Journal of Leukocyte Biology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
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 Grimminger, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hattar, K.
Right arrow Articles by Grimminger, F.
(Journal of Leukocyte Biology. 2001;69:89-97.)
© 2001 by Society for Leukocyte Biology

Subthreshold concentrations of anti-proteinase 3 antibodies (c-ANCA) specifically prime human neutrophils for fMLP-induced leukotriene synthesis and chemotaxis

Katja Hattar*, Ulf Sibelius*, Annette Bickenbach*, Elena Csernok{dagger}, Werner Seeger* and Friedrich Grimminger*

* Department of Internal Medicine, Justus-Liebig-Universität, Giessen
{dagger} Department of Rheumatology, Medical University of Lübeck, Germany

Correspondence: F. Grimminger, M.D., Ph.D., Department of Internal Medicine, Justus-Liebig University Giessen, Klinikstrasse 36, D-35392 Giessen, Germany. E-mail: friedrich.grimminger{at}innere.med.uni-giessen.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Anti-neutrophil cytoplasmic antibodies (ANCA) targeting proteinase 3 (PR3) possess a high sensitivity and specificity for Wegener’s granulomatosis. Due to their capacity of directly activating neutrophils, a pathogenetic role for these autoantibodies has been proposed. We investigated the impact of subthreshold concentrations of monoclonal anti-PR3 antibodies (anti-PR3; 0.1 µg/mL) on neutrophil activation elicited by a secondary agent. Preincubation with anti-PR3 resulted in a massive amplification of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced leukotriene (LT) generation, with a marked increase in the liberation of LTB4, LTA4, and 5-hydroxyeicosatetraenoic acid (5-HETE). This priming commenced within 2.5 min, with a maximum after 5–7.5 min. Moreover, anti-PR3 pretreatment markedly enhanced PMN movement toward fMLP. The priming effect of anti-PR3 toward fMLP challenge was reproduced by c-ANCA, but not by F(ab)2 fragments of the antibodies and isotype-matched control IgG. Generation of superoxide anion and release of elastase were suppressed in anti-PR3-pretreated neutrophils undergoing fMLP challenge. In contrast, neutrophil activation by platelet-activating factor (PAF) or the calcium ionophore A23187 remained unaffected. We conclude that subthreshold concentrations of anti-PR3 antibodies selectively modify neutrophil responses to fMLP, with enhancement of leukotriene generation and chemotaxis, but suppression of respiratory burst and degranulation. Such priming might contribute to localized neutrophil accumulation together with blunted host defense in Wegener’s granulomatosis.

Key Words: anti-neutrophil-cytoplasmic antibodies • neutrophil activation • chemotactic peptide • Wegener’s granulomatosis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Anti-neutrophil-cytoplasmic antibodies (ANCA) targeting proteinase 3 (PR3) are a sensitive and specific marker for Wegener’s granulomatosis (WG) [1 ], a systemic vasculitis that may affect several organs and has a poor prognosis in full-blown cases. Histologically, WG is characterized by segmental fibrinoid necrosis of small vessels, accompanied by neutrophil infiltration [2 ]. There is now good evidence that, in addition to being a seromarker, PR3-ANCA play an active role in the pathogenesis of this disease [3 , 4 ]. Their interaction with neutrophils has been well characterized in vitro: in the presence of proinflammatory cytokines, the target antigen PR3 is translocated from the azurophilic granules to the neutrophil membrane [5 ], and subsequent autoantibody binding induces direct activation of a wide variety of inflammatory neutrophil functions such as degranulation, respiratory burst, and liberation of lipid mediators and cytokines [6 7 8 9 ]. Moreover, in leukocyte-endothelial cocultures, ANCA-induced polymorphonuclear neutrophil (PMN) activation is followed by lysis of endothelial cells [10 ], and in situ neutrophil activation correlates with the degree of tissue injury [11 ]. Thus, activated neutrophils seem to be major effector cells in the development of vasculitic lesions in WG.

In addition to direct activation of secretory neutrophil functions, sensitizing (priming) of neutrophils for subsequent activation plays an important role in neutrophil-mediated tissue injury. In vitro, when primed with bacterial lipopolysaccharides or proinflammatory cytokines, neutrophils show an enhanced secretory response upon subsequent stimulation with naturally occurring agonists such as bacterial n-formylated peptides or artificial stimuli like calcium ionophores [12 13 14 15 ]. In vivo, if adequately controlled, this enhanced responsiveness of primed neutrophils toward bacterial products can enhance resistance to bacterial infections. However, such up-regulation of PMN responsiveness may also result in progressive tissue destruction under pathophysiological conditions.

Against this background, we investigated whether subthreshold concentrations of anti-PR3-antibodies are capable of sensitizing neutrophils for enhanced responsiveness to proinflammatory agents. Our interest was particularly centered upon bacterial n-formylated peptides because exposure of PMN to these potent agonists is very likely to occur in active WG, with exacerbations of this disease being closely related to infections [16 , 17 ]. In essence, we found that upon preincubation with low doses of anti-PR3 antibodies, neutrophils displayed a dramatically altered response to subsequent stimulation with the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP), but not to the calcium ionophore A23187 or platelet-activating factor (PAF), with marked amplification of the fMLP-induced leukotriene generation and enhanced chemotactic movement toward this peptide. In contrast, elastase secretion and O2- generation were blunted in anti-PR3-pretreated PMN subsequently undergoing fMLP challenge. These anti-PR3-induced alterations of inflammatory neutrophil behavior may contribute to disturbances in host-defense capacity and mechanisms of tissue injury in WG.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Ficoll-Paque and a protein G column were purchased from Pharmacia (Uppsala, Sweden). Arachidonic acid (AA), cytochrome c, superoxide dismutase, fMLP, and isotype control mouse IgG1 (MOPC-21) were obtained from Sigma (Deisenhofen, Germany). A23187 and PAF-C18 were purchased from Calbiochem (La Jolla, CA), while recombinant human tumor necrosis factor {alpha} (TNF-{alpha}) was from R & D Systems (Wiesbaden, Germany). Phosphate-buffered saline (PBS) was obtained from GIBCO Laboratories (Grand Island, NY). The kinetic-OLC Limulus amebocyte cell lysate test for the detection of endotoxin was from Chromogenix (Mölndal, Sweden), and enzyme-linked immunosorbent assay (ELISA) kits for the detection of PR3-ANCA were from Orgentec (Mainz, Germany). S-2484, a substrate for neutrophil elastase, was purchased from Kabi-Vitrum (Stockholm, Sweden). fMLP-lys-FITC, a fluoresceinated peptide that retains fMLP activity and binding characteristics, was from Peninsula Laboratories (Belmont, CA). The leukotrienes (LT) LTC4, LTD4, LTE4, LTB4, 20-OH- and 20-COOH-LTB4, and the synthetic LTA4-methyl ester were generous gifts from Dr. J. Rokach (Merck Frosst, Toronto, Ontario, Canada). Additional LTs were graciously supplied by Dr. W. Bartmann (Hoechst, Frankfurt, Germany). 5-, 8-, 9-, 11-, 12-, And 15-hydroxyeicosatetraenoic acid (HETE), 5(S),12(S)-diHETE, 5,15-diHETE, and 12-HHT, as well as the non-enzymatic hydrolysis products of LTA4 (6-trans diasteromeric pairs of LTB4 and 5,6-diHETEs) were obtained from Paesel (Frankfurt, Germany). All LTs were checked for purity and quantified spectrophotometrically before use, as described [19 ]. Chromatographic supplies included an analytical high-performance liquid chromatography (HPLC) column (length x inner diameter = 250 mm x 4 mm; Shandon, Astmoor, UK) filled with ODS-Hypersil (particle size 3 µm; pore size 100 ;anA; Machery Nagel, Düren, Germany). HPLC-grade solvents, distilled in glass, were purchased from Fluka (Heidelberg, Germany). All other biochemicals were obtained from Merck (Darmstadt, Germany).

Anti-PR3-antibodies
Murine monoclonal antibodies targeting PR3 were prepared by hybridoma technology, as previously described [18 ]. The clone WGM2 (IgG1) was chosen for further experiments. F(ab)2 fragments were generated by digestion with pepsin in 0.1 NaAc for 16 h at 37°C. After dialysis against PBS, purity was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). ANCA-IgG was isolated from the sera of three patients with monospecific anti-PR3-positive WG, whereas normal IgG was purified from the sera of healthy volunteers, through the use of protein G columns, as described [18 ]. Specificity for PR3 of the human and murine monoclonal antibodies, as well as the ability of F(ab)2 fragments to recognize PR3 after the digestion procedure, was assessed in a commercial antigen-specific ELISA. Endotoxin contamination of the human and murine antibodies was below 15 pg/mL, as assessed by the kinetic-OLC Limulus amebocyte lysate (LAL) test.

Isolation of human neutrophils
Neutrophils were isolated from venous blood of healthy donors by centrifugation over a Ficoll-Paque gradient as previously described [8 ]. 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 1% polyvinyl alcohol. Residual erythrocytes were removed by hypotonic lysis, cells were washed twice in Ca2+/Mg2+-free PBS, and finally resuspended in PBS containing CaCl2 (1 mmol/L) and MgSO4 (1 mmol/L) at 5 x 106 PMN/mL (leukotriene synthesis, respiratory burst, degranulation) or at 5 x 107 PMN/mL (chemotaxis). Cell purity was >97%, as quantified by flow cytometry, and cell viability was >96%, as assessed by trypan blue dye exclusion.

Measurement of leukotrienes
LTs and HETEs were extracted from cell supernatants by octadecylsilyl solid-phase extraction columns, as described [8 , 19 ]. Reversed-phase HPLC was carried out on octadecylsilyl columns (Hypersil, 5-µm particles), with a mobile phase of methanol/water/acetic acid (72:28:0.16, pH 4.9) [19 ]. In addition to the conventional UV detection at 270 nm (LTs) and 237 nm (HETEs), a photodiode array detector (Waters model 990) was used, which provided full UV spectra (190–600 nm) of eluting compounds and allowed checking for peak purity and subtraction of possible coeluting material. All data obtained by the different analytical procedures were corrected for the respective recoveries and are given in picomoles per milliliter throughout the experiments. Recovery was determined by separate recovery experiments using different quantities of the individual compounds in the appropriate concentration range. Factors for recovery were further confirmed by addition of 0.2 µCi of [3H]LTB4 and 5-[3H]-HETE to buffer medium as internal standards in selected experiments. For quantification of LTs and 5-HETE, correspondence of values calculated from UV absorbency in two different chromatographic procedures was required (deviation <10%).

Superoxide anion generation
Neutrophil O2- generation was assessed as superoxide dismutase-inhibitable reduction of cytochrome c according to Cohen [20 ]. Duplicate reaction mixtures containing neutrophils (5 x 106/mL) and 75 µM ferricytochrome c were incubated at 37°C in the presence or absence of 10 µg/mL superoxide dismutase. Incubations were terminated by centrifugation at 4°C at 1200 g. O2- release was quantified as nanomoles of cytochrome c reduction, using an extinction coefficient of 21 mM-1 • cm-1 at 550 nm in a Uvicon Spectrophotometer.

Release of granular constituents
Elastase was taken as marker for neutrophil degranulation, and enzyme activity in the cell supernatant was measured by monitoring the turnover of L-pyroglutamyl-L-propyl-L-valine-p-nitro-anilide at 405 nm according to the method described by Kramps et al. [21 ].

Chemotaxis
The chemotactic response of neutrophils to a gradient of fMLP was measured under agarose according to Nelson et al. [22 ]. In brief, 1.5 mL of a 1% electrophoresis-grade agarose in medium 199 with 0.5% gelatin, 200 mM glutamine, and 10% fetal calf serum was adjusted to pH 7.4 and plated on a 35 x 10-mm-diameter disposable plastic Petri dish. Four wells, 2.5 mm in diameter, were made in the agarose plate with a template punch, and plugs were removed with suction. Three concentric wells were spaced 5 mm apart to one central well. Neutrophil migration was determined by placing 10 µL of the chemotactic agent in one (chemotaxin well) and the appropriate quantity of saline in the remaining two control wells. Ten microliters of the neutrophil suspension (5 x 107/mL) were added to the central well. The agarose plates were incubated at 37°C in a 5% CO2 atmosphere for various time periods and finally fixed with 2.5% glutaraldehyde for 1 h at room temperature. The agarose layers were subsequently removed from each slide, and the cells were stained with Wright’s stain. Counting was facilitated by using a slide projector at x40 magnification. The migratory distance was expressed as the chemotactic index: directed cellular migration toward the chemotaxin well divided by random cell migration toward saline. All assays were done in triplicate.

Flow cytometry
For the determination of surface expression of fMLP receptors, flow cytometry was performed. In brief, isolated neutrophils (5 x 106/mL) were resuspended in PBS and incubated with murine monoclonal anti-PR3-antibodies (0.1 µg/mL), isotype-matched mouse control-IgG (0.1 µg/mL), or sham-incubated for various time periods (1–20 min). Cells were pelleted at 4°C, and resuspended in PBS containing 0.1% BSA and 0.02% sodium acid. Then, 2 x 105 cells were distributed to each well of flexible round-bottom microtiter plates, washed, and incubated with 20 µL of fMLP-lys-FITC (1 µM) for 30 min. After three final washes, cells were resuspended in PBS and kept on ice until flow cytometric analysis. Flow cytometry was performed in a FACScan (Becton-Dickinson, Mountain View, CA) using forward and orthogonal light scatter to select viable cells. Background fluorescence was quantified by the addition of 1000-fold excess unlabeled fMLP along with the fluoresceinated analog. CellQuest® research software (Becton-Dickinson) was used to analyze the generated data.

Experimental protocols
In the standard protocol, neutrophils were at 5 x 106 PMN/mL and incubated with TNF-{alpha} (0.5 ng/mL, 15 min) to induce surface expression of PR3. Then, neutrophils were preincubated with murine monoclonal anti-PR3-antibodies, isotype-matched control mouse IgG, F(ab)2 fragments of the anti-PR3-antibody, human ANCA-IgG, or normal IgG. Monoclonal antibodies were used at 0.1 µg/mL, and ANCA-IgG or normal IgG was used at 1 µg/mL. After various preincubation periods (2–20 min), neutrophils were stimulated with fMLP (1 µM), PAF (5 µM), or A23187 (1 µM) for various time periods (5–20 min). For induction of leukotriene synthesis, exogenous AA (10 µM) was supplied together with fMLP and PAF. Reactions were stopped by centrifugation at 4°C (13.000 g, 5 min), and the respective mediators were analyzed in the cell supernatant.

Statistics
For statistical comparison, one-way analysis of variance (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 fMLP-induced leukotriene generation
Incubation of neutrophils with 1 µM fMLP and 10 µM AA for 5–20 min resulted in a progressive accumulation of LTB4 and its omega-oxidation products ({varpi}-OH-LTB4 and {varpi}-COOH-LTB4, summed up as {varpi}-Ox-LTB4) in the cell supernatant (Fig. 1 ). In parallel, decay products arising from the non-enzymatic hydrolysis of LTB4 (6t-LTB4, 6t,12e-LTB4, 5S,6R-DiHETE, 5S,6S-DiHETE; summed up as LTA4 decay) as well as 5-HETE were liberated from fMLP-stimulated neutrophils (Fig. 1B and 1C) . The kinetics of liberation differed between these metabolites: while the sum of LTB4 and its oxidation products, as well as the LTA4 decay products, displayed progressive accumulation over the entire incubation period, the release of 5-HETE peaked at 5 min, with subsequent rapid decline. When neutrophils were pretreated with monoclonal anti-PR3-antibodies (anti-PR3; 0.1 µg/mL) for 5 min, and subsequently exposed to fMLP and AA, the fMLP-induced liberation of leukotrienes was markedly amplified. In fact, anti-PR3-pretreated neutrophils released about 3.5- to 4-fold more 5-LO-mediators than mono-fMLP-stimulated neutrophils. Preincubation of PMN with equal concentrations of the anti-PR3 F(ab)2 fragments was entirely ineffective. Similarly, exposure of PMN to equal concentrations of an isotype-matched control-IgG had no affect on the fMLP-induced leukotriene generation. The 5-min anti-PR3 pretreatment per se, with subsequent mono-exposure to AA, did not induce any activation of neutrophil leukotriene synthesis under the present experimental conditions (data not shown).



View larger version (14K):
[in this window]
[in a new window]
  		Figure 1. Effect of anti-PR3-antibodies on fMLP-induced leukotriene-synthesis. Neutrophils (5 x 106/mL) were preincubated with murine monoclonal anti-PR3-antibodies (anti-PR3), isotype-matched mouse control IgG (IgG), or F(ab)2 fragments of the anti-PR3-antibodies [anti-PR3 F(ab)2] for 5 min, or sham incubation was performed. All antibodies were used at 0.1 µg/mL. After the preincubation period, neutrophils were stimulated with fMLP (1 µM) and AA (10 µM) for various time periods (5–20 min), and cell supernatants were subjected to solid-phase-extraction and HPLC. 5-LO-metabolites are given in picomoles per milliliter. Nonenzymatic hydrolysis products of LTA (6t-LTB4, 6t,12e-LTB4, 5S,6R-DiHETE, 5S,6S-DiHETE) are summed up as LTA4-decay, and products derived from the {varpi}-oxidation pathway of LTB4 (20-OH-LTB4 and 20-COOH-LTB4) are indicated as {varpi}-Ox-LTB4. Means ± SEM of at least four experiments each are given. *Significantly different from sham-pretreated, fMLP-stimulated PMN.

 
As depicted for the LTB4 release, analysis of kinetics, performed by preincubating neutrophils for various time periods with anti-PR3 antibodies, showed that up-regulation of the fMLP-induced leukotriene generation was evident within 2.5 min of preincubation with the antibodies and peaked after 5–7.5 min of antibody pretreatment (Fig. 2 ). The amplifying effect of the anti-PR3-antibodies was, however, transient, since it was largely lost after a preincubation period of 20 min or longer. Due to these kinetics, a 5-min preincubation time was chosen for further experiments.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 2. Time-dependence of the anti-PR3-related priming of leukotriene synthesis in response to fMLP. Neutrophils (5 x 106/mL) were preincubated for different time periods (0–20 min) with 0.1 µg/mL murine monoclonal anti-PR3-antibodies (anti-PR3), isotype-matched mouse control IgG (IgG), or sham incubation was performed. After the preincubation period, neutrophils were stimulated with fMLP (1 µM) and AA (10 µM) for 10 min. 5-LO-metabolites were extracted from the cell supernatant and LTB4 release, as exemplarily depicted, is given in picomoles per milliliter. Means ± SEM of at least four experiments each are given. *Significantly different from sham-pretreated, fMLP-stimulated PMN.

 
Anti-PR3 antibodies enhance the chemotactic movement of neutrophils toward fMLP
Next, the effect of anti-PR3 pretreatment on the migratory behavior of neutrophils toward fMLP was studied. Neutrophils were preincubated with intact anti-PR3-antibodies (0.1 µg/mL), F(ab)2 fragments of the anti-PR3-antibodies, equal concentrations of an isotype-matched control-IgG, ANCA-IgG from anti-PR3-positive patients (1 µg/mL) or normal IgG (1 µg/mL) or sole buffer fluid (sham pretreatment). Subsequently, PMN were incubated in agarose-containing dishes in the presence of a gradient of 0.1 µM fMLP for various time periods (1–4 h). A massive enhancement of the migratory response toward the formylated peptide was noted for the anti-PR3-pretreated PMN, whereas neither isotype-matched control IgG nor F(ab)2 fragments of the anti-PR3-antibodies exerted any influence on the chemotactic response (Fig. 3 ). It is important to note that the enhancement of neutrophil migration upon pretreatment with murine monoclonal anti-PR3-antibodies was reproduced by ANCA-IgG, but not by normal human IgG.



View larger version (16K):
[in this window]
[in a new window]
  		Figure 3. Influence of anti-PR3 antibodies on neutrophil migratory behavior toward fMLP. Neutrophils (5 x 107/mL) were preincubated with murine monoclonal anti-PR3 antibodies (anti-PR3, 0.1 µg/mL), equal concentrations of an isotype-matched mouse control IgG (IgGm), F(ab)2 fragments of the anti-PR3 antibodies [anti-PR3 F(ab)2, 0.1 µg/mL), ANCA-IgG from anti-PR3-postive patients (1 µg/mL; PR3-ANCA) or normal human IgG (IgGh) or sham-incubated for 5 min. Then, 10 µl of the neutrophil suspension (50,000 cells) were placed on agarose plates and exposed to 0.1 µM fMLP (chemotaxin well) or assay buffer (control well). After various times of incubation (1–4 h) in a 5% CO2 atmosphere, cells were fixed and stained with Wright’s stain. Migratory distance is expressed as chemotactic index. Means ± SEM of at least three independent experiments, each with values performed in triplicate, are given. *Significantly different from sham-incubated PMN.

 
Anti-PR3-antibodies suppress fMLP-induced superoxide anion release and degranulation
To explore whether anti-PR3-antibodies affect further fMLP-mediated neutrophil functions, their influence on fMLP-induced superoxide anion release and degranulation was studied. For this purpose, neutrophils were pretreated with anti-PR3-antibodies or control-IgG, or were sham-incubated for 5 min after stimulation with fMLP (1 µM, 10 min). Surprisingly, both the fMLP-induced O2- liberation as well as the elastase liberation were markedly reduced in anti-PR3-pretreated neutrophils (Fig. 4 ), although the anti-PR3 preincubation per se was followed by a moderate activation of these neutrophil functions. The suppressive effect of anti-PR3-antibodies was unlikely to be a toxic effect, since LDH release from anti-PR3-treated PMN was constantly below <5% of the total cellular content. Control-IgG, in contrast to anti-PR3, did not affect neutrophil respiratory burst and degranulation in response to fMLP.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 4. Effect of anti-PR3 antibodies on neutrophil respiratory burst and degranulation induced by fMLP. PMN (5 x 106 PMN/mL) were preincubated with murine monoclonal anti-PR3-antibodies (anti-PR3, 0.1 µg/mL), equal concentrations of an isotype-matched mouse control IgG (IgG), or sham-incubated for 5 min. Then, cells were exposed to fMLP (1 µM, filled columns) or sham-stimulated (shaded columns) for an additional 10 min, and secretion products were analyzed. Superoxide generation is expressed as nmol O2-/5 x 106 PMN (A), and elastase is given in units per liter (B). Means ± SEM of at least four independent experiments each are given. *Significantly different from sham-incubated PMN.

 
The anti-PR3-induced alterations of inflammatory neutrophil functions are specific for stimulation with fMLP
To determine whether the anti-PR3-related alterations of neutrophil responses to fMLP are also true for other agonists, PMN were preincubated with anti-PR3 antibodies for 5 min and subsequently exposed to PAF (5 µM) or to the calcium ionophore A23187 (1 µM). Neither the amplifying effect of the autoantibodies on the fMLP-mediated leukotriene response, nor the inhibition of O2- and elastase secretion were reproduced for PAF- and A23187-induced PMN activation (Fig. 5A-C ), indicating that the priming effect of anti-PR3 antibodies was specific for fMLP-mediated neutrophil responses. Also at shorter (2 min) or longer (10 and 20 min) preincubation periods with anti-PR3, no priming effect was observed for calcium ionophore- or PAF-induced neutrophil activation (Fig. 6 ).



View larger version (30K):
[in this window]
[in a new window]
  		Figure 5. Effect of anti-PR3 antibodies on neutrophil activation with other agonists than fMLP. Neutrophils (5 x 106/mL) were pretreated with murine monoclonal anti-PR3 antibodies (anti-PR3, 0.1 µg/mL) or sham pretreated for 5 min, and subsequently exposed to fMLP (1 µM; filled columns), PAF (5 µM; shaded columns), or A23187 (1 µM; open columns). For induction of leukotriene synthesis, AA (10 µM) was added simultaneously with fMLP or PAF. After an additional 10 min of incubation, secretion products were analyzed in the cell supernatant (A, LTB4 release; B, O2- secretion; C, release of elastase) and are expressed as percentage of sham-pretreated PMN. Means ± SEM of at least three independent experiments each are given. *Significantly different from sham-incubated PMN.

 


View larger version (17K):
[in this window]
[in a new window]
  		Figure 6. Time course of anti-PR3 preincubation for activation with other agonists than fMLP. Neutrophils (5 x 106/mL) were preincubated for different time periods (0–20 min) with 0.1 µg/mL murine monoclonal anti-PR3-antibodies (anti-PR3), or sham incubation was performed. After the preincubation period, neutrophils were stimulated with A23187 (1 µM) or PAF (5 µM) and AA (10 µM) for 10 min. 5-LO-metabolites were extracted from the cell supernatant and LTB4 release, as exemplarily depicted, is given in picomoles per milliliter. Means ± SEM of at least four experiments each are given. No significant differences were observed between anti-PR3 and sham-pretreated PMN.

 
Anti-PR3 antibodies do not affect the number of fMLP receptors expressed on the neutrophil cell surface
Because the anti-PR3-mediated alterations of inflammatory neutrophil behavior were found to be specific for fMLP-induced PMN activation, we speculated that the autoantibodies might modulate the fMLP response at a receptor level. Therefore, the binding of FITC-fMLP to anti-PR3- versus sham-preincubated neutrophils was assessed by flow cytometry. However, no significant changes in the density of surface fMLP receptors in neutrophils pretreated with anti-PR3-antibodies for various time periods (1–20 min) were observed (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In Wegener’s granulomatosis, a pathogenetic role of PR3-ANCA has been proposed due to the capacity of these autoantibodies to directly activate inflammatory neutrophil functions [6 7 8 9 10 11 ]. In this study, an alternative approach was made to define the influence of PR3-ANCA on neutrophil behavior. By preincubating PMN with sub-stimulatory concentrations of anti-PR3 antibodies, we evaluated their effect on neutrophil activation with defined agonists, such as the receptor-operated ligands fMLP and PAF and the calcium ionophore A23187. This experimental approach was chosen for several reasons. In vivo, neutrophils exist in different states of activation, ranging from dormant via primed to fully activated. A wide variety of agents targeting neutrophils, LPS, and proinflammatory cytokines, among others, do not elicit secretory neutrophil responses by themselves, but cause a sensitization of this leukocyte type toward subsequent activation with bacterial or endogenous stimuli [12 13 14 15 ]. Circulating neutrophils from patients with active WG show an increased expression of activation markers (CD66b, CD64, CD63), attributable to a state of cellular preactivation [23 ]. This priming of neutrophils could be related to local or systemic elevation of proinflammatory cytokines in WG [24 , 25 ]. We hypothesized that interaction of neutrophils with subthreshold concentrations of anti-PR3 antibodies might also induce a priming of PMN, thereby enhancing their responsiveness to subsequent stimulation with microbial or endogenous inflammatory agents. Exposure of neutrophils to these agonists may very likely occur in active WG because exacerbations of the disease are closely related to intercurrent infections [16 , 17 ]. The low anti-PR3 concentrations used may well reflect the ANCA concentration at the onset of disease, with neutrophils being primed, but not fully activated by ANCA.

Indeed, when employed for pre-incubation of neutrophils in vitro, low doses of anti-PR3 antibodies caused a most impressive priming of the neutrophil response to the tripeptide fMLP, which represents a prototype of the family of bacterial N-formylated peptides [26 , 27 ]. Upon pre-exposure of neutrophils to a murine monoclonal anti-PR3 antibody, but not to an isotype-matched control IgG, a massive amplification of fMLP-induced leukotriene and 5-HETE synthesis was noted, along with an enhanced chemotactic movement toward the peptide. This effect was reproduced by ANCA-IgG from patients with monospecific anti-PR3-positive WG. In contrast, the fMLP-elicited microbicidal neutrophil functions, namely the respiratory burst and the release of proteolytic enzymes such as elastase, were diminished upon pretreatment with anti-PR3 antibodies. This pattern of anti-PR3-related preactivation of neutrophils thus displays some distinct features previously not described for "classic" PMN priming agents, such as LPS, granulocyte-macrophage colony-stimulating factor, or TNF-{alpha}. (1) Priming by the autoantibodies was a very rapid event, with a maximum after 5–7.5 min, which contrasts with the more long-lasting priming efficacy of LPS and the above-mentioned cytokines [12 13 14 15 ]. (2) Moreover, the anti-PR3-induced priming of the fMLP response appeared to be transient because it was no longer observed after incubation periods of 20 min or longer. In general, neutrophil priming is not an irreversible process. The phenomenon of reversible neutrophil priming has already been observed upon exposure to PAF or after hypotonic treatment [28 , 29 ], however, the mechanisms underlying the transient feature of neutrophil priming remain largely to be elucidated. As for the anti-PR3-induced priming, it is possible that the duration of the priming signal or its rate of onset are key determinants, or that the dynamic feature of ligand-receptor interactions [30 ] are responsible for the kinetics of the anti-PR3-induced priming for the fMLP response. (3) Priming did not occur when PAF or the calcium ionophore A23187 were used as neutrophil agonists, thus suggesting a mechanism of pre-activation specific for the chemotactic peptide. To the best of our knowledge, such selectivity of neutrophil priming for formylated peptides has hitherto not been described for a circulating agent. Clearly, the currently described priming phenomenon was not due to any contamination of the autoantibodies with LPS because endotoxin-induced priming of PMN presents with completely different features [12 13 14 15 ], and no LPS was detected in the anti-PR3 preparation when assayed with the kinetic LAL test.

The molecular mechanisms underlying the anti-PR3-induced priming of neutrophil responsiveness to fMLP largely remain to be elucidated. Because both the F(ab)2 fragments of the anti-PR3 antibodies and sole targeting of Fc{gamma}-receptors (Fc{gamma}Rs) by an isotype-matched control-IgG were noted to be entirely ineffective, engagement of both PR3 and Fc{gamma}Rs is apparently a prerequisite for the priming phenomenon. This is in line with previous investigations, addressing direct activation of secretory neutrophil responses by sufficiently high autoantibody titers [7 , 31 ]. One possible explanation for the priming phenomenon might be an increase in the overall number of neutrophil fMLP receptors in response to anti-PR3 pretreatment, however, this was excluded in the FACS studies measuring the binding of FITC-labeled fMLP. Alternatively, a change in the receptor affinity for N-formylated-peptides might be operative. The neutrophil fMLP receptor exists in two affinity states that are regulated dynamically, and changes in receptor affinity and function are closely related [32 , 33 ]. Various agonists have been reported to affect the neutrophil response toward N-formylated peptides by modifying the fMLP-receptor affinity in vitro [34 , 35 ]. Consistent with the theory that high-affinity receptors mediate chemotaxis, whereas low-affinity receptors play a role in secretion of superoxide anion and granule components [36 37 38 ], the anti-PR3-induced alterations of the fMLP response, priming of chemotactic movement, and leukotriene generation with a decrease in secretion of O2- and elastase, might reflect a switch toward predominance of high-affinity fMLP receptors. This is well in line with previous studies demonstrating that chemotactic movement and secretory functions induced by fMLP are regulated independently [36 37 38 ]. Furthermore, changes in characterized post-fMLP receptor signaling pathways [38 39 40 ] might be involved in the anti-PR3-related priming event. It has already been described that preincubation of human neutrophils with PR3-ANCA affects fMLP-related generation of inositolphosphates and membrane translocation of protein kinase C [41 ], which is of interest in view of the role of these signaling pathways in exocytosis and respiratory burst [40 , 42 ]. Although at first glance contradicting the previous findings describing a direct activation of these neutrophil functions by sufficiently high concentrations of c-ANCA [6 7 8 , 43 ], the presently observed down-regulation of fMLP-induced neutrophil degranulation and respiratory burst by low concentrations of monoclonal anti-PR3 antibodies might reflect an interference of commonly used signaling pathways between anti-PR3 and fMLP. In this regard, the recent demonstration that c-ANCA do possess the capacity to directly activate PKC [44 ] is of particular interest. It may be speculated that previous activation of PKC by anti-PR3 antibodies may desensitize this signaling pathway for subsequent engagement with formylated peptides, as known for well-characterized PKC activators such as phorbol esters [45 ]. Clearly, further studies are mandatory to clarify the molecular mechanisms underlying the neutrophil priming by subthreshold doses of anti-PR3 in detail.

In conclusion, low doses of anti-PR3 antibodies, being ineffective per se in causing PMN stimulation, exert a major impact on the human neutrophil responsiveness to fMLP, but not to PAF and A23187. Although generation of leukotrienes as well as the chemotactic response toward the peptide are massively enhanced, respiratory burst and elastase secretion are blunted. On the one hand, such priming may promote neutrophil accumulation at sites of infections via direct (cell migration) and indirect (leukotriene synthesis) mechanisms, and on the other hand, anti-microbicidal capacity may be suppressed. It may be speculated that such an altered response profile of neutrophils under the influence of circulating autoantibodies might contribute to perivascular neutrophil accumulation, persistence of infections, and granuloma formation in Wegener’s granulomatosis.


   ACKNOWLEDGEMENTS
 
This work was supported by the Deutsche Forschungsgemeinschaft (GR 534). Katja Hattar and Ulf Sibelius contributed equally to this work.

Received May 15, 2000; revised August 21, 2000; accepted August 22, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. 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: a tool for diagnosis and marker of disease activity in Wegener’s granulomatosis Lancet 1,425-429[Medline]
  2. Jennette, J. C. (1991) Antineutrophil cytoplasmic autoantibody-associated diseases: a pathologist’s perpsective Am. J. Kidney Dis. 18,164-170[Medline]
  3. Kallenberg, C. G. M., Heeringa, P., Cohen-Tervaert, J. W. C. (1999) Are antineutrophil cytoplasmic antibodies pathogenic in Wegener’s granulomatosis? Lessons from in vitro and in vivo experimental findings Shoenfeld, Y. eds. The Decade of Autoimmunity ,227-234 Elsevier Amsterdam, The Netherlands.
  4. Gross, W. L., Csernok, E., Flesch, B. K. (1993) "Classic" Anti-neutrophil cytoplasmic autoantibodies (ANCA), Wegener’s autoantigen, and their immunpathogenetic role in Wegener’s Granulomatosis J. Autoimmun. 6,171-184[Medline]
  5. Csernok, E., Ernst, M., Schmitt, W., Bainton, D. F., Gross, W. L. (1994) Activated neutrophils express proteinase 3 on their plasma membrane in vitro and in vivo Clin. Exp. Immunol. 95,244-250[Medline]
  6. 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]
  7. Porges, A. J., Redecha, P. B., Kimberley, W. T., Csernok, E., Gross, W. L., Kimberely, R. P. (1994) Anti-neutrophil cytoplasmic antibodies engage and activate human neutrophils via Fc{gamma}RIIa J. Immunol. 153,1271-1280[Abstract]
  8. 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]
  9. Brooks, C. J., King, W. J., Radford, D. J., Adu, D., McGrath, M., Savage, C. O. S. (1996) Il-1beta production by human polymorphonuclear leukocytes stimulated by anti-neutrophil cytoplasm ic autoantibodies: Relevance to systemic vasculitis Clin. Exp. Immunol. 106,273-279[Medline]
  10. 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]
  11. 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]
  12. 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 lipopolysacharide. Evidence for increased activity of the superoxide-producing enzyme J. Exp. Med. 160,1656-1671[Abstract/Free Full Text]
  13. Doerfler, M. E., Danner, R. L., Shelhammer, J. H., Parillo, J. E. (1989) Bacterial lipopolysaccharides prime human neutrophils for enhanced production of leukotriene B4 J. Clin Invest. 83,970-977
  14. Surette, M. E., Palmiantier, R., Gosselin, J., Borgeat, P. (1993) Lipopolysaccharides prime whole blood and isolated neutrophils for the increased synthesis of 5-lipoxygenase products by enhancing arachidonic acid availability: involvement of the CD14 antigen J. Exp. Med. 178,1347-1355[Abstract/Free Full Text]
  15. Steinbeck, M. J., Roth, J. A. (1989) Neutrophil activation by recombinant cytokines Rev. Infect. Dis. 11,549-568[Medline]
  16. Pinching, A. J., Rees, A. J., Pussel, B. A., Lockwood, C. M., Mitchison, R. S., Peters, D. K. (1980) Relapses in Wegener’s Granulomatosis: The role of infections Br. J. Med. 281,836-838
  17. Stegemann, C. A., Cohen Tervaert, J. W., Slutter, 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
  18. 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]
  19. Grimminger, F., Becker, G., Seeger, W. (1988) High yield enzymatic conversion of intravasal leukotriene A4 in blood-free perfused lungs J. Immunol. 141,2431-2436[Abstract]
  20. Cohen, H. J., Chovaniec, M. E. (1978) Superoxide generation by digitonin-stimulated guinea pig granulocytes J. Clin. Invest. 61,1081-1087
  21. Kramps, J. A. (1983) L-Pyroglutamyl-L-propyl-L-valine-p-nitro-anilide, a highly specific substrate for granulocyte elastase Scand. J. Clin. Lab. Invest. 43,427-432[Medline]
  22. Nelson, R. D., Quie, P. G., Simmons, R. L. (1975) Chemotaxis under agarose: a new and simple method for measuring chemotaxis and spontaneous migration of human polymorphonuclear leukocytes and monocytes J. Immunol. 115,1650-1656[Abstract/Free Full Text]
  23. 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[Medline]
  24. Noronha, I. L., Krüger, C., Andrassy, K., Ritz, E., Waldherr, R. (1993) In situ production of TNF-{alpha}, IL-1ß, and IL-2R in ANCA-positive glomerulonephritis Kidney Int 43,682-693[Medline]
  25. Arimura, Y., Minoshima, Y., Kamiya, Y., Tanaka, U., Nakabayashi, K., Kitamoto, K., Nagasawa, T., Sasaki, T., Suzuki, K. (1993) Serum myeloperoxidase and serum cytokines in anti-myeloperoxidase antibody-associated glomerulonephritis Clin. Nephrol. 40,256-264[Medline]
  26. Schiffmann, E., Corcoran, B. A., Wahl, S. M. (1975) N-formyl-methionyl peptides as chemoattractants for leukocytes Proc. Natl. Acad. Sci. USA 72,1059-1062[Abstract/Free Full Text]
  27. Marasco, W. A., Phan, S. H., Krutzsch, H., Showell, H. J., Feltner, D. E., Nairn, R., Becker, E. L., Ward, P. A. (1984) Purification and identification of formyl-methionyl-leucyl-phenylalanine as the major peptide neutrophil chemotactic factor produced by Escherichia coli J. Biol. Chem. 259,5430-5439[Abstract/Free Full Text]
  28. Kitchen, E., Rossi, A. G., Condliffe, A. M., Haslett, C., Chilvers, E. R. (1996) Demonstration of reversible priming of human neutrophils using platelet-activating factor Blood 88,4330-4337[Abstract/Free Full Text]
  29. Edashige, K., Watanabe, Y., Sato, E. F., Takehara, Y., Utsumi, K. (1993) Reversible priming and protein-tyrosol phosphorylation in human peripheral neutrophils under hypotonic conditions Arch. Biochem. Biophys. 302,343-349[Medline]
  30. Sklar, L., Hyslop, P. A., Oades, Z. G., Omann, G. M., Jesaitis, A. J., Painter, R. G., Cochrane, C. G. (1985) Signal transduction and ligand-receptor dynamics in the human neutrophil: Transient responses and occupancy-response relations at the formyl peptide receptor J. Biol. Chem. 260,11461-11467[Abstract/Free Full Text]
  31. Kocher, M., Edberg, J. C., Fleit, H. B., Kimberley, R. P. (1998) Antineutrophil cytolasmic antibodies preferentially engage Fc gamma RIIIb on human neutrophils J. Immunol. 161,6909-6914[Abstract/Free Full Text]
  32. Koo, C., Lefkowtiz, R. J., Snyderman, R. (1982) The oligopeptide chemotactic factor receptor on human polymorphonuclear leukocyte membranes exists in two affinity states Biochem. Biophys. Res. Commun. 106,442-449[Medline]
  33. Mackin, W. M., Huang, C. K., Becker, E. L. (1982) The formylpeptide chemotactic receptor on rabbit peritoneal neutrophils. 1. Evidence for two binding sites with different affinities J. Immunol. 129,1608-1611[Abstract]
  34. Koo, C., Lefkowitz, R. J., Snyderman, R. (1983) Guanine nucleotides modulate the binding affinity of the oligopeptide chemoattractant receptor on human polymorphonuclear leukocytes J. Clin. Invest. 72,748-753
  35. Yuli, I., Tomonaga, A., Snyderman, R. (1982) Chemoattractant receptor function in human polymorphonuclear leukocytes are divergently altered by membrane fluidizers Proc. Natl. Acad. Sci. USA 79,5906-5911[Abstract/Free Full Text]
  36. Kreutzer, D. L., O’Flaherty, J. T., Orr, W., Showell, H. J., Ward, P. A., Becker, E. L. (1978) Quantitative comparisons of various biological responses of neutrophils to different active and inactive chemotactic factors Immunopharmacol 1,39-47[Medline]
  37. Snyderman, R., Pike, M. C. (1984) Chemoattractant receptors on phagocyte cells Annu. Rev. Immunol. 2,257-281[Medline]
  38. Prossnitz, E. R., Ye, R. D. (1997) The N-formyl peptide receptor: a model for the study of chemoattractant receptor structure and function Pharmacol. Ther. 74,73-102[Medline]
  39. Yasui, K., Yamazaki, M., Miyabayshi, M., Tsuno, T., Komiyama, A. (1995) Signal transduction pathway in human polymorphonuclear leukocytes for chemotaxis induced by a chemotactic factor. Distinct from the pathway for superoxide anion production J. Immunol. 152,5922-5929[Abstract]
  40. Cockcroft, S. (1994) Receptor-mediated signal transduction pathways in neutrophils: regulatory mechanisms that control phospholipases C, D and A2 Hellewell, P. G. Williams, T. J. eds. Immunopharmacology of Neutrophils ,160-193 Academic San Diego, CA.
  41. Lockwood, C. M., Lai, K. N. (1991) The effect of anti-neutrophil cytoplasm autoantibodies on the signal transduction in human neutrophils Clin. Exp. Immunol. 85,396-410[Medline]
  42. Thelen, M., Dewald, B., Bagglioni, M. (1993) Neutrophil signal transduction and activation of the respiratory burst Physiol. Rev. 73,797-821[Free Full Text]
  43. Franssen, C. F., Huitema, M. G., Muller Kobold, A. C., Oost-Kort, W. W., Limburg, P. C., Tiebosch, A., Stegman, C. A., Kallenberg, C. G., Tervaert, J. W. (1999) In vitro neutrophil activation by antibodies to proteinase 3 and myeloperoxidase from patients with crescentic glomerulonephritis J. Am. Soc. Nephrol. 10,1506-1515[Abstract/Free Full Text]
  44. Radford, D. J., Lord, J. M., Savage, C. O. S. (1999) The activation of the neutrophil respiratory burst by anti-neutrophil cytoplasmic autoantibody (ANCA) from patients with systemic vasculitis requires tyrosine kinases and protein kinase C activation Clin. Exp. Immunol. 118,171-179[Medline]
  45. Naccache, P. H., Molski, T. F., Borgeat, P., White, J. R., Sha’afi, R. I. (1985) Phorbol esters inhibit the fMet-Leu-Phe and leukotriene B4-stimulated calcium mobilization and enzyme secretion in rabbit neutrophils J. Biol. Chem. 260,2125-2131[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Leukoc. Biol.Home page
K. Hattar, S. van Burck, A. Bickenbach, U. Grandel, U. Maus, J. Lohmeyer, E. Csernok, T. Hartung, W. Seeger, F. Grimminger, et al.
Anti-proteinase 3 antibodies (c-ANCA) prime CD14-dependent leukocyte activation
J. Leukoc. Biol., October 1, 2005; 78(4): 992 - 1000.
[Abstract] [Full Text] [PDF]

Home page
 Eur Respir JHome page
A. Linden, M. Laan, and G. P. Anderson
Neutrophils, interleukin-17A and lung disease
Eur. Respir. J., January 1, 2005; 25(1): 159 - 172.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
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 Grimminger, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hattar, K.
Right arrow Articles by Grimminger, F.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS