help button home button
AJRCMB
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on March 1, 2007, doi:10.1165/rcmb.2006-0457OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2006-0457OCv1
37/1/85    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 Wong, C. K.
Right arrow Articles by Lam, C. W. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wong, C. K.
Right arrow Articles by Lam, C. W. K.
American Journal of Respiratory Cell and Molecular Biology. Vol. 37, pp. 85-96, 2007
© 2007 American Thoracic Society
DOI: 10.1165/rcmb.2006-0457OC

Intracellular Signaling Mechanisms Regulating Toll-Like Receptor–Mediated Activation of Eosinophils

Chun K. Wong, Phyllis F. Y. Cheung, Wai K. Ip and Christopher W. K. Lam

Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong; and the Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Macau

Correspondence and requests for reprints should be addressed to Professor C. W. K. Lam, Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, N.T., Hong Kong. E-mail: waikeilam{at}cuhk.edu.hk


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Activation of eosinophils by microbe-derived molecules via Toll-like receptors (TLR) potentially provides the link between microbe-induced innate immune responses and the exacerbation of allergic inflammation. We investigated the expression of TLRs and the effect of their ligands on human eosinophils. Expression of TLR1–9 was detected by Western blot and flow cytometry. Adhesion molecules, cytokines, superoxides, and eosinophlilic cationic protein (ECP) were assessed by flow cytometry, enzyme-linked immunosorbent assay, chemiluminescent method, and fluorescence immunoassay, respectively. Human eosinophils differentially expressed TLR1, -2, -4, -5, -6, -7, and -9. Peptidoglycan (PGN) (TLR2 ligand), flagellin (TLR5 ligand), and Imiquimod R837 (TLR7 ligand) could significantly upregulate cell surface expression of intercellular adhesion molecule (ICAM)-1 and CD18, and induce the release of IL-1beta, IL-6, IL-8, growth-related oncogene (GRO)-{alpha}, and superoxides of eosinophils. Only PGN could induce the degranulation for ECP release. However, ds poly I-C (TLR3 ligand), LPS (TLR4 ligand), ssRNA (TLR8 ligand), and CpG-DNA (TLR9 ligand) were much less effective or inactive. PGN, flagellin, and R837 could activate both nuclear factor (NF)-{kappa}B and extracellular signal–regulated protein kinase (ERK). PGN could activate phosphatidylinositol 3-kinase (PI3K)-Akt, and R837 both PI3K-Akt and p38 mitogen-activated protein kinase (MAPK). The induction of the release of IL-1beta, IL-6, IL-8, GRO-{alpha}, superoxides, and ECP by PGN, flagellin, and R837 was found to be differentially regulated by NF-{kappa}B, ERK, PI3K-Akt, and p38 MAPK. The above results therefore support that microbial infection may lead to the exacerbation of allergic inflammation.

Key Words: Toll-like receptors • eosinophils • cytokines • chemokines • signal transduction



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Results demonstrate the intracellular mechanisms for the Toll-like receptor–mediated activation of eosinophils, thereby providing new postulates for immunopathologic mechanisms and drug development of respiratory tract infection–mediating allergic inflammation.

 
Allergic diseases such as asthma are prevalent in developed countries and have been increasing worldwide (1). Eosinophils are the principal effector cells of allergic inflammation, which is characterized by an accumulation and infiltration of eosinophils in tissues mediated by specific eosinophil chemokine eotaxin, and adhesion molecules vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1 on epithelial cells (2), and E-selectin on the endothelium (3). The activated eosinophils release cytotoxic molecules such as major basic protein, eosinophil peroxidase, eosinophilic cationic protein (ECP), and superoxides, as well as cytokines and chemokines that cause tissue damage and inflammatory reaction and consequently the manifestation of allergic diseases such as allergic asthma (4). Eosinophils are also capable of producing and releasing a variety of proinflammatory cytokines such as IL-3, IL-4, IL-5, IL-6, IL-16, TNF-{alpha}, and granulocyte-macrophage colony-stimulating factor (GM-CSF), together with chemokines including eotaxin, IL-8, macrophage inflammatory protein (MIP)-1, monocyte chemoattractant protein (MCP), regulated upon activation, normal T cell expressed and secreted (RANTES), and growth-related oncogene (GRO)-{alpha} (5).

Toll-like receptors (TLR) are functionally important receptors for the recognition of conserved motifs in pathogens termed pathogen-associated molecular patterns (6). TLR1–11 are essential for the recognition of microbial pathogens to activate intracellular signaling pathways for distinct pattern of gene expression that result in innate immune response against microbial infections and the development of antigen-specific acquired immunity (7). TLRs have been reported to play important roles in the modulation of allergic response by regulating dendritic cells, T cells, and mast cells (8). There has been accumulating evidence for the role of TLR polymorphisms in the pathogenesis of allergic asthma (9). TLR7 ligand R-848 was previously shown to activate eosinophils in term of superoxide release and cell survival, but TLR4 ligand LPS was reported to exert conflicting effects on eosinophils (10–12).

Regarding the general TLR-mediated intracellular signal transduction, after ligand binding, two central adapter proteins: MyD88 and Toll/IL-1 receptor (TIR) domain-containing adapter inducing IFN-beta (TRIF), propagate TLR signal transduction by interacting with TLRs via their respective TIR components and recruiting downstream enzymes (e.g., IL-1R–associated kinases, TNF receptor–associated factor 6) through their "death" domains (13, 14). Apart from the MyD88-dependent pathway involving the early phase of NF-{kappa}B activation for inflammatory cytokine release, the induction of IFN-{alpha}, beta expression by TLR3,4 occurs through a MyD88-independent pathway that leads to the activation of interferon regulatory factor 3 (IRF3) involving the late phase of NF-{kappa}B activation (14). However, the detailed TLR-mediated intracellular signal transductions, particularly the mitogen-activated protein kinases (MAPK) and phosphatidylinositol 3-kinase (PI3K)-AKT cascades of eosinophils for degranulation and cytokine release, have not been elucidated.

We investigated the potential link between the innate immune response and the exacerbation of allergic inflammation, multiple intracellular signal transduction pathways regulating the various TLR-mediated induction of cytokines and chemokines, degranulation for the release of superoxides and ECP, and cell surface expression of adhesion molecules including ICAM-1, leukocyte function–associated antigen (LFA)-1 (CD11a/CD18), and macrophage antigen (Mac)-1 (CD11b/CD18) of eosinophils.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Reagents
For Western blot, monoclonal antibodies of rabbit anti-human TLR1 (clone GD2.F4), rabbit anti-human TLR5 (clone 19D1885.2.1), rabbit anti-human TLR6 (clone 86B1153), mouse anti-human TLR8 (clone 44C143), and mouse anti-human TLR9 (26C593), as well as polyclonal rabbit anti-human TLR7, were purchased from Imgenex Corp. (San Diego, CA). Monoclonal antibodies of mouse anti-human TLR2 (clone TL2.1) and anti-human TLR3 (clone TLR3.7), as well as polyclonal rabbit anti-human TLR4 antibodies were, purchased from eBioscience Corp. (San Diego, CA). Antibodies for flow analysis of TLR1–9 were purchased from Imgenex. Ultra-purified lipopolysaccharide (LPS) from Escherichia coli K12 strain without any contamination by lipoprotein, R837 (Imiquimod, a synthetic antiviral molecule), ssRNA, and CpG DNA, for TLR4, -7, -8, and –9. were purchased from InvivoGen Corp. (San Diego, CA), while flagellin for TLR5 was from Calbiochem Corp. (San Diego, CA). Poly I-C (TLR3 ligand) was purchased from Sigma-Aldrich Co. (St. Louis, MO), and peptidoglycan (PGN) for TLR2 from Fluka Chemie GmbH (Buchs, Switzerland). Neutralizing antibodies for TLR2 and TLR4 were from eBiosciences Corp. Janus kinase (JAK) inhibitor AG490, PI3K inhibitor LY294002, I{kappa}B-{alpha} phosphorylation inhibitor BAY11–7082, extracellular signal–regulated protein kinase (ERK) inhibitor PD98059, c-Jun N-terminal activated kinase (JNK) inhibitor SP600125, and p38 MAPK inhibitor SB203580 were purchased from Calbiochem. SB203580 was dissolved in water, while AG490, LY294002, BAY11–7082, PD98059, and SP600125 were dissolved in dimethyl sulfoxide (DMSO). GM-CSF was purchased from Peprotech Ltd (London, UK). In all studies, the concentration of DMSO was 0.1% (vol/vol).

Isolation of Human Blood Eosinophils and Neutrophils from Buffy Coat
Fresh human buffy coat obtained from the Hong Kong Red Cross Blood Transfusion Service was diluted 1:2 with phosphate-buffered saline (PBS) at 4°C and centrifuged using an isotonic Percoll solution (density 1.082 g/ml; Amersham and Pharmacia Biotech, Uppsala, Sweden) for 30 min at 1,000 x g. The eosinophil-rich granulocyte fraction was collected and washed twice with cold PBS containing 2% fetal bovine serum (FBS). Cells were then incubated with anti-CD16 and anti-CD14 magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) at 4°C for 45 min. CD16- and CD14-positive cells were depleted by passing through a LS+ column (Miltenyi Biotec) within a magnetic field. With this preparation, the drop-through fraction contained eosinophils without any other cell types as assessed by Hemacolor rapid blood smear stain (E Merck Diagnostica, Darmstadt, Germany). CD16-positive neutrophils were collected by positive selection using LS+ column. All studies were performed with freshly isolated eosinophils without being further maintained in culture because of the short lifespan of eosinophils after isolation.

Endotoxin-Free Solutions
Eosinophil culture medium (RPMI 1640 medium supplemented with 10% FBS and 20% mM Hepes) was purchased from Gibco (Grand Island, NY), free of detectable LPS (< 0.1 EU/ml). All other solutions were prepared using pyrogen-free water and sterile polypropylene plastic ware. No solution and TLR ligands except ultra-purified LPS contained detectable LPS, as determined by the Limulus amoebocyte lyase assay (sensitivity limit 12 pg/ml; BioWhittaker Inc., Walkersville, MD).

Protein Array Analysis of Chemokines and Cytokines in Culture of Eosinophils
The expression profile of 79 different cytokines in culture supernatant of eosinophils was assessed semiquantitatively using antibody-based RayBio human cytokine array V (RayBiotech Inc., Norcross, GA) (5).

L-1beta, IL-6, IL-8 and GRO-{alpha} Assays
Concentrations of proinflammatory cytokines IL-1beta and IL-6, and chemokine IL-8, in culture supernatant were measured by enzyme-linked immunosorbent assay (ELISA; BD Pharmingen, San Diego, CA). Concentration of GRO-{alpha} was measured by ELISA (R&D Systems, Minneapolis, MN).

Assay for Release of Superoxide Anions and ECP in Culture Supernatant
The generation of superoxide in culture supernatant was measured using a chemiluminescence assay (Calbiochem), and ECP by fluorescence enzyme immunoassay (AutoCAP analyzer; Pharmacia Diagnostics AB, Uppsala, Sweden).

Flow Cytometry of the Expression of TLRs and Adhesion Molecules
Eosinophils (5 x 105 cells) were resuspended with cold PBS supplemented with 0.5% bovine serum albumin (BSA). After blocking with 2% human pooled serum for 20 min at 4°C and washing with PBS supplemented with 0.5% BSA, cells were incubated with fluorescein isothiocyanate (FITC)-conjugated mouse anti-human TLR1–9, ICAM-1, CD18, mouse IgG1 or rabbit IgG2a isotype for 30 min at 4°C in the dark. After washing, the cells were resuspended in 1% paraformaldehyde as fixative. To determine the intracellular expression of TLR1–9, eosinophils were fixed with 4% paraformaldehyde for 10 min at 37°C. After washing once, cells were permeabilized in ice-cold 0.5% saponin for 30 min. After another washing, cells were stained with FITC-conjugated mouse anti-human TLR1–9 or mouse IgG1 for 30 min at 4°C in the dark. Cells were washed again, resuspended, and subjected to flow analysis. Expressions of TLRs and adhesion molecules on 10,000 viable cells were then gated and analyzed by flow cytometry (FACSCalibur, Becton Dickinson Biosciences Corp., San Jose, CA) as mean fluorescence intensity (MFI), which included both the changes of adhesion molecule expression on individual cell and the percentage of cells expressing the adhesion molecules.

Western Blot Analysis
Cells (1 x 107) were washed with ice-cold PBS and lysed in 0.2 ml lysis buffer (20 mM Tris-HCl, pH 8.0, 120 mM NaCl, 1% Triton X-100, 10 mM EDTA, 1 mM EGTA, 0.05% 2-mercaptoethanol, 1x protease inhibitors). Cell debris was removed by centrifugation at 14,000 x g for 15 min, and the supernatant was boiled in Laemmli sample buffer (Bio-Rad Laboratory, Hercules, CA) for 5 min. An equal amount of proteins was subjected to sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis before blotting onto a PVDF membrane (Amersham and Pharmacia Biotech). The membrane was blocked with 5% skimmed milk in Tris-buffered saline with 0.05% Tween 20, pH 7.6 for 1 h at room temperature, and probed with primary anti-human TLR1–9, anti-human phospho-ERK and total ERK, anti-human phospho- and total p38 MAPK, anti-human phospho- and total I{kappa}B-{alpha}, and anti-human phospho- and total Akt antibodies (Cell Signaling Technology Inc., Beverly, MA) at 4°C overnight. After washing, membrane was incubated with corresponding secondary donkey anti-rabbit or sheep anti-mouse antibodies coupled to horseradish peroxidase (Amersham and Pharmacia Biotech) for 1 h at room temperature. Antibody–antigen complexes were then detected using ECL chemiluminescent detection system according to the manufacturer's instructions (Amersham and Pharmacia Biotech) (5). The Western blots after phosphorylated protein analysis were reprobed with antibodies against the total p38 MAPK, Akt, ERK, and I{kappa}B{alpha} to demonstrate equal protein loading.

Electrophoretic Mobility Shift Assay
Eosinophils were harvested and washed, and nuclear proteins were extracted with NE-PER nuclear and cytoplasmic extraction reagents (Pierce Chemical Co., Rockford, IL) according to the manufacturer's instructions. Equal amounts of nuclear protein extracts were subjected to a test for NF-{kappa}B protein/DNA binding using LightShift chemiluminescent electrophoretic mobility shift assay (EMSA) kit (Pierce) with a biotin end-labeled NF-{kappa}B oligonucleotide (5'-AGT TGA GGG GAC TTT CCC AGG C-3') (Research Genetics Invitrogen Co., Huntsville, AL). Briefly, nuclear extracts were incubated with biotin end-labeled NF-{kappa}B oligonucleotide for 20 min at room temperature to allow DNA/protein binding. The DNA/protein complexes were then resolved by a 6% native polyacrylamide gel electrophoresis and transferred to a Hybond-N+ membrane (Amersham and Pharmacia Biotech). The biotin end-labeled DNA was detected using a streptavidin–horseradish peroxidase conjugate and a chemiluminescent substrate (5).

Statistical Analysis
All data were expressed as mean ± SD. Differences between groups were assessed by one-way ANOVA analysis. P < 0.05 was considered significantly different. All analyses were performed using the Statistical Package for the Social Sciences (SPSS) statistical software for Windows, version 10.1.4 (SPSS Inc., Chicago, IL).


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Expression of TLRs of Eosinophils and Neutrophils
As shown in Figure 1A, human peripheral blood eosinophils expressed TLR1, -2, -4, -5, -6, -7, and –9, while neutrophils expressed TLR1, -2, -3, -4, -5, -6, -7, and -9. The protein expression profile of Western blots (Figure 1A) is similar to that assessed using flow cytometry for the cell surface and intracellular expression of TLRs in both eosinophils and neutrophils (Figure 1B).


Figure 1
Figure 1
View larger version (70K):
[in this window]
[in a new window]

  		Figure 1. Protein expressions of TLRs of eosinophils and neutrophils. (A) Total protein was extracted from eosinophils (eos) and neutrophils (neu) (1 x 107 cells), and equal protein amounts were analyzed using Western blot for TLR1–9. beta-actin was used as loading control. Molecular weights are as follows (in kD): TLR1, 87; TLR2, 90; TLR3, 120; TLR4, 100; TLR5, 97; TLR6, 92; TLR7, TLR8, and TLR9, ~ 120; beta-actin, 43. (B) Surface (top panel) and intracellular (bottom panel) expression of TLRs of eosinophils (solid bars) and neutrophils (shaded bars) (5 x 105 cells) was determined by flow cytometry. TLR1, -2, -3, -4, -5, -6, -7, -8, and -9 expression was shown as MFI subtracting corresponding isotypic control and is expressed as the arithmetic mean plus SD of three independent experiments.

 
Effects of TLR Ligands on the Induction of Cytokines, Superoxides, and ECP from Eosinophils
We have first assessed the cytokine expression profile of eosinophils activated by the seven TLR ligands PGN, LPS, flagellin, R837, poly I-C, ssRNA, and CpG (1 µg/ml) using antibody-based RayBio human cytokine array V (RayBiotech) (5). Results indicated that PGN, flagellin, and R837 (1 µg/ml) for TLR2, -5, and -7, respectively, could conspicuously induce protein expressions of cytokine IL-1beta, IL-6, CXC chemokine IL-8, and GRO among the 79 different cytokines being screened (Figure 2). PGN, flagellin, and R837, but not poly I-C, LPS, ssRNA, or CpG, could induce the release of IL-1beta, IL-6, IL-8, GRO-{alpha}, superoxides, and ECP (Figure 3). GM-CSF was used as a positive control. As shown in Figure 4, PGN, flagellin, and R837 (0.05–5 µg/ml) could activate eosinophils to release IL-1beta, IL-6, IL-8, and GRO-{alpha} dose dependently at 24 h. On the other hand, LPS, poly I-C, ssRNA, and CpG could not induce any cytokines, even at concentrations of 5 µg/ml (data not shown).


Figure 2
Figure 2
View larger version (115K):
[in this window]
[in a new window]

  		Figure 2. Representative profile of the release of cytokines from eosinophils activated by TLR ligands. Eosinophils (1 x 106 cells) were cultured with or without different TLR ligands (all 1 µg/ml) for 24 h in a 24-well plate. Cell-free culture supernatant was then harvested and 79 different cytokines in culture supernatant were semi-quantitated using antibody-based RayBio human cytokine array V. Positive and negative controls were designated at (1a, 1b, 1c, 1 d, 8j, 8k) and (1e, 1f, 8i), respectively. Triplicate experiments were performed with essentially identical results. Table lists the format of antibodies on the cytokine membrane array.

 

Figure 3
Figure 3
View larger version (26K):
[in this window]
[in a new window]

  		Figure 3. The induction of cytokines, superoxides, and ECP from eosinophils activated by TLR ligands. (A) Releases of IL-1beta, IL-6, IL-8, and GRO-{alpha} from eosinophils (1 x 106/well) activated by TLR ligands (all 1 µg/ml) for 24 h in a 24-well plate were determined by ELISA. (B) Release of superoxides from eosinophils (1 x 105 cells) activated by TLR ligands (all 1 µg/ml) for 30 min in a 96-well plate was determined using chemiluminescence-based assay kit. (C) Release of ECP from eosinophils (1 x 106 cells) activated by TLR ligands (all 1 µg/ml) for 6 h in a 96-well plate was determined by FEIA. Results are expressed as the arithmetic mean ± SD from three independent experiments. *P < 0.05 and **P < 0.01 when compared with the medium control. GM-CSF (50 ng/ml) was acted as a positive control.

 

Figure 4
Figure 4
View larger version (22K):
[in this window]
[in a new window]

  		Figure 4. Release of IL-1beta, IL-6, IL-8, and GRO-{alpha} from eosinophils activated by PGN, flagellin, and R837. Eosinophils (1 x 106/well) were cultured with or without PGN, flagellin, or R837 (0.05–5 µg/ml) for 24 h in a 24-well plate. Cytokines released into the culture supernatant were determined using ELISA. Results are expressed as the arithmetic mean ± SD from three independent experiments. *P < 0.05 and **P < 0.01 when compared with the medium control.

 
Effects of Anti-TLR2 and TLR4-Neutralizing Antibodies on the PGN-Induced Cytokines of Eosinophils
Results in Figure 5 indicate that TLR2-neutralizing antibody could completely suppress the PGN-mediated induction of IL-1beta, IL-8, IL-6, and GRO-{alpha} from eosinophils. On the other hand, TLR4-neutralizing antibody showed no significant effect on the PGN-induced IL-1beta, IL-8, IL-6, and GRO-{alpha} from eosinophils. These findings illustrate that the TLR2 ligand PGN can specifically bind to TLR2 but not other TLRs such as TLR4.


Figure 5
View larger version (14K):
[in this window]
[in a new window]

  		Figure 5. Effects of anti-TLR2– and anti-TLR4–neutralizing antibodies on the PGN-induced cytokine release of eosinophils. Eosinophils (1 x 106 cells) were cultured with or without PGN (1 µg/ml) for 24 h in a 24-well plate in the presence of anti-TLR2 antibody (5 µg/ml), anti-TLR4 antibody (5 µg/ml), or mouse IgG control antibody (5 µg/ml). Cytokines released into the culture supernatant were determined by ELISA. Results are expressed as the arithmetic mean plus SD from three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 when compared with the medium control.

 
Effects of PGN, Flagellin, and R837 on the Cell Surface Expression of Adhesion Molecule ICAM-1 and CD18
Treatment of eosinophils (5 x 105 cells) with PGN, flagellin, or R837 (5 µg/ml) for 16 h could up-regulate the cell surface expression of ICAM-1 (MFI: medium versus PGN, 2.88 ± 0.20 versus 6.63 ± 0.16, medium versus flagellin, 2.37 ± 0.17 versus 4.90 ± 0.81, medium versus R837, 2.58 ± 0.08 versus 3.69 ± 0.39, all P < 0.05) and CD18 (MFI: medium versus PGN, 11.31 ± 1.11 versus 22.75 ± 2.81, medium versus flagellin, 10.40 ± 0.40 versus 18.45 ± 0.45, medium versus R837, 11.53 ± 0.59 versus 19.98 ± 1.04, all P < 0.05) on eosinophils in triplicate experiments.

Effects of TLR Ligands on the Activation of Intracellular Signaling Molecules ERK, p38 MAPK, PI3K, and NF-{kappa}B
Figure 6 shows that PGN, flagellin, and R837 could activate eosinophils by rapidly inducing the phosphorylation of ERK1 and I{kappa}B-{alpha} within 5 min (Figures 6A and 6D). However, phosphorylation of p38 MAPK and Akt could only be activated by R837, and PGN and R837, respectively (Figures 6B and 6C). Results therefore indicated the differential activation of intracellular ERK1, p38 MAPK, PI3K-Akt, and NF-{kappa}B activity in eosinophils by PGN, flagellin, and R837. We have performed the Western blot analysis of phosphorylated ERK1 and ERK2. Only the expression of phosphorylated ERK1 was increased in eosinophils after TLR ligand activation. Increased phosphorylation of Akt implied elevated PI3K activity, since Akt is a direct downstream substrate of PI3K. Total (phosphorylated and nonphosphorylated) protein was used as control to ensure that an equal protein amount was loaded at different treatments. However, the three TLR ligands did not exhibit any effect on JAK and JNK activity (data not shown). Figure 7 illustrates that untreated eosinophils showed a low shifted band, thereby indicating a basal activity of NF-{kappa}B. Eosinophils treated with PGN, flagellin, or R837 (1 µg/ml) for 4 h were shown to activate NF-{kappa}B for subsequent gene transcription because there were significant increases in band shift. Competitive control at lane 2 using excessive unlabeled p50 NF-{kappa}B–binding DNA to totally suppress the PGN-induced band shift confirmed the specificity of NF-{kappa}B–DNA interaction.


Figure 6
Figure 6
View larger version (103K):
[in this window]
[in a new window]

  		Figure 6. Activation of ERK, p38 MAPK, Akt, and NF-{kappa}B activities in eosinophils by TLR ligands. Eosinophils (1 x 107 cells) were cultured with different TLR ligands (all 1 µg/ml) for different indicated incubation times. Total cellular proteins were extracted from eosinophils for the measurement of (A) phosphorylated ERK1 and total ERK1, (B) phosphorylated and total p38 MAPK, (C) phosphorylated and total Akt, and (D) phosphorylated and total I{kappa}B{alpha} by Western blot analysis. Experiments were performed in three independent replicates with essentially identical results, and representative results are shown.

 

Figure 7
View larger version (19K):
[in this window]
[in a new window]

  		Figure 7. Effect of PGN, flagellin, and R837 on NF-{kappa}B activity in eosinophils. Eosinophils (1 x 107/well) were cultured with or without PGN, flagellin, and R837 (all 1 µg/ml) for 4 h. Nuclear proteins were extracted from eosinophils, 5 µg protein was then subjected to EMSA, and relative intensity of shifted band was detected by densitometry. Experiments were performed in three independent replicates with essentially identical results, and representative results are shown. Lane 1, negative control; lane 2, competitive control + PGN (competitive control with unlabeled p50 NF-{kappa}B–binding DNA); lane 3, medium control; lane 4, PGN; lane 5, flagellin; lane 6, R837.

 
Effects of Signaling Molecule Inhibitors AG-490, BAY11–7082, PD98059, SP600125, LY294002, and SB203580 on TLR Ligand–Induced Release of IL-1beta, IL-6, IL-8, and GRO-{alpha} from Eosinophils
As shown in Figure 8A, pretreatment of eosinophils with NF-{kappa}B inhibitor BAY11–7082, ERK inhibitor PD98059, and PI3K inhibitor LY294002 for 1 h could significantly suppress the PGN-induced release of IL-1beta, IL-8, IL-6, and GRO-{alpha} (all P < 0.001). Figures 8B and 8C show that only BAY11–7082 and PD98059 could significantly suppress the flagellin-induced release of cytokines, while BAY11–7082, PD98059, LY294002, and SB203580 could significantly suppress the R837-induced release of cytokines from eosinophils (all P < 0.001). Results were therefore compatible with that of Figures 6 and 7. However, JAK inhibitor AG490 and JNK inhibitor SP600125 did not exhibit any effect on the TLR ligand–induced release of cytokines.


Figure 8
Figure 8
Figure 8
View larger version (52K):
[in this window]
[in a new window]

  		Figure 8. Effects of AG490, BAY11–7082, PD98059, LY294002, SB203580, and SP600125 on the TLR ligands induced release of IL-1beta, IL-6, IL-8, and GRO-{alpha} from eosinophils. Eosinophils (1 x 106 cells) were pretreated with AG490 (3 µM), BAY11–7082 (1 µM), PD98059 (10 µM), LY294002 (5 µM), SB203580 (7.5 µM), or SP600125 (3 µM) for 45 min, followed by incubation with or without (A) PGN, (B) flagellin, and (C) R837 (all 1 µg/ml) in the presence of inhibitors for further 24 h. Release of cytokines in the culture supernatant was determined by ELISA. Results are expressed as the arithmetic mean plus SD from three independent experiments. DMSO (0.1%) was used as the DMSO control. *P < 0.05, **P < 0.01, ***P < 0.001 when compared with the medium control (CTL). AG, AG 490; BAY, BAY11–7082; PD, PD98059; LY, LY 294002; SB, SB203580; SP, SP600125; Flag, flagellin.

 
Effects of BAY11–7082, PD98059, LY294002, and SB203580 on PGN-, Flagellin-, and R837-Induced Superoxide and ECP Release from Eosinophils
Figure 9 shows that PGN, flagellin, and R837 (1 µg/ml) could significantly activate eosinophils to release superoxide anion at 30 min (all P < 0.05). Similar to that of cytokines induction, BAY11–7082 and PD98059 could significantly inhibit PGN-, flagellin-, and R837-induced release of superoxides (all P < 0.05). LY294002 and SB203580 could significantly suppress PGN- and R837-, and R837-induced release of superoxides, respectively. LPS, Poly I-C, ssRNA, and CpG-DNA (1 µg/ml), and all four inhibitors alone, did not exhibit any significant effect on the superoxide release (all P > 0.05). Moreover, anti-TLR2 but not -TLR4 antibody could completely suppress the PGN-induced superoxide release from eosinophils (data not shown).


Figure 9
View larger version (14K):
[in this window]
[in a new window]

  		Figure 9. Effects of BAY11–7082, PD98059, LY294002, and SB203580 on TLR ligands induced release of superoxides from eosinophils. Eosinophils (1 x 105 cells) were pretreated with BAY11–7082 (1 µM), PD98059 (10 µM), LY294002 (5 µM), or SB203580 (7.5 µM) for 45 min, followed by incubation with or without (A) PGN, (B) flagellin, and (C) R837 (all 1 µg/ml) in the presence of inhibitors for further 30 min. Superoxide released into the culture supernatant was determined using chemiluminescence-based assay kit. Results are expressed as the arithmetic mean plus SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 when compared with the medium control (CTL). Flag, flagellin.

 
Inhibition study revealed that the PGN-induced degranulation for ECP release was inhibited by BAY11–7082, LY294002, and PD98059 (Figure 10). Moreover, anti-TLR2 but not TLR4 antibody could completely suppress the PGN-induced ECP release from eosinophils (data not shown).


Figure 10
View larger version (9K):
[in this window]
[in a new window]

  		Figure 10. Effects of BAY11–7082, PD98059, LY294002, and SB203580 on TLR ligands induced degranulation for the release of ECP from eosinophils. Eosinophils (1 x 106 cells) were pretreated with BAY11–7082 (1 µM), PD98059 (10 µM), LY294002 (5 µM), or SB203580 (7.5 µM) for 45 min, followed by incubation with or without PGN (1 µg/ml) in the presence of inhibitors for further 6 h. ECP released into the culture supernatant was determined by FEIA. Results are expressed as the arithmetic mean plus SD from three independent experiments. *P < 0.05 and **P < 0.01 when compared with the medium control (CTL). BAY, BAY11–7082; PD, PD98059; LY, LY294002; SB, SB203580.

 

   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
The inflammatory and airway remodeling processes in allergic asthma are the consequence of highly complex interactions among inflammatory cells such as eosinophils, activated T lymphocytes, mast cells, and macrophages with structural tissue cells including the bronchial epithelium, endothelial cells, and fibroblasts (15). It has been shown that the viral or bacterial infections precede the exacerbation of allergic asthma and atopic dermatitis (16, 17). TLR2-, -4–, -5–, -7–, and -9–mediated activation has previously been shown to involve in the pathogenesis of allergic inflammatory diseases (12, 18–21).

In previous studies, the expression of TLRs of esoinophils has generally been investigated using RT-PCR (10–12). Controversial and contradictory results have been reported for the TLR4- and CD14-mediated activation of eosinophils (10–12, 22, 23). Although some studies reported no observed gene expression of TLR2 and TLR4 (11, 12), many other reports confirmed mRNA expression of TLR2 and TLR4 as well as the positive immunofluorescent surface staining of TLR4 on eosinophils (10, 22, 23). In the present study, the significant protein expression of TLR1, -2, -4, -5, -6, -7, and -9 of human eosinophils has been confirmed using Western blot and flow cytometry. The TLR expression profiles were found to be different between eosinophils and neutrophils, implying different responses of eosinophils and neutrophils toward the activation by viral or bacterial agents. We found TLR2, -4, -5, and -6 were expressed both on cell surface and at intracellular compartment of eosinophils and neutrophils. Actually, intracellular expressions of TLR2, -4, -5, and -6 in different cell types including human macrophages, dendritic cells, neutrophils, and lymphocytes have been previously reported (24–26).

Using an animal model, intranasal application of PGN (TLR2 ligand) was found to exaggerate the airway allergen hypersensitivities (27). The present results of our cytokine study (Figure 2) confirmed that ligands of TLR2, -5, and -7 (PGN, flagellin, and R837, respectively) could selectively induce the significant in vitro release from eosinophils of proinflammatory cytokine IL-1beta and IL-6, and CXC chemokine IL-8 and GRO-{alpha} for neutrophils in a time- and dose-dependent manner. The above three TLR ligands but not LPS, Poly I-C, ssRNA, or CpG DNA could also enhance the viability of eosinophils by ~ 50% compared with negative control (data not shown). Among the three TLR ligands, TLR2-mediated cytokine release was found to be the most potent, while TLR7 was relatively weak. It may be due to the nature of the synthetic compound of TLR7 ligand R837. Cytokines IL-1 and IL-8 can induce the recruitment of eosinophils and adhesion on endothelium at inflammatory sites (2). Moreover, neutrophil migration in response to IL-8 and GRO-{alpha} may also lead eosinophils to accumulate in the airways of asthma and aggravate the disease (28). Consequently, TLR-mediated activation of eosinophils could induce proinflammatory cytokines for the activation of macrophages, lymphocytes, and chemokines for the recruitment and infiltration of neutrophils and eosinophils at inflammatory sites in the bronchial airway, thereby amplifying inflammatory responses during allergic asthma or dermatitis.

A previous study has shown that TLR7 ligand R848 could induce release of superoxides from eosinophils (12). We found that TLR ligands PGN, flagellin, and R837 could also up-regulate the cell surface adhesion molecules including ICAM and CD18, a component of LFA-1 and Mac-1, and induce the degranulation of eosinophils for the release of toxic superoxides and ECP. Our results therefore imply that both viral and bacterial infection could enhance the adhesion of eosinophils onto bronchial epithelial cells, a crucial step for activation and transendothelial migration of eosinophils, and trigger the degranulation of eosinophils, causing tissue damage. These have led us to hypothesize that eosinophils can play a crucial role in linking the innate immune response upon bacterial and viral infection and the initiation of allergic inflammation via adhesion, the release of cytokines, chemokines, superoxides, and granular toxic proteins.

TLR9 is the receptor for both bacterial and viral DNA for inducing Th1 response and suppressing Th2 response (29). The expression and functions of TLR9 have been characterized in epithelial cells, B cells, and plasmacytoid dendritic cells (29). Although we could not observe the TLR9-mediated cytokine release or degranulation of eosinophils upon the activation by TLR9 ligand CpG-DNA, our result is actually in concordance with the previous publication (12). Moreover, eosinophils can only express relatively low level of TLR9. Furthermore, eosinophils are mainly related to Th2 immune response in allergic inflammation; it is therefore reasonable that Th1-related TLR9 ligand cannot activate eosinophils.

With regard to the intracellular mechanisms regulating the induction of cytokines and chemokines, and degranulation, this should be the first report showing the differential activation of intracellular NF-{kappa}B, ERK, and PI3K-Akt of eosinophils by TLR2, NF-{kappa}B, and ERK by TLR5, and NF-{kappa}B, ERK, p38 MAPK, and PI3K-Akt by TLR7 (Figure 6). Our former studies have shown that p38 MAPK and NF-{kappa}B play crucial roles in the IL-25–mediated expression of cytokines and chemokines, adhesion molecules of eosinophils (5). We have used inhibitors AG490, BAY11–7082, PD98059, SP600125, LY294002, and SB203580 to elucidate the intracellular signaling mechanisms regulating the induction of cytokines, chemokines, and degranulation. Following previous publications (5, 30), we used the optimal concentration of AG490 (3 µM), BAY11–7082 (1 µM), PD98059 (10 µM), LY294002 (5 µM), SP600125 (3 µM), and SB203580 (7.5 µM) with the highest inhibitory effect without any significant cell toxicity. The inhibition experiments demonstrated that the production of IL-1beta, IL-6, IL-8, and GRO-{alpha} induced by PGN, flagellin, and R837 was differentially mediated by the intracellular ERK, p38 MAPK, PI3K-Akt, and NF-{kappa}B but not JNK and JAK-STAT activities, which were consistent with the results of Western blots. The intracellular signaling mechanisms regulating the PGN-, flagellin-, and R837-induced superoxide release and PGN-induced degranulation for the release of ECP were also found to be the same to that of the release of cytokines and chemokines. In fact, a previous study has shown that degranulation and chemotaxis of eosinophils are regulated by ERK and p38 MAPK (31). Moreover, p38 MAPK and NF-{kappa}B are common signal transduction molecules by which the expression of cytokines and chemokines can be regulated in eosinophils (5, 30). It is therefore reasonable that the molecular mechanism by which the TLR2, -5, and -7 mediated release of IL-1beta, IL-6, IL-8, GRO-{alpha}, and superoxides, and degranulation of eosinophils share the above common signaling molecules. There is increasing evidence showing that activation of MAPKs is required for NF-{kappa}B–dependent gene expression, and there is cross-talk between discrete intracellular signaling pathways (32). We are performing further experiments to confirm this type of regulatory mechanism upon different TLR ligand activation in eosinophils.

In conclusion, our present study has demonstrated the intracellular signaling molecules regulating the TLR-mediated activation of eosinophils for the release of cytokines, chemokines, superoxides, and granular proteins for the inflammatory responses. Our results suggest that TLR2–, -5–, and -7–induced activation in eosinophils is mediated by the combined activation of ERK and NF-{kappa}B pathways, thereby providing new postulates for immunopathologic mechanisms of respiratory tract infection mediating the exacerbation of eosinophil-related allergic inflammation in pulmonary disorders such as allergic asthma (16, 17). Together with other studies on the immunologic roles of TLRs in infection and immunity (33), our results imply that TLR manipulation, particularly TLR2, -5, and 7, might be a potential therapeutic target for allergic diseases such as asthma and atopic dermatitis. In view of recent advances in the application of Akt, MAPK, and NF-{kappa}B inhibitors as potential anti-inflammatory agents in asthma (34, 35), our study of TLR-mediated activation on eosinophils might provide new insights on the development of therapeutic intervention for allergic disorders.


   Footnotes
 
This study was supported by a Direct Grant for Research, The Chinese University of Hong Kong (2005.1.016).

Originally Published in Press as DOI: 10.1165/rcmb.2006-0457OC on March 1, 2007

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form December 11, 2006

Accepted in final form February 14, 2007


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

  1. Lucas SR, Platts-Mills TA. Physical activity and exercise in asthma: relevance to etiology and treatment. J Allergy Clin Immunol 2005;115:928–934.[CrossRef][Medline]
  2. Lampinen M, Carlson M, Hakansson LD, Venge P. Cytokine-regulated accumulation of eosinophils in inflammatory disease. Allergy 2004;59:793–805.[CrossRef][Medline]
  3. Ulfman LH, Joosten DP, van der Linden JA, Lammers JW, Zwaginga JJ, Koenderman L. IL-8 induces a transient arrest of rolling eosinophils on human endothelial cells. J Immunol 2001;166:588–595.[Abstract/Free Full Text]
  4. Adamko DJ, Odemuyiwa SO, Vethanayagam D, Moqbel R. The rise of the phoenix: the expanding role of the eosinophil in health and disease. Allergy 2005;60:13–22.[CrossRef][Medline]
  5. Wong CK, Cheung PF, Ip WK, Lam CWK. IL-25 induced chemokines and IL-6 release from eosinophils is mediated by p38 MAPK, JNK and NF-{kappa}B. Am J Respir Cell Mol Biol 2005;33:186–194.[Abstract/Free Full Text]
  6. Gangloff SC, Guenounou M. Toll-like receptors and immune response in allergic disease. Clin Rev Allergy Immunol 2004;26:115–125.[Medline]
  7. Akira S, Takeda K, Kaisho T. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat Immunol 2001;2:675–680.[CrossRef][Medline]
  8. Bellou A, Schaub B, Ting L, Finn PW. Toll receptors modulate allergic responses: interaction with dendritic cells, T cells and mast cells. Curr Opin Allergy Clin Immunol 2003;3:487–494.[CrossRef][Medline]
  9. Yang IA, Fong KM, Holgate ST, Holloway JW. The role of Toll-like receptors and related receptors of the innate immune system in asthma. Curr Opin Allergy Clin Immunol 2006;6:23–28.[Medline]
  10. Plotz SG, Lentschat A, Behrendt H, Plotz W, Hamann L, Ring J, Rietschel ET, Flad HD, Ulmer AJ. The interaction of human peripheral blood eosinophils with bacterial lipopolysaccharide is CD14 dependent. Blood 2001;97:235–241.[Abstract/Free Full Text]
  11. Sabroe I, Jones EC, Usher LR, Whyte MK, Dower SK. Toll-like receptor (TLR)2 and TLR4 in human peripheral blood granulocytes: a critical role for monocytes in leukocyte lipopolysaccharide responses. J Immunol 2002;168:4701–4710.[Abstract/Free Full Text]
  12. Nagase H, Okugawa S, Ota Y, Yamaguchi M, Tomizawa H, Matsushima K, Ohta K, Yamamoto K, Hirai K. Expression and function of Toll-like receptors in eosinophils: activation by Toll-like receptor 7 ligand. J Immunol 2003;171:3977–3982.[Abstract/Free Full Text]
  13. Hopkins PA, Sriskandan S. Mammalian Toll-like receptors: to immunity and beyond. Clin Exp Immunol 2005;140:395–407.[CrossRef][Medline]
  14. O'Neill LA. How Toll-like receptors signal: what we know and what we don't know. Curr Opin Immunol 2006;18:3–9.[CrossRef][Medline]
  15. Chiappara G, Gagliardo R, Siena A, Bonsignore MR, Bousquet J, Bonsignore G, Vignola AM. Airway remodelling in the pathogenesis of asthma. Curr Opin Allergy Clin Immunol 2001;1:85–93.[CrossRef][Medline]
  16. Gern JE, Busse WW. Relationship of viral infections to wheezing illnesses and asthma. Nat Rev Immunol 2002;2:132–138.[CrossRef][Medline]
  17. Bunikowski R, Mielke ME, Skarabis H, Worm M, Anagnostopoulos I, Kolde G, Wahn U, Renz H. Evidence for a disease-promoting effect of Staphylococcus aureus-derived exotoxins in atopic dermatitis. J Allergy Clin Immunol 2000;105:814–819.[CrossRef][Medline]
  18. Redecke V, Hacker H, Datta SK, Fermin A, Pitha PM, Broide DH, Raz E. Cutting edge: activation of Toll-like receptor 2 induces a Th2 immune response and promotes experimental asthma. J Immunol 2004;172:2739–2743.[Abstract/Free Full Text]
  19. Eisenbarth SC, Piggott DA, Huleatt JW, Visintin I, Herrick CA, Bottomly K. Lipopolysaccharide-enhanced, toll-like receptor 4-dependent T helper cell type 2 responses to inhaled antigen. J Exp Med 2002;196:1645–1651.[Abstract/Free Full Text]
  20. Didierlaurent A, Ferrero I, Otten LA, Dubois B, Reinhardt M, Carlsen H, Blomhoff R, Akira S, Kraehenbuhl JP, Sirard JC. Flagellin promotes myeloid differentiation factor 88-dependent development of Th2-type response. J Immunol 2004;172:6922–6930.[Abstract/Free Full Text]
  21. Wild JS, Sur S. CpG oligonucleotide modulation of allergic inflammation. Allergy 2001;56:365–376.[CrossRef][Medline]
  22. Bonini S, Micera A, Iovieno A, Lambiase A, Bonini S. Expression of Toll-like receptors in healthy and allergic conjunctiva. Ophthalmology 2005;112:1528–1534.[Medline]
  23. Komiya A, Nagase H, Okugawa S, Ota Y, Suzukawa M, Kawakami A, Sekiya T, Matsushima K, Ohta K, Hirai K, et al. Expression and function of toll-like receptors in human basophils. Int Arch Allergy Immunol 2006;140:23–27.[CrossRef][Medline]
  24. Ueta M, Nochi T, Jang MH, Park EJ, Igarashi O, Hino A, Kawasaki S, Shikina T, Hiroi T, Kinoshita S, et al. Intracellularly expressed TLR2s and TLR4s contribution to an immunosilent environment at the ocular mucosal epithelium. J Immunol 2004;173:3337–3347.[Abstract/Free Full Text]
  25. Crellin NK, Garcia RV, Hadisfar O, Allan SE, Steiner TS, Levings MK. Human CD4+ T cells express TLR5 and its ligand flagellin enhances the suppressive capacity and expression of FOXP3 in CD4+CD25+ T regulatory cells. J Immunol 2005;175:8051–8059.[Abstract/Free Full Text]
  26. Jablonska E, Marcinczyk M, Jablonski J. Toll-like receptors types 2 and 6 and the apoptotic process in human neutrophils. Arch Immunol Ther Exp (Warsz) 2006;54:137–142.[CrossRef][Medline]
  27. Chisholm D, Libet L, Hayashi T, Horner AA. Airway peptidoglycan and immunostimulatory DNA exposures have divergent effects on the development of airway allergen hypersensitivities. J Allergy Clin Immunol 2004;113:448–454.[CrossRef][Medline]
  28. Kikuchi I, Kikuchi S, Kobayashi T, Hagiwara K, Sakamoto Y, Kanazawa M, Nagata M. Eosinophil trans-basement membrane migration induced by interleukin-8 and neutrophils. Am J Respir Cell Mol Biol 2006;34:760–765.[Abstract/Free Full Text]
  29. Cristofaro P, Opal SM. Role of Toll-like receptors in infection and immunity: clinical implications. Drugs 2006;66:15–29.[CrossRef][Medline]
  30. Wong CK, Ip WK, Lam CWK. IL-3, 5 and GM-CSF-induced adhesion molecule expression on eosinophils by p38 MAPK and NF-{kappa}B. Am J Respir Cell Mol Biol 2003;29:133–147.[Abstract/Free Full Text]
  31. Kampen GT, Stafford S, Adachi T, Jinquan T, Quan S, Grant JA, Skov PS, Poulsen LK, Alam R. Eotaxin induces degranulation and chemotaxis of eosinophils through the activation of ERK2 and p38 mitogen-activated protein kinases. Blood 2000;95:1911–1917.[Abstract/Free Full Text]
  32. Carter AB, Knudtson KL, Monick MM, Hunninghake GW. The p38 mitogen-activated protein kinase is required for NF-{kappa}B-dependent gene expression. The role of TATA-binding protein (TBP). J Biol Chem 1999;274:30858–30863.[Abstract/Free Full Text]
  33. Cristofaro P, Opal SM. Role of Toll-like receptors in infection and immunity: clinical implications. Drugs 2006;66:15–29.[CrossRef][Medline]
  34. Kaminska B. MAPK signalling pathways as molecular targets for anti-inflammatory therapy–from molecular mechanisms to therapeutic benefits. Biochim Biophys Acta 2005;1754:253–262.[Medline]
  35. Makarov SS. NF-kappaB as a therapeutic target in chronic inflammation: recent advances. Mol Med Today 2000;6:441–448.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
P. F. Y. Cheung, C. K. Wong, and C. W. K. Lam
Molecular Mechanisms of Cytokine and Chemokine Release from Eosinophils Activated by IL-17A, IL-17F, and IL-23: Implication for Th17 Lymphocytes-Mediated Allergic Inflammation
J. Immunol., April 15, 2008; 180(8): 5625 - 5635.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
L. Xu, H. Fang, and D. M. Frucht
Anthrax Lethal Toxin Increases Superoxide Production in Murine Neutrophils via Differential Effects on MAPK Signaling Pathways
J. Immunol., March 15, 2008; 180(6): 4139 - 4147.
[Abstract] [Full Text] [PDF]

Home page
 Int ImmunolHome page
P. F.-Y. Cheung, C.-K. Wong, W.-K. Ip, and C. W.-K. Lam
FAK-mediated activation of ERK for eosinophil migration: a novel mechanism for infection-induced allergic inflammation
Int. Immunol., March 1, 2008; 20(3): 353 - 363.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2006-0457OCv1
37/1/85    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 Wong, C. K.
Right arrow Articles by Lam, C. W. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wong, C. K.
Right arrow Articles by Lam, C. W. K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Crit. Care Med.
Copyright © 2007 American Thoracic Society.