [18 , 19 ], we investigated whether the increased leukocyte response to bacterial factors corresponded with an increased expression of these molecules on the leukocyte surface. As monocytes, as opposed to neutrophils, represent the prototype of a TLR-expressing cell [32 ], studies about TLR expression were done in these cells. However, preatreatment with anti-PR3 antibodies did not affect the expression of TLR2 and TLR4 on monocytes (Table 2) .


View this table:
[in this window]
[in a new window]
  		Table 2. Lack of Regulation of TLR2 or TLR4 Expression by Anti-PR3 Antibodies

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Among the ANCA, those targeting PR3 have a strong and specific association with WG [20 ]. Besides their significance as a seromarker, a pathogenetic role has been proposed for these autoantibodies as a result of their capacity to directly activate leukocytes in vitro [22 23 24 25 ]. In the present study, an alternative approach was chosen to define the effect of PR3 ANCA on inflammatory leukocyte functions: Preincubation of isolated monocytes and TNF-pretreated neutrophils with substimulatory concentrations of anti-PR3 antibodies were performed, and the impact of this priming on subsequent leukocyte activation with bacterial cell-wall components was evaluated. Generation of IL-8, which is recognized to play a pathogenetic role in ANCA-associated vasculitis [28 ], and other proinflammatory cytokines, such as TNF-{alpha} and IL-6, were noted to be strongly up-regulated by anti-PR3 priming in monocytes and neutrophils.

The background for this experimental rationale is based on the fact that disease activation in WG, which is paralleled by a rising c-ANCA titer [20 , 21 ], appears to be triggered by bacterial infections [7 8 9 10 ]. Therefore, simultaneous exposure of leukocytes to proinflammatory cytokines, inducing surface expression of the ANCA-target antigen PR3, such as TNF-{alpha} used for neutrophil pretreatment in the current study circulating anti-PR3 antibodies and bacterial cell-wall components, actively secreted or liberated as a result of antibiotic treatment [33 ], may likely occur in active WG. In vivo, the leukocyte response to bacterial pathogens depends on the state of cellular activation, varying from "dormant" via "primed" to "fully activated." Priming is a key mechanism involved in the regulation of the leukocyte-dependent host defense: Although not directly activating secretory neutrophil and monocyte functions, priming agents induce a "sensitization" of the leukocytes for subsequent stimulation with naturally occurring agonists, such as bacterial-derived products [34 , 35 ]. In case of local or systemic microbial invasion, full-blown leukocyte activation would then follow priming. As a result of its important role in the regulation of inflammatory leukocyte functions, the phenomenon of leukocyte priming may also be relevant to the pathogenesis of vasculitis. Indeed, in active WG, neutrophils and monocytes display a phenotype attributable to a state of cellular preactivation with enhanced surface expression of activation markers such as CD11b and CD64 [36 , 37 ].

In the present study, we hypothesized that c-ANCA, apart from their direct prosecretory capacity, may induce leukocyte priming and sensitize these effector cells for subsequent activation with cell-wall components of Gram-negative (LPS) and Gram-positive (LTA) bacteria. After a 6-h pre-exposure of both leukocyte types to two different murine monoclonal anti-PR3 antibodies, markedly enhanced generation of IL-8 in response to stimulation with LPS was indeed noted. This phenomenon was not observed when isotype-matched IgGc was used in place of the anti-PR3 antibodies. Moreover, in isolated monocytes, an impressive priming of TNF-{alpha} and IL-6 synthesis was also observed. The priming response for IL-8 was reproduced by ANCA IgG, using IgG preparations from three different patients with monospecific, anti-PR3-positive WG but not with IgG from healthy donors. Therefore, specific targeting of PR3, present on the surface of isolated leukocytes, most likely underlies the ANCA-mediated priming response. Although anti-PR3 antibodies have previously been described to directly induce neutrophil [28 ] and monocyte [26 ] cytokine generation, the presently observed, strong increase in agonist-induced chemokine generation after anti-PR3 pretreatment was clearly not merely a result of an additive effect but rather, was caused by a real priming phenomenon. When compared with LPS-induced leukocyte activation, resulting in the liberation of large amounts of IL-8 from monocytes (~150 ng/ml) and neutrophils (~10 ng/ml), the quantities of IL-8 release elicited by the currently used anti-PR3 antibodies per se were comparatively low (~20 ng/ml for monocytes and ~1 ng/ml for neutrophils), corresponding well to previous investigations [26 , 28 ]. It is important that endotoxin contamination of the murine and human anti-PR3 antibodies, which has to be considered, as even low doses of LPS are capable of inducing leukocyte priming, was excluded in the highly sensitive kinetic Limulus assay system.

When attempting to reproduce the ANCA-induced priming by using agonists other than endotoxin, it became evident that the priming response was restricted to leukocyte activation by bacterial cell-wall components. After anti-PR3 pretreatment, a marked amplification of leukocyte IL-8 generation was noted upon stimulation with LTA derived from S. aureus, whereas chemokine synthesis elicited by TNF-{alpha} (and other cytokines, such as IL-1ß and granulocyte macrophage-colony stimulating factor; data not given) was not influenced by the anti-PR3 antibodies.

It is interesting that in this context, we have recently described a short-term priming effect by anti-PR3 antibodies on neutrophil activation with the bacterial-derived chemotactic peptide formyl-Met-Leu-Phe (fMLP) [38 ], which resulted in enhanced leukotriene synthesis and chemotactic movement toward the peptide. It is important that the priming response to LPS and LTA stimulation, which we describe here, displays some fundamental differences from our previous data. Priming for subsequent stimulation with fMLP required lower concentrations of anti-PR3 antibodies and had a rapid and transient character, occurring within minutes of anti-PR3 challenge. These differences may be a result of the different mechanisms underlying the priming phenomena. Changes in the affinity of the formyl peptide receptor most probably affect the priming toward the chemotactic peptide [38 ], which takes place within seconds to minutes, and up-regulation of CD14 requires mobilization from intracellular pools and/or activation of transcriptional processes.

In contrast to TNF-{alpha}-mediated IL-8 secretion, which involves ligation of TNF receptor p55 and p75, LPS- and LTA-induced leukocyte activation is known to proceed via initial binding to the CD14 receptor, expressed abundantly by monocytes and by a subset of neutrophils [39 ]. In the present study, the CD14 dependence of leukocyte activation by the bacterial cell-wall components was again proven by the inhibitory capacity of the function-blocking antibody MY-4. In contrast, MY-4 did not inhibit TNF-{alpha}-induced leukocyte activation, thus arguing against any nonspecific inhibitory effect of the CD-14 antibody.

As LPS- and LTA-mediated leukocyte activation was largely dependent on CD14, whereas TNF-{alpha}-elicited cellular activation was an independent event, we speculated that the anti-PR3 antibodies modified the leukocyte response to the bacterial cell-wall components at the receptor level. Indeed, when analyzed for CD14 membrane expression after a 6-h anti-PR3 pretreatment versus IgGc, an up-regulation of this receptor molecule was observed on neutrophils and monocytes.

CD14, initially described as a marker to identify cells of the monocyte/macrophage lineage, was identified in 1990 as a receptor for endotoxin [16 ]. Besides recognizing LPS, recent studies demonstrated that CD14 equally confers cellular responsiveness to Gram-positive cell-wall components, including LTA, peptidoglycan [17 , 19 , 40 ], and recently isolated molecules derived from S. aureus [41 ]. Corresponding to its receptor function, the expression of this GPI-linked protein is not stationary but subject to distinct regulatory processes, and several agents have been described to up-regulate membrane CD14 expression by de novo synthesis or by mobilization from preformed intracellular stores [42 , 43 ]. In monocytes, de novo synthesis of CD14 is believed to be the more important pathway involved in the regulation of CD14 membrane expression, whereas in neutrophils, CD14 is stored in an intracellular compartment, thus rapidly available for translocation to the membrane surface [44 ]. The different kinetics of the leukocyte responses to anti-PR3 priming with a rapid reaction by neutrophils and a more prolonged response by monocytes may thus be attributable to translocation of CD14 from an intracellular store in neutrophils and a de novo synthesis of this molecule in monocytes. In line with this reasoning, Nowack et al. [45 ] recently described an increase in CD14 mRNA in monocytes following incubation with c-ANCA with comparable kinetics. Besides acting as a surface receptor for bacterial wall components, CD14 has been implicated in the regulation of adherence and apoptosis in leukocytes, and an increase is associated with a proadhesive state and prolonged survival [46 , 47 ]. Such events might additionally promote the persistence of inflammatory events and granuloma formation.

However, the observation that anti-CD14 antibody MY-4 blocked LPS-induced IL-8 secretion by ~70% but not completely may reflect the fact that other surface molecules in addition to CD14 may confer leukocyte responsiveness to bacterial cell-wall components. Therefore, from the current data, displaying an approximate twofold increase in surface CD14 on monocytes and a weaker up-regulation on neutrophils but a constant two- to threefold augmentation of the cytokine response, one could speculate that CD14 is not the only cell surface receptors for bacterial cell-wall components to be up-regulated by anti-PR3 antibodies. An up-regulation of TLR2 and TLR4, key molecules conferring cellular responsiveness to LPS and LTA, has been excluded in the current study. It is interesting that up-regulation of CD18, which has been implicated as a LPS receptor [48 ], was recently demonstrated to occur in ANCA-stimulated monocytes [45 ]. Alternatively, post-CD14 receptor signal transduction pathways, such as the activation of protein kinases, may be stimulated by ANCA [49 , 50 ].

In conclusion, c-ANCA, being only a weak, direct activator of monocyte and neutrophil cytokine release per se, exert a major priming effect on these leukocytes, enhancing their responsiveness to secondary stimulation with bacterial cell-wall components of Gram-positive and Gram-negative bacteria. Up-regulation of the CD14 molecule, acting as a cellular receptor for LPS and LTA, was characterized as one mechanism underlying the anti-PR3-elicited priming response. Such cooperation between anti-PR3 antibodies and bacterial cell-wall components may well trigger exacerbations of disease activity during infections and contribute to the persistence of inflammatory lesions and granuloma formation in WG.


   ACKNOWLEDGEMENTS
 
This work was supported by the Deutsche Forschungsgemeinschaft (GR 534; SFB 547/B8).

Received September 8, 2002; revised March 9, 2005; accepted March 26, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Jennette, J. C. (1991) Antineutrophil cytoplasmic autoantibody-associated diseases: a pathologist’s perspective Am. J. Kidney Dis. 18,164-170[Medline]
  2. Rastaldi, M. P., Ferrario, F., Tunesi, S., Yang, L., D’Amicio, G. (1996) Intraglomerular and interstitial leukocyte infiltration, adhesion molecules, and interleukin 1{alpha} expression in 15 cases of antineutrophil cytoplasmic autoantibody-associated renal vasculitis Am. J. Kidney Dis. 27,48-57[Medline]
  3. Brouwer, E., Huitema, M. G., Mulder, A. H. L., Heeringa, P., van Goor, H., Cohen Tervaert, J. W., Weening, J. J., Kallenberg, C. G. M. (1994) Neutrophil activation in vitro and in vivo in Wegener’s granulomatosis Kindey Int. 45,1120-1131[Medline]
  4. Muller Kobold, A. C., Kallenberg, C. G., Tervaert, J. W. (1999) Monocyte activation in patients with Wegener’s granulomatosis Ann. Rheum. Dis. 58,237-245[Abstract/Free Full Text]
  5. Rastaldi, M. P., Ferrario, F., Crippa, A., Dell’Antonio, G., Casartelli, D., Grillo, C., D‘Amicio, G. (2000) Glomerular monocyte-macrophage features in ANCA-positive renal vasculitis and glomerulonephritis J. Am. Soc. Nephrol. 11,2036-2043[Abstract/Free Full Text]
  6. Cockwell, P., Savage, C. O. S. (2000) Role of leukocytes in the immunopathogenesis of ANCA-associated glomerulonephritis Nephron 85,287-306[CrossRef][Medline]
  7. Falk, R. J., Hogan, S., Carey, T. S., Jennette, J. C. (1990) Clinical course of anti-neutrophil cytoplasmic autoantibodies-associated glomerulonephritis and systemic vasculitis Ann. Intern. Med. 113,656-663
  8. Stegeman, C. A., Cohen-Tervaert, J. W., de Jong, P. E., Kallenberg, C. G. M. (1996) Tripethoprim-sulfamethoxazole (co-trimoxazole) for the prevention of relapses of Wegener’s granulomatosis. Dutch Co-Trimoxazole Wegener Study Group N. Engl. J. Med. 335,16-20[Abstract/Free Full Text]
  9. DeRemee, R. A. (1988) The treatment of Wegener’s granulomatosis with trimethoprim/sulfamethoxazol: illusion or vision? Arthritis Rheum. 31,1068-1072[Medline]
  10. Stegemann, C. A., Cohen Tervaert, J. W., Sluiter, W. J., Manson, W., De Jong, P. E., Kallenberg, C. G. M. (1994) Association of chronic nasal carriage of Staphylococcus aureus and higher relapse rates in Wegener’s granulomatosis Ann. Intern. Med. 113,12-17
  11. Fischer, W., Mannsfeld, T., Hagen, G. (1990) On the basic structure of poly (glycerophosphate) lipoteichoic acids Biochem. Cell Biol. 68,33-43[Medline]
  12. DeLeo, F. R., Renee, J., McCormick, S., Nakamura, M., Apichella, M., Weiss, J., Naussee, W. M. (1998) Neutrophils exposed to bacterial lipopolysaccharide upregulate NADPH oxidase assembly J. Clin. Invest. 101,455-463[Medline]
  13. Peveri, P., Walz, A., Dewald, B., Baggiolini, M. (1988) A novel neutrophil-activating factor produced by human mononuclear phagocytes J. Exp. Med. 167,1547-1559[Abstract/Free Full Text]
  14. Kawamura, N., Imanishi, N., Koike, H., Nakahara, H., Phillips, L., Morooka, S. (1995) Lipoteichoic acid-induced neutrophil adhesion via E-selectin to human umbilical vein endothelial cells (HUVECs) Biochem. Biophys. Res. Commun. 217,1208-1215[CrossRef][Medline]
  15. Standiford, T. J., Arenberg, D. A., Danforth, J. M., Kunkel, S. L., VanOtteren, G. M., Strieter, R. M. (1994) Lipoteichoic acid induces secretion of interleukin-8 from human blood monocytes: a cellular and molecular analysis Infect. Immun. 62,119-125[Abstract/Free Full Text]
  16. Wright, S. D., Ramos, R. A., Tobias, P. S., Ulevitch, R. J., Mathison, J. C. (1990) CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein Science 249,1431-1433[Abstract/Free Full Text]
  17. Cleveland, M. G., Gorham, J. D., Murphy, T. L., Tuomanen, E., Murphy, K. M. (1996) Lipoteichoic acid preparations of gram-positive bacteria induce interleukin-12 through a CD14-dependent pathway Infect. Immun. 64,1906-1912[Abstract]
  18. Qureshi, S. T., Lariviere, L., Leveque, G., Clermont, S., Moore, K. J., Gros, P., Malo, D. (1999) Endotoxin-tolerant mice have mutations in Toll-like receptor 4 J. Exp. Med. 189,615-619[Abstract/Free Full Text]
  19. Schwandner, R., Dziarski, R., Wesche, H., Rothe, M., Kirschning, C. J. (1999) Peptidyglycan- and lipoteichoic acid-induced cell activation is mediated by Toll-like receptor 2 J. Biol. Chem. 274,17406-17409[Abstract/Free Full Text]
  20. van der Woude, F. J., Rasmussen, N., Lobatto, S., Wiik, A., Permin, H., van Es, L. A., van der Giessen, M., van der Hem, G. K., The, T. H. (1985) Autoantibodies against neutrophils and monocytes: tool for diagnosis and marker of disease activity in Wegener’s granulomatosis Lancet 1,425-429[Medline]
  21. Tervaert, J. W., van der Woude, F. J., Fauci, A. S., Ambrus, J. L., Velosa, J., Keane, W. F., Meijer, S., van der Giessen, M., van der Hem, G. K., The, T. H., et al (1989) Association between active Wegener’s granulomatosis and anticytoplasmic antibodies Arch. Intern. Med. 149,2461-2465[Abstract]
  22. Falk, R. J., Terrell, R. S., Charles, L. A., Jennette, J. C. (1990) Anti-neutrophil cytoplasmic autoantibodies induce neutrophils to degranulate and produce oxygen radicals in vitro Proc. Natl. Acad. Sci. USA 87,4115-4119[Abstract/Free Full Text]
  23. Grimminger, F., Hattar, K., Papavassilis, C., Temmesfeld, B., Csernok, E., Gross, W. L., Seeger, W., Sibelius, U. (1996) Neutrophil activation by anti-proteinase 3 antibodies in Wegener’s granulomatosis: role of exogenous arachidonic acid and leukotriene B4 generation J. Exp. Med. 184,1567-1572[Abstract/Free Full Text]
  24. Savage, C. O. S., Pottinger, B. E., Gaskin, G., Pusey, C. D., Pearson, J. D. (1992) Autoantibodies developing to myeloperoxidase and proteinase 3 in systemic vasculitis stimulate neutrophil cytotoxicity towards cultured endothelial cells Am. J. Pathol. 141,335-342[Abstract]
  25. Muller Kobold, A. C., van der Geld, Y., Limburg, P. C., Tervaert, J. W., Kallenberg, C. G. (1999) Pathophysiology of ANCA-associated glomerulonephritis Nephrol. Dial. Transplant. 14,1366-1375[Abstract/Free Full Text]
  26. Ralston, D. R., Marsh, C. B., Lowe, M. P., Wewers, M. D. (1997) Antineutrophil cytoplasmic antibodies induce monocyte IL-8 release J. Clin. Invest. 100,1416-1424[Medline]
  27. Casselman, B. L., Kilgore, K. S., Miller, B. F., Warren, J. S. (1995) Antibodies to neutrophil cytoplasmic antigens induce monocyte chemoattractant protein-1 secretion from human monocytes J. Lab. Clin. Med. 126,495-502[Medline]
  28. Cockwell, P., Brooks, C. J., Adu, D., Savage, C. O. S. (1999) Interleukin-8: a pathogenetic role in antineutrophil cytoplasmic autoantibody-associated glomerulonephritis Kidney Int. 55,852-863[CrossRef][Medline]
  29. Morath, S., Geyer, A., Hartung, T. (2001) Structure-function relationship of cytokine induction by lipoteichoic acids from Staphylococcus aureus J. Exp. Med. 193,393-397[Abstract/Free Full Text]
  30. Csernok, E., Lüdemann, J., Gross, W. L., Bainton, D. F. (1990) Ultrastructural localization of proteinase 3, the target antigen of anti-cytoplasmic antibodies circulating in Wegener’s granulomatosis Am. J. Pathol. 137,1113-1120[Abstract]
  31. Moosig, F., Csernok, E., Kumanovics, G., Gross, W. L. (2000) Opsonization of apoptotic neutrophils by anti-neutrophil cytoplasmic antibodies (ANCA) leads to enhanced uptake by macrophages and increased release of tumor necrosis factor-{alpha} (TNF-{alpha}) Clin. Exp. Immunol. 122,499-503[CrossRef][Medline]
  32. Sabroe, I., Jones, E. C., Usher, L. R., Whyte, M. K. B., Dower, S. K. (2002) Toll-like receptor (TLR) 2 and TLR4 in human peripheral blood granulocytes: a critical role for monocytes in leukocyte lipopolysachharide responses J. Immunol. 168,4701-4710[Abstract/Free Full Text]
  33. Alkan, M. L., Beachey, E. H. (1978) Excretion of lipoteichoic acid by group A streptococci. Influence of penicillin on excretion and loss of ability to adhere to human oral mucosal cells J. Clin. Invest. 61,671-677
  34. Condliffe, A. M., Kitchen, E., Chilvers, E. R. (1998) Neutrophils priming: pathophysiological consequences and underlying mechanisms Clin. Sci. (Lond.) 94,461-471[Medline]
  35. Hallett, M. B., Lloyds, D. (1995) Neutrophil priming: the cellular signals that say amber, but not green Immunol. Today 16,264-268[CrossRef][Medline]
  36. Muller-Kobold, A. C., Kallenberg, C. G. M., Cohen-Tervaert, J. W. (1999) Monocyte activation in patients with Wegener’s granulomatosis Ann. Rheum. Dis. 58,237-245
  37. Muller-Kobold, A. C., Mesander, G., Stegman, C. A., Kallenberg, C. G. M., Cohen-Tervaert, J. W. (1998) Are circulating neutrophils intravascularly activated in patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis? Clin. Exp. Immunol. 114,491-499[CrossRef][Medline]
  38. Hattar, K., Sibelius, U., Bickenbach, A., Csernok, E., Seeger, W., Grimminger, F. (2001) Subthreshold concentrations of anti-proteinase 3 antibodies (c-ANCA) specifically prime human neutrophils for fMLP-induced leukotriene synthesis and chemotaxis J. Leukoc. Biol. 69,89-97[Abstract/Free Full Text]
  39. Landmann, R., Muller, B., Zimmerli, W. (2000) CD14, new aspects of ligand and signal diversity Microbes Infect. 2,295-304[CrossRef][Medline]
  40. Dziarski, R., Ulmer, A. J., Gupta, D. (2000) Interactions of CD14 with components of gram-positive bacteria Chem. Immunol. 74,83-107[Medline]
  41. Kusunoki, T., Hailman, E., Juan, T. S. C., 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]
  42. Cosentino, G., Soprana, E., Thienes, C. P., Siccardi, A. G., Viale, G., Vercelli, D. (1995) IL-13 down-regulates CD14 expression and TNF-a secretion in normal human monocytes J. Immunol. 155,3145-3151[Abstract]
  43. Marchant, A., Duchow, J., Deville, J. P., Goldmann, M. (1992) Lipopolysaccharide induced up-regulation of CD14 molecules on monocytes in human whole blood Eur. J. Immunol. 22,1663-1665[Medline]
  44. Detmers, P. A., Zhou, D., Powell, D., Lichenstein, H., Kellley, M., Pironkova, R. (1995) Endotoxin receptors (CD14) are found with CD16 (Fc{gamma}RIII) in an intracellular compartment of neutrophils that contains alkaline phosphatase J. Immunol. 155,2085-2095[Abstract]
  45. Nowack, R., Schwalbe, K., Flores-Suarez, L. F., Yard, B., van der Woude, F. J. (2000) Upregulation of CD14 and CD18 on monocytes in vitro by antineutrophil cytoplasmic autoantibodies J. Am. Soc. Nephrol. 11,1639-1646[Abstract/Free Full Text]
  46. Beekhuizen, H., Blokland, I., Corel-van Tilburg, A. J., Koning, F., van Furth, R. (1991) CD14 contributes to the adherence of human monocytes to cytokine-stimulated endothelial cells J. Immunol. 147,3761-3765[Abstract]
  47. Heidenreich, S., Schmidt, M., August, C., Cullen, P., Rademaekers, A., Pauels, H. G. (1997) Regulation of human monocyte apoptosis by the CD14 molecule J. Immunol. 159,3178-3188[Abstract]
  48. Perera, P. Y., Mayadas, T. N., Takeuchi, O., Akira, S., Zaks-Zilbermann, M., Goyert, S. M., Vogel, S. N. (2001) CD11b/CD18 acts in concert with CD14 and Toll-like receptors (TLR) to elicit full lipopolysaccharide and taxol-inducible gene expression J. Immunol. 166,574-581[Abstract/Free Full Text]
  49. Kettritz, R., Schreiber, A., Luft, F. C., Haller, H. (2001) Role of mitogen-activated protein kinases in activation of human neutrophils by antineutrophil cytoplasmic antibodies J. Am. Soc. Nephrol. 12,37-46[Abstract/Free Full Text]
  50. Hewins, P., Williams, J. M., Wakelam, M. J., Savage, C. O. (2004) Activation of Syk in neutrophils by antineutrophil cytoplasm antibodies occurs via Fc{gamma} receptors and CD18 J. Am. Soc. Nephrol. 15,796-808[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 CVIHome page
A. Uehara, Y. Hirabayashi, and H. Takada
Antibodies to Proteinase 3 Prime Human Oral, Lung, and Kidney Epithelial Cells To Secrete Proinflammatory Cytokines upon Stimulation with Agonists to Various Toll-Like Receptors, NOD1, and NOD2
Clin. Vaccine Immunol., July 1, 2008; 15(7): 1060 - 1066.
[Abstract] [Full Text] [PDF]

Home page
 Rheumatology (Oxford)Home page
C. Iking-Konert, T. Vogl, B. Prior, C. Wagner, O. Sander, E. Bleck, B. Ostendorf, M. Schneider, K. Andrassy, and G. M. Hansch
T lymphocytes in patients with primary vasculitis: expansion of CD8+ T cells with the propensity to activate polymorphonuclear neutrophils
Rheumatology, May 1, 2008; 47(5): 609 - 616.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0902442v1
78/4/992    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 Hattar, K.
Right arrow Articles by Sibelius, U.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hattar, K.
Right arrow Articles by Sibelius, U.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS