Role of ERK1/2 Mitogen-Activated Protein Kinases and NF- B
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| |
Abstract |
|---|
|
|
|---|
Human alveolar macrophages (HAM) express Fc
R receptors
for immunoglobulin (Ig)A which could link humoral and cellular branches of lung immunity. Here, we investigate the effects of polymeric (p-IgA) and secretory (S-IgA) IgA interaction
with Fc
R on lipopolysaccharide (LPS)- and phorbol myristate
acetate (PMA)-activated respiratory burst and TNF-
release
by HAM. Activation of HAM with LPS and PMA increases the
respiratory burst and TNF-
release through activation of the
extracellular signal-related protein kinases 1 and 2 (ERK1/2)
pathway, because these effects are inhibited by treatment of
HAM with PD98059, a selective inhibitor of mitogen-activated protein (MAP)/ERK kinases (MEK) pathway. S-IgA and p-IgA
downregulate the LPS-increased respiratory burst in HAM
through an inhibition of ERK1/2 activity. In contrast, p- and S-IgA
induce an increase in the respiratory burst of PMA-treated
HAM. This effect is associated with an upregulation by IgA of
the PMA-induced phosphorylation of ERK1/2 and is also inhibited by PD98059. Moreover, p-IgA and S-IgA enhance TNF-
release by HAM through an alternative pathway distinct from
ERK1/2. Because LPS is known to activate nuclear factor-
B
(NF-
B) in HAM, we evaluate the effect of IgA on NF-
B. Treatment of HAM with LPS, p- and S-IgA, but not PMA, induces NF-
B
activation through I
B
phosphorylation and subsequent proteolysis. Antioxidants, namely N-acetylcysteine (NAC) and glutathione (GSH), have no effects on IgA-mediated NF-
B nuclear
translocation and only a minor and late effect on that of LPS, suggesting that reactive oxygen intermediates (ROI) play a minor
role in HAM activation through NF-
B. TNF-
release by LPS-activated HAM is sensitive to NF-
B inhibition and only partly
to oxidant scavenging. In contrast, TNF-
release by IgA-treated
HAM is not dependent on oxidants and only partly dependent
on NF-
B. Our results show a differential HAM regulation by IgA
through both dependent and independent modulation of ERK
pathway. In addition, IgA activates NF-
B and this effect was
independent on oxidants. These data may help to understand
the role of IgA in both lung protection and inflammation.
| |
Introduction |
|---|
|
|
|---|
Immunoglobulin (Ig)A is the predominant Ig isotype in
secretory fluids such as gastrointestinal, respiratory, and
urogenital secretions. In secretions, IgA is predominantly
polymeric (p-IgA) and is synthesized by local mucosal plasma
cells before it is transported through epithelial cells by
their polymeric Ig receptor (pIgR) (1) and secreted into
the mucosal fluids as secretory IgA (S-IgA), which consists
of dimeric or p-IgA complex containing a joining J chain
and secretory component. In contrast, serum IgA is largely
monomeric and derived from plasma cells in the bone marrow (2). Polymeric and especially S-IgA, which constitutes the main mediator of specific humoral immunity at mucosal
surfaces, neutralize bacterial toxins and viral particles (3)
and inhibit adherence of bacteria to epithelial cells (4). Despite some well-recognized anti-infectious properties of IgA
antibodies (Abs), the mechanisms of IgA-derived cell responses still remain poorly understood. The interaction of
IgA with pIgR on epithelial cells and with IgA-specific Fc
high affinity receptors (Fc
R [CD89]) expressed on a wide
variety of blood and tissue myeloid cells (5, 6) may provide
additional elements to explain the effector functions of IgA.
Fc
R are heavily but variably glycosylated transmembrane proteins on leukocytes (5). They are expressed on the cell surface in association with the common FcR
-chain homodimer (7). The FcR
-chain is recognized as a signaling
molecule, which, upon Fc
R crosslinking, is phosphorylated
on its immunoreceptor tyrosine-based activation motif (ITAM)
and leads to the activation of several sets of cytoplasmic
protein tyrosine kinases, such as those of the Src family,
and further downstream proteins such as protein kinase C
(PKC) and phospholipase C (PLC) (8). Crosslinking of Fc
R
can trigger several biologic responses, including phagocytosis of IgA immune complexes (9, 10), killing of IgA-opsonized bacteria (11), generation of reactive oxygen intermediates
(ROI) (12), and release of inflammatory cytokines such as
tumor necrosis factor (TNF)-
and interleukin (IL)-6 (13).
However, the interaction of Fc
R with IgA is not exclusively associated with the activation of proinflammatory
processes. Other studies have shown that IgA can also downregulate the oxidative burst and the release of proinflammatory cytokines such as TNF-
and IL-6 by monocytes
and neutrophils (14, 15). This suggests that IgA could also
to some extent control mucosal and systemic inflammatory responses, thereby protecting tissues from injury.
These observations suggest that the ability of IgA to activate leukocytes through Fc
R does not necessarily conflict with its proposed anti-inflammatory activity, and that IgA-mediated cell responses depend not only on the type
of costimulatory signals and the nature of the effector cell
triggered but also on the molecular form of IgA. Even
though the biology of Fc
R has been widely investigated in
monocytes, neutrophils and eosinophils, the molecular mechanisms of IgA-mediated cellular responses still remain poorly understood.
Fc
R are constitutively well expressed on human alveolar macrophages (HAM) (16), an essential cell population at the interface of respiratory mucosal and systemic
immunity, representing therefore the first line of defense
in the lung (19). Fc
R of HAM bind both IgA1 and IgA2,
although the IgA1 subclass predominates in lung secretions. However, upon deglycosylation, Fc
R of HAM are resolved to a protein core of 28 kD (18) instead of 32 kD
in monocytes (5). Indeed, HAM express at their surface a
protein product of an alternatively spliced Fc
R transcript
(18). The biologic importance and the IgA-mediated signaling through these receptors, however, remain unknown.
In this study, we have investigated the effect of HAM's
Fc
R binding by p- and S-IgA, the two main and abundant forms of IgA in mucosal secretions, on the oxidative
burst and the production of the inflammatory cytokine
TNF-
, as well as the potential involvement of extracellular signal-related protein kinases 1 and 2 (ERK1/2) and
NF-
B activation in the modulation of Fc
R function in
resting, lipopolysaccharide (LPS)-, and phorbol myristate
acetate (PMA)-activated HAM. The mitogen-activated protein (MAP) kinases superfamily represents an important
signaling pathway for many cell functions (20, 21). NF-
B,
which is held in an inactivated state in the cytoplasm of unstimulated cells through interaction with a class inhibitory proteins of I
B family (22), translocates into the nucleus in activated cells upon I
B
phosphorylation, polyubiquitination, and subsequent degradation (23). NF-
B controls
the expression of several genes, especially cytokines such
as TNF-
and IL-8, at their level of gene transcription.
We found that the ERK1/2 pathway plays a central role
in the IgA-modulated respiratory burst in both LPS- and
PMA-stimulated HAM. Activated ERK kinases also control the production of TNF-
in both LPS (CD14 receptor-dependent) and PMA (receptor-independent) membrane
activation of HAM, but not in IgA-treated HAM. Both
LPS and IgA, but not PMA, activate NF-
B nuclear translocation through the classic pathway of I
B
phosphorylation and proteolysis. Activated NF-
B plays an important
role in TNF-
release by LPS-activated HAM and only a
minor role in that by IgA-treated HAM, suggesting that an
alternative pathway distinct from ERK1/2 and NF-
B is implicated in the IgA-mediated upregulation of TNF-
secretion. Oxidants do not play an important role in NF-
B
activation and play a minor role in LPS-induced TNF-
release. In contrast, PMA do not activate NF-
B and activates
HAM essentially through ERK1/2 MAP kinase pathway.
| |
Materials and Methods |
|---|
|
|
|---|
Reagents and Antibodies
Recombinant human TNF-
(rh-TNF-
, specific activity > 1 × 108 U/mg) was purchased from Boehringer (Boehringer Mannheim
GmbH, Mannheim, Germany). LPS from Salmonella typhimurium, PMA, N-acetylcysteine (NAC), glutathione (GSH), cytochrome c, and superoxide dismutase (SOD) were purchased from
Sigma (Sigma Chemical Co., St. Louis, MO). PD98059, SB203580,
and MG 132 were purchased from BioMol (Plymouth Meeting,
PA). The mouse monoclonal antibodies (mAbs) A77, A59, and
A62 specific for Fc
R were prepared as described previously
(24). Polyclonal rabbit anti-ERK1/2 MAP Kinases, anti-phospho
ERK1/2 (pERK1/2) MAP Kinases, anti-p38 and anti-phospho p38 MAP kinases, anti-I
B
and anti-phospho I
B
Abs were
purchased from New England Biolabs (Beverly, MA). The
mouse anti-human CD14 mAb conjugated to fluorescein isothiocyanate (FITC) was purchased from Becton Dickinson (San Jose,
CA), the swine anti-rabbit (SAR) IgG conjugated to horseradish
peroxidase (HRP) was purchased from Dako (Glostrup, Denmark) and the F(ab')2 sheep anti-mouse (SAM) IgG conjugated
to FITC was from Sigma. Other materials were reagent grade.
Isolation of HAM
Bronchoalveolar lavages were obtained from normal nonsmoking volunteers as described previously (16) and approved by the local ethics committee. The lavage fluid was passed through a layer of sterile gauze to remove gross mucus and then centrifuged at 500 rpm for 10 min at 4°C to separate cells from fluid. The cell pellet was resuspended and washed twice in complete culture medium: i.e., RPMI-1640 medium (Bio-Whittaker, Walkersville, MD) supplemented with 10% decomplemented (56°C, 30 min) fetal calf serum (FCS), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. HAM were > 95% pure and contained < 1% of neutrophils and monocytes; the remaining cells were lymphocytes as determined by morphologic characteristics. HAM were cultured in complete culture medium and they were stimulated either with LPS (1 µg/ml) or PMA (0.1 µg/ml). The pharmacologic inhibitor of MEK/ERK pathways, PD98059, added 1 h before stimulation of HAM, was used at 50 µM. This concentration inhibited all detectable pERK1/2 MAP Kinases in Western blots and was not toxic to HAM as determined by trypan blue staining.
Purification of IgA
Pure polymers of monoclonal IgA1 were isolated from myeloma sera containing high levels of monoclonal IgA with a large proportion of p-IgA as described by Vaerman and colleagues (25). Polyclonal milk S-IgA was prepared as described previously (26). Purity of IgA was checked by immunoelectrophoresis and/or Ouchterlony analysis using a battery of monospecific antisera against serum proteins as described previously (27). Size distribution of IgA preparations was assessed by sucrose density gradient ultracentrifugation and by gradient polyacrylamide gel electrophoresis (PAGE) with and without sodium dodecyl sulfate (SDS) (25). Polymeric IgA was conjugated to FITC as described previously (16) and the F/P ratio was 2.5.
Anti-IgA Abs Preparations and Fluorescent Conjugates
Rabbit anti-IgA isotype (iso)-specific and idiotype (Id)-specific
IgG Abs were prepared as described by Sibille and coworkers (17). To prevent the Fc portion of the anti-IgA Abs from binding to Fc
R, F(ab')2 fragments were prepared by pepsin digestion of rabbit IgG for 16-20 h at 37°C with an enzyme/substrate weight ratio of 1/50 in 0.1 M Na acetate buffer, pH 4.5. F(ab')2 fragments were separated by Ultrogel AcA 44 and their purity was checked by their lack of precipitation with a goat antiserum specific to
rabbit Fc
in Ouchterlony immunodiffusion and by SDS-PAGE. F(ab')2 were conjugated with FITC as described previously (16).
Flow Cytometric Analysis
Surface expression of Fc
R on HAM was assayed by indirect immunofluorescence. To mask Fc
R, cells (3 × 105) were first incubated with aggregated human IgG (10 mg/ml) in flow cytometric
analysis (FCA) buffer (Hanks' balanced salt solution [HBSS]
with 3% FCS and 0.05% sodium azide) for 30 min at 4°C. Cells
were then washed and thereafter incubated with mouse anti-Fc
R A59, A62, or A77 mAbs (0.1 mg/ml) for 1 h at 4°C. After
four washes, cells were exposed to FITC-labeled F(ab')2 of SAM
IgG Ab (1%) for 1 h at 4°C. Cells were then washed and fixed in
2% (vol/vol) formaldehyde in HBSS. Cell-associated fluorescence was analyzed by flow cytometry using a FACScan (Becton
Dickinson, Mountainview, CA). In parallel, autofluorescence and
nonspecific binding of the secondary Ab were assessed. Surface
expression of CD14 LPS receptors on HAM was assessed by direct immunofluorescence using an anti-CD14 IgG mAb conjugated with FITC.
For IgA binding, assays were performed by incubating cells (3 × 105) at 4°C for 1 h with p- or S-IgA at 1.5 mg/ml in FCA buffer. Cells were then washed and incubated at 4°C for 1 h with FITC-labeled F(ab')2 of rabbit anti (id)-IgA (10 µg/ml) to reveal idiotypic p-IgA, or with FITC-labeled F(ab')2 of rabbit anti(iso)-IgA (10 µg/ml) for detecting S-IgA. Cells were thereafter washed and cell-associated fluorescence analyzed by FACScan.
Confocal Microscopy
The internalization of surface-bound IgA was investigated by
confocal microscopy. HAM seeded on coverslips were incubated on ice with FITC-labeled p-IgA or with S-IgA at the indicated concentrations in complete culture medium for 1 h in the presence of 10 µg/ml of human transferrin conjugated to Texas-Red
(Molecular Probes, Eugene, OR). Transferrin was used to assess
intracellular colocalization of IgA/Fc
R complexes within the
transferrin receptor recycling endosomes. Cells were then washed
with cold culture medium before incubation at 37°C, 5% CO2 for
different time intervals. At each time, cells were washed and
fixed with 3.7% (vol/vol) formaldehyde. For the intracellular
staining of S-IgA, cells were permeabilized for 10 min with 0.2%
Triton X-100 in phosphate-buffered saline (PBS) and incubated
for 1 h with 10 µg/ml of FITC-labeled F(ab')2 anti(iso)-IgA. Coverslips were mounted in 2.5% 1,4-diacylbicyclo-(2,2,2)octane (DABCO; Sigma) in Mowiol (Calbiochem-Novabiochem International Inc, Darmstadt, Germany). Observations were made under oil immersion with a ×63 objective with an MRC1024 (Bio-Rad Laboratories, Richmond, CA) confocal microscope. Images
were digitally recorded with a Focus Graphics Image Recorder
and used for direct computer-assisted reproduction with an ink-jet photo printer.
Measurements of Respiratory Burst
Fluorometric assay.The formation of ROI was assessed in LPS- or PMA-activated and nonactivated HAM by measuring the oxidation of dichlorofluorescein diacetate (DCFH; Sigma) to its fluorescent analog dichlorofluorescein (DCF) by fluorometry adapted for use in microplates as described by Wan and associates (28). Briefly, treated and untreated HAM (3 × 105 cells/ well) were loaded with DCFH (15 µM) for 15 min at 37°C, 5% CO2 in the dark. The DCF-specific fluorescence resulting from DCFH oxidation via the respiratory burst was assessed by fluorometry using a fluorimetric plate reader (Packard Instruments, Downers Grove, IL) with excitation and emission wavelengths of 485 nm and 538 nm, respectively. Results are expressed as nmol of DCF (calculated from a standard curve with defined amounts of fluorescent DCF) per mg of cell proteins. Total cell protein was assessed using a BCA protein assay reagent (Pierce, Rockford, IL.). The autofluorescence of HAM was so negligible and did not contribute to these calculations. To assess the effect of antioxidants or MAP kinase inhibitor, HAM were pretreated for 1 h with NAC, GSH, or PD98059, respectively, and then treated or not for 1 h with IgA and finally stimulated or not with LPS for 2 h or with PMA for 1 h.
Measurement of superoxide anion (O2
) generation.O2
production by HAM was measured by the superoxide dismutase (SOD)-
inhibitable reduction of cytochrome c assay as previously reported (29). HAM (1 × 105/well) seeded in 100 µl of complete
culture medium on 96-well flat-bottom tissue culture plates were
used to measure O2
production. Cells were loaded with 160 µM
cytochrome c in HBSS and the optical density (OD) at 550 nm
was measured with a microplate autoreader (Labsystems, Brussels, Belgium) at different times of incubation. Plates were incubated at 37°C, 5% CO2 between OD measurements. Control reactions were conducted with 300 U/ml of SOD.
Western Blot Analysis of Total and Phosphorylated
ERK1/2 MAP Kinases and I
B
Treated and untreated HAM were washed with ice-cold PBS and
then lysed in lysis buffer (150 mM NaCl, 20 mM Tris-HCl pH 7.4, 5 mM EDTA, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate) supplemented with 1 mM phenyl methyl sulfonyl fluoride (PMSF), 10 µg/ml of each protease inhibitor (leupeptin,
aprotinin, pepstatin, trypsin inhibitor), 1 mM Na3VO4, and 50 mM
NaF. Lysates were cleared by centrifugation at 13,000 rpm for 15 min at 4°C and protein concentrations were determined using a
BCA protein assay reagent (Pierce). Equal amounts of total extract proteins were resuspended in 2× Laemmli buffer under reducing conditions, boiled for 5 min, subjected to 12% SDS-PAGE,
and then transferred to Hybond-nitrocellulose membranes (Amersham Life Sciences, Little Chalfont, UK). Blots were blocked with
5% bovine serum albumin (BSA) in Tris-buffered saline supplemented with Tween-20 (TBST: 50 mM Tris-HCl pH 7.5, 0.15 M
NaCl, and 0.1% Tween-20) for 1 h. Membranes were then washed three times with TBST and probed overnight with specific anti- phospho-ERK1/2 rabbit Ab (1/1,000) for pERK1/2, anti-phospho-I
B
rabbit Ab (1/1,000) for pI
B
, respectively, or with
anti-ERK1/2 rabbit Ab (1/1,000) for total ERK1/2 or anti-I
B
rabbit Ab (1/1,000) for total I
B
proteins, respectively, in TBST
with 5% BSA. After three washes, membranes were incubated
with HRP-conjugated SAR IgG secondary Ab (1/2,000 in TBST-5% BSA) for 1 h. Blots were washed and immunoreactive bands
were visualized with the Supersignal Enhanced Chemiluminescence detection reagents (Pierce). Phosphorylated ERK2 protein
and extracts from TNF-
-stimulated Hela cells were used as positive controls for ERK1/2 and I
B
revelation, respectively.
Phospho-Specific Antibody Cell-Based ELISA
Quantitation of pERK1/2 was performed by phospho-specific antibody cell-based ELISA (PACE) assay. HAM (1 × 105/well) were seeded on 96-well flat-bottom tissue culture plates. Following stimulation, HAM were fixed with 8% formaldehyde in PBS for 20 min. Cells were washed with 0.1% Triton X-100 in PBS (PBS-Tx) and then incubated for 20 min in PBS-Tx with 0.1% azide and 1% H2O2 to quench endogenous peroxidases. After three washes with PBS-Tx, cells were blocked with 10% FCS in PBS-Tx for 1 h and then incubated with specific anti-phospho ERK1/2 rabbit Ab (1/250) overnight at 4°C. Cells were then washed three times (5 min each) and incubated with HRP-conjugated SAR IgG secondary Ab (1/500) for 1 h. After three washes, 50 µL of 0.04% o-phenylendiamine (OPD) and 0.015% H2O2 in OPD-buffer (35 mM citric acid in 66 mM Na2HPO4, pH 5.6) were added to the wells, and plates were incubated on a shaker at 200 rpm/min for 15 min in the dark. Reactions were stopped by addition of 25 µl/well of 1 M H2SO4 and OD measured on microplate reader at 492 nm. Results were standardized by cell number quantification using crystal violet as described by Scragg and colleagues (30) and OD reading at 595 nm. Results are expressed as arbitrary units (OD492 nm/OD 595 nm).
Nuclear Extracts and Electrophoretic Mobility Shift Assay
Nuclear extracts were prepared from HAM as described by Carter
and coworkers (31) with minor modifications. HAM were washed with cold PBS and then resuspended in 100 µl of ice-cold electrophoretic mobility shift assay (EMSA) lysis buffer (10 mM Hepes
pH 7.9, 10 mM KCl, 2 mM MgCl2, 2 mM EDTA, 1 mM DTT, 1 mM
PMSF) supplemented with protease inhibitors and incubated on
ice for 15 min. Nonidet P-40 (NP-40, 10%) was added to lyse the
cells and cells were vortexed and centrifuged at 4°C at 13,000 rpm.
Nuclei were resuspended in extraction buffer (50 mM Hepes pH
7.9, 10% glycerol, 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 1 mM
DTT, 0.5 mM PMSF and protease inhibitors) for 20 min on ice.
The nuclear suspension was then centrifuged at 13,000 rpm and
supernatant stored at
70°C.
EMSA was performed using the Promega (Madison, WI) gel
shift assay system. NF-
B consensus oligonucleotide (5'AGTTGA
GGGGACTTTCCCAGGC3') was end-labeled with [
-32P]ATP
(Amersham Pharmacia Biotech) using T4 polynucleotide kinase for 10 min at 37°C in a final volume of 10 µl. The reaction was stopped by addition of 1 µl of 0.5 M EDTA and labeled oligonucleotides were purified by chromatography through chromaspin
columns (Clontech Laboratories, Inc., Palo Alto, CA). EMSA
were performed at room temperature for 30 min with 5 µg of nuclear extract proteins and the 32P-labeled oligonucleotides as described in the gel shift assay system (Promega E3300 kit). Binding specificity was conducted with specific competitor used at
100-fold excess of unlabeled NF-
B consensus sequence oligonucleotides added 10 min before the addition of the labeled probe.
The protein-DNA complexes were separated on 5% nondenaturing polyacrylamide gel in 0.5 × Tris-EDTA-borate. The gels
were dried and exposed to autoradiographic films at -70°C.
TNF-
Assay
TNF-
produced by HAM was measured by its cytotoxicity
against the highly sensitive WEHI 164 clone 13 (WEHI) fibrosarcoma cells as described previously (32) with minor modifications. WEHI cells (5 × 104/well) in complete RPMI-1640 culture medium were plated in 96-well flat-bottom plates and incubated for
3 h at 37°C, 5% CO2. WEHI cells then received 100 µl of serial dilutions of test supernatant samples and incubated for 24 h at 37°C,
5% CO2. After 24 h, 20 µl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) at 5 mg/ml in PBS were
added to each well and plates were incubated for an additional
4 h at 37°C, 5% CO2. Cells were then lysed (100 µl/well) with
SDS/HCl solution (15% SDS in 0.015 M HCl) and the plates further incubated overnight to dissolve the blue formazan crystals
and bleach the phenol red color of the culture medium. The
amount of MTT taken up and reduced was detected by OD reading at 550 nm. Serial dilutions of known concentrations of rh-TNF-
were used to quantify HAM secreted TNF-
. Results are
expressed as pg of TNF-
per mg of cell proteins.
Statistical Analysis
Experiments were performed in triplicate and repeated at least three times. Results are expressed as means ± SD. When appropriate, the statistical significance of the differences observed between treated groups and controls or between pertinent groups was analyzed by the Student t test with a probability value of P < 0.05 considered to be significant.
| |
Results |
|---|
|
|
|---|
HAM Express Fc
R, Bind and Internalize p- and S-IgA
Indirect immunofluorescence analysis of Fc
R expression on
HAM was performed by staining HAM cells with anti-Fc
R
mAbs. Figure 1A shows representative flow cytometric profiles of HAM from normal nonsmoking volunteers stained
with anti-Fc
R A59 or A62 mAbs. Moreover, and as reported previously (18), Fc
R was immunoprecipitated with
A77 anti-Fc
R mAb from metabolically labeled HAM (data
not shown). Immunofluorescence assay for IgA-binding to
HAM carried with purified p- and S-IgA shows that HAM
efficiently bound both p- (Figure 1B) and S-IgA (Figure 1C).
|
Endocytosis of HAM Surface-Bound IgA
Confocal microscopy shows that at 4°C, p- and S-IgA were distributed homogeneously on the cell surface of HAM (Figure 2A). Incubation at 37°C induced rapid internalization of p- and S-IgA, which, after 1 min, were already intracellularly localized, as shown by numerous intracellular vesicles containing IgA (Figure 2A). Colocalization experiments revealed that IgA was largely found within vesicles that stained positively for transferrin. However, and as shown in Figure 2A, p-IgA is preferentially perinuclear while S-IgA is widely distributed in the cytoplasm. These intracellular distributions were maintained from 5 to 30 min after incubation at 37°C, and IgA still clearly colocalized within the recycling transferrin receptor endosomes, as illustrated by the yellow fluorescent dots due to colocalization of the two markers. Concentrations as low as 0.25 mg/ml induced a significant internalization of both p- and S-IgA and only a slight increase of IgA-endocytosis was observed with concentrations ranging from 0.5 to 2 mg/ml (Figure 2B).
|
LPS-Induced Increase of HAM Respiratory Burst Depends on Activated ERK1/2 MAP Kinases
HAM were first analyzed for the expression of CD14, the specific binding sites of LPS/LPS binding protein. Immunofluorescence analysis of HAM directly stained with FITC- labeled anti-CD14 mAb showed that CD14 were distributed homogeneously on the surface of HAM (Figure 3A). The CD14 basal expression on HAM was, however, lower than that on monocytes (data not shown), as also reported by Hopkins and associates (33). Quantitation of HAM oxidative burst revealed that HAM constitutively exhibited substantial respiratory burst, as demonstrated by intracellular oxidation of loaded DCFH (Figure 4A). Treatment of HAM with LPS induced significant enhancement of DCFH oxidation (Figure 4A). Kinetic studies (not shown) of the LPS-increased respiratory burst revealed that 2 h treatment of HAM with LPS were necessary to induce important enhancement of oxidative burst. The 2-h period of LPS activation was then respected in all experiments with LPS. Pretreatment of HAM with PD98059, a pharmacologic inhibitor of the MEK pathway (the upstream activator of ERK), completely inhibited the LPS-induced increase of DCFH oxidation (Figure 4A). These results indicate that activation of MEK/ERK MAP kinases pathway is of critical importance in the mechanism by which LPS enhances the respiratory burst in HAM.
|
|
Downregulation of LPS-Increased HAM Respiratory Burst by p- and S-IgA: Role of Activated ERK1/2
Both p- and S-IgA, which had no significant effect on the spontaneous respiratory burst of HAM (Figure 4B), downregulated the increase of respiratory burst induced by LPS (Figure 4B). This effect was similar to that obtained with PD98059 treatment of HAM (Figures 4A and 4C), suggesting that p- and S-IgA could modulate the LPS-enhanced respiratory burst through the activated ERK1/2 MAP kinases.
The release of O2
was also investigated to better evaluate effects of IgA on the respiratory burst of HAM. Activation by LPS resulted in
2.5-fold greater release of O2
as
compared with unstimulated HAM (Figure 4D). Treatment
with PD98059 completely abolished this stimulatory effect
of LPS (Figure 4D). Preincubation of HAM with p- or S-IgA
significantly inhibited the LPS-increased O2
release and
also diminished its basal release (Figure 4E). PD98059, together with p- or S-IgA, had pronounced inhibitory effects
on the LPS-induced increase of O2
release (Figure 4F), suggesting that PD98059 might have at least an additive effect
with p- and S-IgA. These data again support the idea that activated ERK1/2 play an important role in both LPS-increased
and IgA-decreased HAM respiratory burst. These effects of
p- and S-IgA on LPS-activated respiratory burst of HAM
were not related to a downregulation of their CD14 surface
expression by IgA (Figures 3B and 3C).
Downregulation of LPS-Induced ERK1/2 Activation in HAM by p- and S-IgA
To further demonstrate the role of activated ERK1/2 in the
LPS-induced increase of HAM respiratory burst and its downregulation by p- and S-IgA, we analyzed the activated (dually
tyrosine/threonine phosphorylated) ERK1/2 by Western blotting and PACE assay using a phospho-specific ERK1/2 Ab.
In comparison with untreated HAM, treatment of HAM
with LPS led to activation of ERK1/2 (Figures 5A, 5B, and
5D). HAM were stimulated with LPS for 30, 60, and 120 min
to determine the kinetic of ERK1/2 phosphorylation. Figure 5A shows that high levels of ERK1/2 phosphorylation were
observed at 30 min, and although it decreased after HAM
stimulation longer than 30 min, there was still significant
ERK1/2 phosphorylation over an extended time course and
as late as 120 min. Total ERK1/2 proteins were not modified
by LPS (data not shown). PD98059, an inhibitor of ERK1/2
phosphorylation, was used to evaluate the role of ERK1/2
pathway in both LPS-induced oxidative burst and TNF-
release in HAM. As shown in Figure 5A, PD98059 inhibited significantly and time-dependently the phosphorylation of
ERK1/2. Moreover, pretreatment of HAM with PD98059 resulted in a dose-dependent suppression of LPS-induced ERK1/2
phosphorylation (Figure 5B) and complete inhibition was observed with PD98059 at 50 µM (Figures 5A and 5J). We also
checked the specificity of the inhibitory effect of PD98059 on
ERK1/2 MAP kinases. As shown in Figure 5K, PD98059 had
no effect on LPS-induced p38 MAP kinase phosphorylation nor on total p38 MAP kinase (Figure 5L). SB203580,
a specific inhibitor of p38 MAP kinase pathway, had no effect on ERK1/2 phosphorylation (Figure 5B). These results,
as reported by Carter and colleagues (21), confirmed the effectiveness and the specificity of PD98059 inhibitor. LPS-
induced activation of ERK1/2 was quantitated by PACE
which showed a 3.5-fold increase as compared with untreated HAM (Figure 5I). To evaluate a potential role of IgA on
LPS-induced oxidative burst through ERK1/2 pathway modulation, the effect of p- and S-IgA on LPS-activated ERK1/2
in HAM was investigated. In untreated HAM, p- and S-IgA
did not change the basal level of ERK1/2 activation (Figure
5C). In contrast, p- and S-IgA inhibited the LPS-induced activation of ERK1/2 (Figures 5D and 5I). The inhibitory effects
observed with p- and S-IgA (Figure 5I) were similar to those
obtained with PD98059 (Figure 5J). S- and p-IgA drastically
inhibited the LPS-activated ERK1/2 as quantitated by the
sensitive PACE assay and no phosphorylated forms were detected by Western blot (Figure 5D). Moreover, kinetic studies showed that p- and S-IgA downregulated the LPS-induced
ERK1/2 phosphorylation as early as 30 min and as late as 120 min after LPS stimulation (data not shown). Total ERK levels were not affected by the LPS, IgA, or PD98059 treatments (Figures 5E-5H). As stated above, LPS activation of
ERK1/2 occurred rapidly, whereas important enhancement of LPS-induced ROI production was delayed to 2 h, suggesting therefore that ERK1/2 phosphorylation is independent of
oxidative burst. Our results (Figure 5M) indeed confirmed
that antioxidant treatment of HAM did not alter the LPS-
induced ERK1/2 activation.
|
Upregulation of TNF-
Production in HAM by p- and
S-IgA is Independent of ERK1/2 Activation
HAM released a significant amount of TNF-
which was
significantly enhanced upon LPS stimulation (Figure 6A).
Pretreatment of HAM with PD98059 inhibited both LPS-activated and basal TNF-
release to the same extent (Figure 6A, and inset) showing that activated ERK1/2 are required for basal and LPS-stimulated TNF-
production.
Moreover, PD98059 inhibited dose-dependently the LPS-induced TNF-
production in HAM (Figure 6B). Addition
of p- or S-IgA to resting HAM stimulated TNF-
production (Figure 6A), and this effect was maintained in the
presence of PD98059. Moreover, IgA-increased TNF-
production was also observed in LPS-stimulated HAM, although IgA inhibited ERK1/2 activation by LPS (Figure
5D). Thus, IgA-stimulated TNF-
release was independent of ERK1/2 activation.
|
NF-
B Activation by LPS in HAM: Role of Oxidants
NF-
B is a crucial transcription factor that regulates the
expression of TNF-
in macrophages. NF-
B activation allows it to translocate from the cytosol into the nucleus and
this occurs following signal-induced phosphorylation and
subsequent degradation of its inhibitors from the I
B family proteins including I
B
(34). Western immunoblots of
cytoplasmic extracts from control and LPS-treated HAM
were performed to determine changes in steady state of
both phosphorylated and total I
B
proteins. In unstimulated HAM, no significant phosphorylation of I
B
could
be detected (Figure 7A) and the level of total I
B
was
higher in comparison with LPS-activated HAM (Figure
7B), indicating that NF-
B is maintained under inactivated
state in control cells. In contrast, LPS induced a rapid phosphorylation (Figure 7A) and subsequent degradation of
I
B
proteins from 5 to 30 min (Figure 7B). This indicates that LPS activated NF-
B in a conventional pathway requiring I
B
proteolysis. At 60 min after activation with
LPS, I
B
was newly present in the cytosol and phosphorylated (Figure 7A), suggesting newly synthesized I
B
,
which indeed is thought to be an NF-
B-driven process.
The phosphorylation of the newly synthesized I
B
decreased again at 120 min, demonstrating continuous activation of NF-
B by LPS. EMSA studies were performed
to assess the NF-
B binding activity in HAM, because
phosphorylation and subsequent degradation of I
B
did
not always correlate with NF-
B nuclear translocation. Figure 8 is a representative gel showing NF-
B binding activity in HAM stimulated with LPS. This stimulation is effectively and significantly inhibited by the proteasome
degradation inhibitor MG 132 (Figure 9), suggesting that
NF-
B activation of HAM by LPS is highly dependent
upon I
B
degradation. MG 132 had no direct effect on
NF-
B binding activity in unstimulated HAM (Figure 10A, lane 1), and competition analysis performed with 100-fold
excess of unlabeled NF-
B consensus sequence oligonucleotide (Figure 10A, lane 2) confirmed the specificity of
the retarded complexes observed in EMSA.
|
|
|
|
ROI have been proposed as activators of NF-
B (35). To
test a potential implication of ROI in LPS-induced NF-
B
activation, HAM were first pretreated with either NAC or
GSH antioxidants at a concentration that suppressed the oxidative burst (Table 1) and subsequently activated with LPS.
Figures 7A and 7B show that NAC had no effect on the kinetic of dual phosphorylation/degradation of I
B
from 5 to
30 min. However, NAC inhibited the newly synthesized I
B
at 60 and 120 min in comparison with LPS alone (Figure 7B). The effect of NAC is in accordance with the fact
that ROI production by LPS-stimulated HAM is delayed to
60 min and increased at 120 min, whereas I
B
phosphorylation occurred as early as 5 min. Thus, ROI could play a minor role (but at late stages) in NF-
B activation by LPS in
HAM. In addition, EMSA studies showed that NAC had no
effect on LPS-induced NF
B binding activity (Figure 10B).
|
Role of NF-
B and Oxidants in LPS-Induced Production
of TNF-
in HAM
The role of NF-
B in LPS-induced TNF-
release in HAM
was investigated using the proteasome inhibitor MG 132, which inhibits NF-
B activation by preventing I
B
degradation. As shown in Figure 11A, preincubation of HAM
for 1 h with 20 µM of MG 132 significantly inhibited TNF-
release in LPS-activated HAM. The effect of MG 132 was
not generalized to all protein synthesis because total proteins from cell lysates were not affected by MG 132. The
effect of MG 132 was specifically related to alteration of
NF-
B activation and of NF-
B nuclear translocation, as
illustrated in Figure 9. Moreover, MG 132 reversed the effects of LPS on I
B
degradation without altering I
B
phosphorylation (Figures 11C and 11D) and EMSA studies demonstrated that MG 132 blocked nuclear translocation of NF-
B. These results also support the idea that
MG 132 works specifically at the level of proteasome.
|
We next investigated the role of ROI in LPS-induced
TNF-
release as LPS stimulated oxidative burst in HAM
(Figures 4A and 4B). Results presented in Figure 11B
show that NAC reduced by ~ 40% the LPS-induced production of TNF-
. These results are in accordance with the
late effects of NAC on NF-
B activation (Figure 7) and
with the delayed production of ROI by LPS.
Effects of IgA on NF-
B and Its Role in IgA-Mediated
TNF-
Production in HAM
Western immunoblots of cytosolic extracts from p- and
S-IgA-treated HAM show that IgA induced phosphorylation of I
B
with different kinetic from that observed with
LPS. Substantial phosphorylation of I
B
was observed
with IgA from 5 to 30 min followed by its depletion but as
late as at 120 min (Figures 12A and 12B). NF-
B nuclear
translocation was also induced by treatment of HAM either with p- or S-IgA (Figures 8 and 9), and the NF-
B
binding activity was increased as much as observed with
LPS activation. The effects of IgA on I
B
phosphorylation and NF-
B nuclear translocation were not affected by
the presence of NAC (Figures 12 and 10B, respectively). IgA, indeed, has no effect on the oxidative burst in unstimulated HAM. Both p- and S-IgA-induced NF-
B binding
activity was reversed by MG 132 (Figure 9). NF-
B activation by IgA could explain the IgA-induced TNF-
production in HAM. Pretreatment of HAM with MG 132 for 1 h
before the addition of IgA reduced but did not completely
inhibit IgA-increased TNF-
production (Figure 13A). In
contrast, NAC treatment of HAM had no effect on TNF-
production by IgA-treated HAM (Figure 13B). This suggests
that IgA-induced production of TNF-
is partly NF-
B-
dependent but oxidant-independent.
|
|
Upregulation of PMA-Induced Increase of HAM O2
Release by p- and S-IgA: Role of Activated ERK1/2
The role of ERK1/2 in the regulation by p- and S-IgA of
receptor-independent activated HAM respiratory burst
was investigated using PMA as stimulus. PMA-stimulated
HAM released 2-fold more O2
than unstimulated HAM
(Figure 14A). PD98059 did not much alter the basal production of O2
but completely inhibited the effect of PMA
(Figure 14A). Treatment of HAM with p- or S-IgA led to
a synergistic increase of O2
release with PMA (Figure 14B)
which was completely abolished by PD98059 (Figure 14B).
Thus, ERK1/2 pathway also plays an important role in receptor-independent stimulated oxidative burst in HAM. Similar results were obtained when the respiratory burst
was assayed through DCFH oxidation (data not shown).
|
Upregulation of PMA-Induced ERK1/2 Activation in HAM by p- and S-IgA
HAM stimulated with PMA showed an increased amount of phosphorylated ERK1/2 in comparison with unstimulated cells as shown by phospho-Western blots (Figures 15A and 15B) and by PACE assay (Figure 15F). The PMA-induced ERK1/2 phosphorylation was detected as early as 2 min and as late as 2 h (data not shown). This effect, which is time dependent, was maximal at 30 min, but demonstrable ERK1/2 activation could still be observed as late as 60 min (Figure 15A). Preincubation of HAM with PD98059 blocked PMA-induced ERK1/2 phosphorylation, and this inhibitory effect, which is dose-dependent (Figure 15B), was maximal at 50 µM of PD98059 (Figures 15A and 15G). As shown for LPS-activated HAM, PD98059 selectively inhibited PMA-induced ERK1/2 phosphorylation without affecting that of p38 MAP kinase (Figures 15H and 15I). In PMA-activated HAM, both p- and S-IgA increased the phosphorylation of ERK1/2 (Figures 15C and 15F), and this IgA-induced upregulation of ERK1/2 was observed as early as 30 min and as late as 60 min after PMA stimulation (data not shown). These effects were also inhibited by PD98059 (Figure 15G). No effect either of PMA, IgA, or PD98059 on total ERK1/2 expression was observed (Figures 15D and 15E). In addition, ERK1/2 phosphorylation in PMA-activated HAM was independent of ROI production, as neither NAC nor GSH antioxidants altered this effect of PMA (Figure 15J).
|
IgA Modulates TNF-
Production in Both Unstimulated
and PMA-Stimulated HAM Independently of
ERK1/2 Activation
HAM stimulated with PMA released more TNF-
than
unstimulated HAM (Figure 16A and inset). Basal and PMA-stimulated productions of TNF-
depended largely on the
ERK1/2 pathway, both being inhibited by PD98059 (Figure 16A and inset). Moreover, inhibition of PMA-induced TNF-
production was PD98059 dose-dependent (Figure
16B). The effect of PMA, however, did not much add to
that of p- and S-IgA, both of which stimulated TNF-
release in resting HAM (Figure 16A). In contrast to PMA,
IgA-mediated increase of TNF-
production was largely
independent of the ERK1/2 pathway (Figure 16A). These results suggest that there is no complete correlation between ERK1/2 activation and TNF-
release and that an
alternative ERK-independent pathway exists for IgA-stimulated TNF-
release in HAM.
|
Role of NF-
B and Oxidants in PMA-Induced TNF-
Production in HAM
In contrast to LPS, treatment of HAM with PMA resulted
in very low phosphorylation of I
B
from 5 to 30 min and
substantial elevation at 60 min (Figure 17A). However,
and surprisingly, the level of total I
B
increased during
PMA stimulation up to 30 min. At 60 min, a low depletion
of I
B
was observed with a level remaining comparable
to that in unstimulated HAM (Figure 17B). EMSA studies
showed no nuclear translocation of NF-
B in PMA-stimulated HAM (Figures 8 and 10A). Antioxidants, NAC, and
GSH had no effect on I
B
phosphorylation/degradation
in PMA-activated HAM (Figures 17A and 17B). In unstimulated HAM, the antioxidant GSH did not show any
significant effect on I
B
proteins (Figure 17). Thus, PMA failed to activate NF-
B despite the small increase in phosphorylation of I
B
. PMA-induced TNF-
release was not
only independent of NF-
B, as no nuclear translocation of
NF-
B occurred subsequent to PMA activation and no
effect of MG 132 on this production was seen (data not
shown), but also was not affected by the presence of NAC
(Figure 17C).
|
| |
Discussion |
|---|
|
|
|---|
HAM represent the major cellular line of defense against
invading microorganisms and inhaled antigens in the lung
(19). Binding sites for IgA were described on HAM (16)
and more recently, the molecular nature of Fc
R in HAM
has been characterized and revealed that HAM express an
Fc
R derived from an alternative splicing of the primary
Fc
R transcript and therefore different from that of neutrophils and monocytes (18). Much progress has been
made regarding the biology of Fc
R on monocytes (5, 7, 36), neutrophils (37), and eosinophils (38, 39). However, the biology and the functional aspects of Fc
R on HAM
remained poorly investigated. Using HAM from healthy
nonsmoking volunteers, we have studied the effects and
the mechanisms by which interaction of their Fc
R with
the predominant IgA forms in mucosal respiratory secretions, namely p- and S-IgA, could modulate LPS receptor- dependent and PMA receptor-independent stimulation of
the respiratory burst and TNF-
release. The IgA concentrations we used are likely to be in the range of those observed in respiratory secretions. Considering that the BAL
procedures induce a dilution factor of roughly 100 (40), we
and others (41, 42) have reported that in vivo IgA concentration in respiratory secretions is in the range of 1 mg/ml
in normal nonsmoking subjects and can increase in disease
states. After first confirming Fc
R expression on HAM, we investigated the IgA-mediated endocytosis and Fc
R-mediated signal transduction in HAM. We showed that
both p- and S-IgA were efficiently and rapidly internalized
by HAM even without crosslinking of IgA by secondary
Abs as reported for the internalization of anti-Fc
R Abs
(43). Intracellular vesicles containing p- and S-IgA were
largely colocalized with those containing the recycling transferrin receptors, suggesting the existence of a recycling pathway for HAM Fc
R.
HAM stimulated either with LPS or PMA show an increased respiratory burst as measured by oxidation of DCFH
and O2
release. Using two methods, DCF (which measures the production of H2O2), and cytochrome c (which
measures the release of O2
), we observed similar increased oxidative burst with stimuli and its decrease with
inhibitors. Both LPS and PMA stimuli were reported to
activate ERK1/2 kinases in HAM (44). We observed that
the inhibition of ERK1/2 activity by its selective inhibitor PD98059 completely abolished both LPS- and PMA-stimulated respiratory burst in HAM, suggesting that activated
ERK1/2 play an important role in the respiratory burst of
HAM. A role of activated ERK1/2 in the stimulation of
the respiratory burst by different proinflammatory stimuli
has been documented for other phagocytic cells (20, 45- 47) but, to our knowledge, this is the first report that the
ERK pathway controls the respiratory burst in HAM.
Incubation of HAM with p- or S-IgA downregulated their LPS-activated respiratory burst. This effect is consistent with our finding that p- and S-IgA, as well as PD98059, downregulated the LPS-induced ERK1/2 activation. These results for the first time demonstrate an effect of IgA on ERK1/2 kinases and suggest that the ERK1/2 pathway is critical in HAM for the modulation of the respiratory burst by both p- and S-IgA. This effect of p- and S-IgA was not related to a downregulation of CD14 expression on HAM by given forms of IgA.
PMA also increased the respiratory burst of HAM as
previously reported (48). We now show in addition that
this effect of PMA is upregulated by both p- and S-IgA, in
contrast to the observed effects of IgA on LPS-activated
respiratory burst. Other contrasting effects on HAM respiratory burst were reported, such as for IL-4 that inhibited
the LPS-induced respiratory burst but increased that induced by IFN-
(49). HAM stimulated with PMA showed
increased amounts of activated ERK1/2 kinases. This effect was further upregulated by both p- and S-IgA and accounts for the enhanced oxidative burst observed in IgA-treated PMA-activated HAM. PD98059 fully inhibited the
increased oxidative burst of HAM induced by both PMA
and IgA. Therefore, activated ERK1/2 are also critical for
the regulation of receptor independent stimulation of respiratory burst in HAM. Altogether, our data suggest that
IgA is an important modulator of the respiratory burst in
HAM via different pathways depending on the nature of
the stimulus.
Our data further suggest that LPS and PMA stimulated
ERK1/2 activity in HAM through different pathways as IgA
differentially modulates LPS- and PMA-stimulated ERK
kinases. The best described pathway of ERK activation is
the Ras-Raf-1-MEK/ERK kinases cascade (50). However,
Raf-1-independent pathways of ERK activation exist as
demonstrated for LPS- (51), TNF-
- (52), and insulin (53)-
regulated ERK kinase pathways. In HAM, Monick and coworkers (44) reported that LPS regulates ERK activation independently of the Raf-1 pathway. These authors showed that
LPS-induced ERK activation can occur either through the
phospatidylcholine-phospholipase C-ceramide-PKC
-MEK
pathway or the phosphatidylinositol-3 kinase-PKC
-MEK
pathway (44, 54). The PKC
therefore plays a central role
in the LPS-stimulation of ERK1/2 pathway in HAM. Monick and associates (44) further demonstrated that PMA, a
strong activator of classical PKC, activates both Raf-1 and
ERK pathways, supporting the idea that PMA activates
ERK through the classical Raf-1-dependent pathway.
Moreover, earlier data (55) showed that in HAM, PMA did
not activate PKC
, as demonstrated by its negligible membrane translocation, suggesting therefore that PMA could
not activate ERK through PKC
-dependent pathway. Thus,
PMA and LPS activate ERK kinases in HAM through distinct pathways, and independently of ROI production. The
contrasting effects of p- and S-IgA on the LPS- and PMA-induced ERK activation in HAM could therefore result
from a differential effect of a given form of IgA on one or
several components of the LPS- and PMA-used pathways.
Additional studies are needed to examine how IgA could
inhibit one or several kinases implicated in the LPS-induced
ERK activation and, in parallel, activate the PMA-induced
Raf-1-dependent ERK pathway.
To produce a respiratory burst, phagocytes must assemble in their plasma or phagolysosomal membranes the constituents of the NADPH oxidase complex. The mechanism through which activated ERK kinases increase the respiratory burst in PMA- and LPS-stimulated HAM could result from the phosphorylation by ERK of cytosolic and/or membrane bound components of the NADPH oxidase. It has been shown that ERK as well as p38 MAP kinases phosphorylate p47phox, a cytosolic factor of the NADPH oxidase system (56).
In our study, both LPS- and PMA-stimulated TNF-
release by HAM through the ERK pathway. Inhibition of
ERK activity by PD98059 completely inhibited LPS- and
PMA-increased TNF-
release. Carter and colleagues (21)
showed that ERK but also p38 MAP kinases were required
for optimal LPS-induced TNF-
gene expression and protein release in HAM. In our system, the effect of LPS and
PMA on TNF-
release were investigated after relatively
short times of stimulation, 2 h and 1 h, respectively, whereas
Carter and associates (21) observed the 24-h effect of LPS.
We may therefore presume that ERK activation is sufficient
to regulate LPS- and PMA-increased production of TNF-
for short times, whereas both ERK and p38 MAP kinases
would be required for a more persistent production of TNF-
.
Analysis of p38 MAP kinase in our system showed that p38
MAP kinase is highly activated by LPS and IgA, and to a
low extent by PMA. Thus, IgA-induced TNF-
could be
mediated through p38 MAP kinase. Further studies are
therefore required to determine either a cooperative or antagonistic effect between ERK and p38 MAP kinases.
The different effects of LPS and PMA on NF-
B activation are another demonstration that these stimulators
used different pathways to activate HAM.
Our data confirm that LPS induced NF-
B activation in
HAM as reported earlier (31). Now, we further demonstrate that NF-
B activation by LPS is highly dependent
upon classical pathway phosphorylation and subsequent
degradation of I
B
. This effect was indeed reversed by
the proteasome inhibitor MG 132. Production of TNF-
,
like that of other cytokines such as IL-6 and IL-8, is, at
least in part, controlled at the level of their gene transcription. Our data show in addition that inhibition of NF-
B
activation via MG 132 results in altered TNF-
release in
LPS-stimulated HAM. Others have shown that the proteasome inhibitor MG 132 inhibits TNF-
release by LPS-
activated HAM and that this effect correlated with the
blockade of NF-
B activation (57). Thus, both ERK1/2 and NF-
B play crucial roles in LPS-increased TNF-
secretion in HAM. Several studies have shown that NF-
B
activation may be regulated by a variety of agents, including cytokines, mitogens, and oxygen radicals (58, 59).
However, other studies (60, 61) have now questioned this
concept and limited the effect of oxidants on NF-
B to
only certain cell types such as human breast (62) and T cell
lines (63), but not monocytic cell lines (64), epidermal cells
(60), or epithelial cell lines (61). Our results using NAC (a
precursor of GSH) and GSH demonstrate that antioxidants efficiently inhibited ROI production as reported for
other macrophages (65), and that ROI play a minor and
delayed role in LPS-induced NF-
B activation in HAM.
This effect is, in addition, consistent with the partial inhibition of TNF-
release by antioxidants. Reduced TNF-
production by NAC and GSH in LPS-activated HAM was
reported previously (66). Thus, the intracellular glutathione redox status plays a minor role in the control of TNF-
production in HAM. Paradoxically, antioxidants such as
NAC can increase NF-
B activation, as observed in adenocarcinoma cell lines (67). In addition, antioxidants can
modulate NF-
B in a manner that is independent of their
scavenging activity. Although the inhibitory effects of antioxidants on NF-
B but also on other transcription or activated protein factors are often controversial and inconclusive, we can speculate that these effects are limited to
certain cell types. Our data also showed that both p- and
S-IgA activated nuclear translocation of NF-
B in HAM
independently of oxidants. Activation of NF-
B by IgA
contributed partly to the upregulation of TNF-
by IgA,
indicating that other transcription or activated protein factors and/or MAP kinases play an essential role in the TNF-
control by IgA. Incubation of HAM with p- or S-IgA resulted in enhanced TNF-
release. In monocytes, Fc
R
crosslinking and p-IgA binding have been shown to stimulate TNF-
production (13). In our system, treatment of
HAM with PD98059 did not inhibit IgA-mediated TNF-
release in both resting and PMA- or LPS-stimulated HAM. Moreover, IgA-induced TNF-
release was mediated only
partly by NF-
B. These results therefore suggest the existence of alternative pathway(s) for IgA-induced TNF-
release and that the TNF-
promoter may contain MAP kinase-dependent and -independent regulatory elements as
reported previously (68). Our finding that both p- and
S-IgA activated strongly the phosphorylation of p38 MAP
kinase may suggest that p38 MAP kinase plays a critical
role in regulating TNF-
release in HAM. The AP-1 site,
which is present in TNF-
promoter, plays an important
role in the transcriptional regulation of TNF-
. However,
AP-1 activity was reported to be loss in HAM (69), which
therefore suggests that AP-1 do not play an important role in regulating TNF-
, but its activation by IgA could not be
ruled out.
In contrast to LPS and IgA, PMA failed to activate NF-
B
nuclear translocation in HAM, and even slightly increased
I
B
phosphorylation as late as 60 min after stimulation.
Other studies have demonstrated the inability of PMA to activate NF-
B in macrophages (70, 71), supporting the idea
that NF-
B activation is independent of PKC including in
HAM (31).
It is important to outline that the mechanism by which
IgA increased TNF-
production did not add with the ERK-dependent LPS- and PMA-stimulated TNF-
release.
TNF-
produced by IgA-treated HAM was also oxidant-independent because neither p- nor S-IgA modulated
the oxidative burst in unstimulated HAM. In addition,
IgA downregulated the oxidative burst in LPS-activated
HAM through inhibition of ERK1/2 and not by acting as
radical scavengers like antioxidants do.
The ERK1/2 MAP kinase and NF-
B signaling systems
represent two distinct but interactive signal transduction
pathways. LPS activates both ERK1/2 and NF-
B pathways, which play an important role in TNF-
secretion.
However, the two pathways appear to contribute to TNF-
production competitively rather than additively. MAP kinases may act directly on the NF-
B transcription activation complex or through intermediate kinases. This will be
of great interest to investigate to determine if ERK1/2 or
other MAP kinases could modulate NF-
B activation in
HAM, especially through I
B kinase which is the first step
in NF-
B activation. Preliminary data from our laboratory
have demonstrated that inhibition of NF-
B activation by
MG 132 did not block LPS-activated ERK1/2 MAP kinases.
Examples of NF-
B regulation by MEK-ERK pathway
have been described, such as in lung epithelial cells where
ERK regulates negatively, but JNK positively, NF-
B (72).
HAM play an important role in host defense against infectious pathogens. The release of toxic ROI and proinflammatory cytokines following activation with endotoxin
or contact with bacteria is of critical importance in HAM-mediated protective immunity. By contrast, these mediators
may also play an important role in the pathogenesis of inflammatory processes in the lung (73). Regulation of HAM
is therefore of critical importance in controlling lung inflammation, as recurrent activation of HAM could be deleterious for the alveoli and the lower lung mucosal environment.
Our results show a complex HAM regulation by IgA. In
LPS- but not PMA-stimulated HAM, p- and S-IgA could
act as anti-inflammatory modulators by downregulating the
production of ROI. This effect of IgA could protect lung tissues from injury. ROI are indeed implicated in a number of
pulmonary diseases such as the adult respiratory distress syndrome (ARDS) (74). In contrast, IgA could exacerbate
the noxious effect of PMA-related stimuli. The interaction
of both resting and activated HAM with p- and S-IgA leads
to increased production of TNF-
, which could be beneficial to host defense. TNF-
plays a crucial role in the development of protective responses to bacterial and viral pathogens. However, continuous interactions of Fc
R with IgA
could also lead to elevated levels of cytokines such as TNF-
that have also been correlated with lung inflammatory injury as in patients with ARDS. Although p-IgA and in particular S-IgA account for the majority of IgA in mucosal secretions, substantial amounts of monomeric IgA can be
detected (~ 28% of total IgA) (41) in BAL. We are currently investigating whether the size of IgA could influence
the response of HAM. Thus, IgA could act as an important
and balancing modulator in the HAM-mediated host protective pro- and anti-inflammatory responses.
| |
Footnotes |
|---|
Address correspondence to: Dr. Yves Sibille, MEXP Unit, ICP, 74, Avenue Hippocrate, BP. 7430, 1200-Brussels, Belgium. E-mail: sibille{at}mexp.ucl.ac.be
Abbreviations: antibody(ies), Ab(s); bovine serum albumin, BSA; dichlorofluorescein, DCF; electrohoretic mobility shift assay, EMSA; extracellular protein-related kinase 1 and 2, ERK 1/2; Fc high affinity receptor for IgA, Fc
R; fetal calf serum, FCS; flow cytometric analysis, FCA; fluorescein isothiocyanate, FITC; glutathione, GSH; human alveolar macrophages,
HAM; horseradish peroxidase, HRP; immunoglobulin, Ig; lipopolysaccharide, LPS; monoclonal Ab, mAb; mitogen-activated protein kinase, MAP
kinase; N-acetylcysteine, NAC; nuclear factor-kappa B, NF-
B; superoxide anion, O2-; optical density, OD; phospho-specific antibody cell-based
ELISA, PACE; phosphate-buffered saline, PBS; PD98059, PD; phosphorylated ERK1/2, pERK1/2; protein kinase C, PKC; phorbol myristate acetate, PMA; phenyl methyl-sulfonyl fluoride, PMSF; phosphorylated p38 MAP kinase, p-p38; respiratory burst, RB; reactive oxygen intermediates, ROI; sheep anti-mouse, SAM; SB203580, SB; swine anti-rabbit, SAR; secretory IgA, S-IgA; tris-buffered saline-tween, TBST; tumor necrosis
factor-
, TNF-
; WEHI 164 clone 13, WEHI.
Acknowledgments: The authors thank A. Langendries for dedicated assistance in the purification and labeling of IgA and anti-IgA Abs, and P. Staquet (Hematology Unit, Mont-Godinne Hospital, University of Louvain) for skillful help with the flow cytometry experiments. They thank Dr. P. Courtoy (Cell Unit, Institute of Cellular Pathology, University of Louvain) for access to Bio-Rad confocal microscope. This work was supported by the Belgian Fonds de la Recherche Scientifique Médicale (Grant no. 3.4590.99), by the Swiss Lancardis Foundation, and by grant from Glaxo-Wellcome S.A., Brussels, Belgium.
| |
References |
|---|
|
|
|---|
1. Krajci, P., D. Kvale, K. Tasken, and P. Brandtzaeg. 1992. Molecular cloning and exon-intron mapping of the gene encoding human transmembrane secretory component (the poly-Ig receptor). Eur. J. Immunol. 22: 2309-2315 [Medline].
2. Newkirk, M. M., M. H. Klein, A. Katz, M. M. Fisher, and J. J. Underdown. 1983. Estimation of polymeric IgA in human serum: an assay based on binding of radiolabelled human secretory component with applications in the study of IgA nephropathy, IgA monoclonal gammopathy, and liver disease. J. Immunol. 130: 1176-1181 [Abstract].
3.
Mazanec, M. B.,
C. S. Kaetzel,
M. E. Lamm,
D. Fletcher, and
J. G. Nedruf.
1992.
Intracellular neutralization of virus by immunoglobulin A antibodies.
Proc. Natl. Acad. Sci. USA
89:
6901-6905
4. Brandtzaeg, P.. 1992. Humoral immune response patterns of human mucosae: induction and relation to bacterial respiratory tract infections. J. Infect. Dis. 165: 167-176 .
5.
Monteiro, R. C.,
H. Kubagawa, and
M. D. Cooper.
1990.
Cellular distribution, regulation, and biochemical nature of an Fc
receptor in humans.
J.
Exp. Med.
171:
597-613
6.
Maliszewski, C. R.,
C. J. March,
M. A. Schoenborn,
S. Gimpel, and
L. Shen.
1990.
Expression cloning of a human Fc receptor for IgA.
J. Exp. Med.
172:
1665-1672
7.
Morton, H. C.,
I. E. van den Herik-Oudijk,
P. Vossebeld,
A. Snijders,
A. J. Verhoeven,
P. J. A. Capel, and
J. G. J. van de Winkel.
1995.
Functional association between the human myeloid immunoglobulin A Fc receptor
(CD89) and FcR
chain.
J. Biol. Chem.
270:
29781-29787
8. Gomez-Guerrero, C., N. Duque, and J. Egido. 1996. Stimulation of Fc(alpha) receptors induces tyrosine phosphorylation of phospholipase C-gamma(1), phosphatidylinositol phosphate hydrolysis, and Ca2+ mobilization in rat and human mesangial cells. J. Immunol. 156: 4369-4376 [Abstract].
9. Fanger, M. W., S. N. Goldstine, and L. Shen. 1983. Cytofluorographic analysis of receptors for IgA on human polymorphonuclear cells and monocytes and their correlation of receptor expression with phagocytosis. Molec. Immunol. 20: 1019-1027 [Medline].
10.
Van der Pol, W. L.,
G. Vidarsson,
H. A. Vile,
J. G. J. van de Winkel, and
M. E. Rodriguez.
2000.
Pneumococcal capsular polysaccharide-specific
IgA triggers efficient neutrophil effector functions via Fc
RI (CD89).
J.
Infect. Dis.
182:
1139-1145
[Medline].
11.
Lowell, G. H.,
L. F. Smith,
M. S. Artenstein,
G. S. Nash, and
R. P. MacDermott.
1979.
Antibody-dependent cell-mediated antibacterial activity of human mononuclear cells: I. K lymphocytes and monocytes are effective
against meningococci in cooperation with human immune sera.
J. Exp.
Med.
150:
127-137
12. Gorter, A., P. S. Hiemstra, P. C. Leijh, M. E. van der Sluys, M. T. van den Barselaar, L. A. van Es, and M. R. Daha. 1987. IgA- and secretory IgA- opsonized S. aureus induce a respiratory burst and phagocytosis by polymorphonuclear leucocytes. Immunology 61: 303-309 [Medline].
13.
Deviere. J., J. P. Vaerman, J. Content, C. Denys, L. Schandene, P. Vandenbussche, Y. Sibille, and E. Dupont.
1991.
IgA triggers tumor necrosis factor
secretion by monocytes: a study in normal subjects and patients with
alcoholic cirrhosis.
Hepatology
13:
670-675
[Medline].
14.
Wolf, H. M.,
M. B. Fisher,
H. Puhringer,
A. Samstag,
E. Vogel, and
M. M. Eibl.
1994.
Human serum IgA downregulates the release of inflammatory
cytokines (tumor necrosis factor-alpha, interleukin-6) in human monocytes.
Blood
83:
1278-1288
15. Wolf, H. M., E. Vogel, M. B. Fischer, H. Rengs, H. P. Schwarz, and M. M. Eibl. 1994. Inhibition of receptor-dependent and receptor-independent generation of the respiratory burst in human neutrophils and monocytes by human serum IgA. Pediatr. Res. 36: 235-243 [Medline].
16. Sibille, Y., B. Chatelin, P. Staquet, W. W. Merill, D. L. Delacroix, and J. P. Vaerman. 1989. Surface IgA and Fc alpha-receptors on human alveolar macrophages from normal subjects and from patients with sarcoidosis. Am. Rev. Respir. Dis. 139: 740-747 [Medline].
17. Sibille, Y., S. Depelchin, P. Staquet, P. Coulie, L. Shen, C. Vander, Maelen, and J. P. Vaerman. 1994. Fc alpha-receptor expression on the myelomonocytic cell line THP-1: comparison with human alveolar macrophages. Eur. Respir. J. 7: 1111-1119 [Abstract].
18. Patry, C., Y. Sibille, A. Lehuen, and R. C. Monteiro. 1996. Identification of Fc alpha receptor (CD89) isoforms generated by alternative splicing that are differentially expressed between blood monocytes and alveolar macrophages. J. Immunol. 156: 4442-4448 [Abstract].
19. Sibille, Y., and H. Y. Reynolds. 1990. Macrophages and polymorphonuclear neutrophils in lung defense and injury. Am. Rev. Respir. Dis. 141: 471-501 [Medline].
20.
Downey, G. P.,
J. R. Butler,
H. Tapper,
L. Fialkow,
A. R. Saltiel,
B. B. Rubin, and
S. Grinstein.
1998.
Importance of MEK in neutrophil microbicidal
responsiveness.
J. Immunol.
160:
434-443
21.
Carter, A. B.,
M. M. Monick, and
G. W. Hunninghake.
1999.
Both Erk and
p38 kinases are necessary for cytokine gene transcription.
Am. J. Respir.
Cell Mol. Biol.
20:
751-758
22.
Baeuerle, P., and
D. Baltimore.
1988.
I
B: A specific inhibitor of the NF-
B
transcription factor.
Science
242:
540-546
23. Finco, T. S., and A. S. Baldwin. 1995. Mechanistic aspects of NF-kappa B regulation: the emerging role of phosphorylation and proteolysis. Immunity. 3: 263-272 [Medline].
24.
Monteiro, R. C.,
M. D. Cooper, and
H. Kubagawa.
1992.
Molecular heterogeneity of Fc
receptors detected by receptor-specific monoclonal antibodies.
J. Immunol.
148:
1764-1770
[Abstract].
25. Vaerman, J. P., C. Vander, Maelen, and A. Langendries. 1995. Homogenous IgA monomers, dimers, trimers and tetramers from two human IgA myeloma sera. Immunol. Invest. 24: 631-641 [Medline].
26. Delacroix, D. L., and J. P. Vaerman. 1981. A solid phase, direct competition, radio-immunoassay for the quantitation of secretory IgA in human serum. J. Immunol. Methods 40: 345-358 [Medline].
27. Delacroix, D. L., H. J. F. Hodgson, A. McPherson, C. Dive, and J. P. Vaerman. 1982. Selective transport of polymeric IgA in bile: quantitative relationships of monomeric and polymeric IgA, IgM, and other proteins in serum, bile and saliva. J. Clin. Invest. 70: 230-241 .
28. Wan, C. P., E. Myung, and B. H. Lau. 1993. An automated micro-fluorometric assay for monitoring oxidative burst activity of phagocytes. J. Immunol. Methods 159: 131-138 [Medline].
29. Pick, E.. 1986. Microassay for superoxide and hydrogen peroxide production and nitroblue tetrazolium reduction using an enzyme immunoassay microplate reader. Methods Enzymol. 132: 407-421 [Medline].
30. Scragg, M. A., and L. R. Ferreira. 1991. Evaluation of different staining procedures for the quantification of fibroblasts cultured in 96-well plates. Ana. Biochem. 198: 80-85 .
31.
Carter, A. B.,
M. M. Monick, and
G. W. Hunninghake.
1998.
Lipopolysaccharide-induced NF-
B activation and cytokine release in human alveolar
macrophages is PKC-independent and TK- and PC-PLC-dependent.
Am.
J. Respir. Cell Mol. Biol.
18:
384-391
32. Espevik, T., and J. Nissen-Meyer. 1986. A highly sensitive cell line, WEHI 164 clone 13, for measuring cytotoxic factor/ tumor necrosis factor from human monocytes. J. Immunol. Methods 95: 99-105 [Medline].
33. Hopkins, H. A., M. M. Monick, and G. W. Hunninghake. 1995. Lipopolysaccharide upregulates surface expression of CD14 on human alveolar macrophages. Am. J. Physiol. 269: 849-854 .
34.
Barnes, P. J., and
M. Karin.
1997.
Nuclear factor-kappaB: a pivotal transcription factor in chronic inflammatory diseases.
N. Engl. J. Med.
336:
1066-1071
35. Kaul, N., J. Choi, and H. J. Forman. 1998. Transmembrane redox signaling activates NF-kappaB in macrophages. Free Radic. Biol. Med. 24: 202-207 [Medline].
36. Shen, L., J. E. Collins, M. A. Schoenborn, and C. R. Maliszewski. 1994. Lipopolysaccharide and cytokine augmentation of human monocyte IgA receptor expression and function. J. Immunol. 152: 4080-4086 [Abstract].
37. Albrechtsen, M., G. R. Yeaman, and M. A. Kerr. 1988. Characterization of the IgA receptor from human polymorphonuclear leucocytes. Immunology 64: 201-205 [Medline].
38. Monteiro, R. C., R. W. Hostoffer, M. D. Cooper, J. R. Bonner, G. L. Gartland, and H. Kubagawa. 1993. Definition of immunoglobulin A receptors on eosinophils and their enhanced expression in allergic individuals. J. Clin. Invest. 92: 1681-1685 .
39.
Bracke, M.,
P. J. Coffer,
J. W. Lammers, and
L. Koenderman.
1998.
Analysis of signal transduction pathways regulating cytokine-mediated Fc receptor activation on human eosinophils.
J. Immunol.
161:
6768-6774
40.
Rennard, S.,
G. Basset,
D. Lecossier,
O. K. Donnell,
P. Martin, and
R. G. Crystal.
1986.
Estimations of the absolute volume of epithelial lining fluid
recovered by bronchoalveolar lavage using urea as an endogenous marker
of dilution.
J. Appl. Physiol.
60:
532-538
41. Delacroix, D. L., F. X. Marchandise, C. Francis, and Y. Sibille. 1985. Alpha-2-macroglobulin, monomeric and polymeric immunoglobulin A, and immunoglobulin M in bronchoalveolar lavage. Am. Rev. Respir. Dis. 132: 829-835 [Medline].
42. Merrill, W. W., G. P. Naegel, J. J. Olchowski, and H. Y. Reynolds. 1985. Immunoglobulin G subclass proteins in serum and lavage fluid of normal subjects. Am. Rev. Respir. Dis. 131: 584-587 [Medline].
43. Silvain, C., C. Patry, P. Launey, A. Lehuen, and R. C. Monteiro. 1995. Altered expression of monocytes IgA Fc receptors is associated with defective endocytosis in patients with alcoholic cirrhosis. J. Immunol. 155: 1606-1618 [Abstract].
44.
Monick, M. M.,
A. B. Carter,
D. M. Flaherty,
M. W. Peterson, and
G. W. Hunninghake.
2000.
Protein kinase C
plays a central role in activation of
the p42/p44 mitogen-activated protein kinase by endotoxin in alveolar
macrophages.
J. Immunol.
165:
4632-4639
45.
Avdi, N. J.,
B. W. Winston,
M. Russell,
S. K. Young,
G. L. Johnson, and
G. S. Worthen.
1996.
Activation of MEKK by formyl-methionyl-leucyl-phenylalanine in human neutrophils: mapping pathways for mitogens-activated protein kinase activation.
J. Biol. Chem.
271:
33598-33606
46. Rane, M. J., S. L. Carrithers, J. M. Arthur, J. B. Klein, and K. R. McLeich. 1997. Formyl peptide receptors are coupled to multiple mitogen-activated protein kinases cascades by distinct signal transduction pathways. J. Immunol. 159: 5070-5078 [Abstract].
47. McLeish, K. R., C. Knall C, R. A. Ward, P. Grewins, P. Y. Coxon, J. B. Kelin, and G. L. Johnson. 1998. Activation of mitogen-activated protein kinase cascades during priming of human neutrophils by TNF-alpha and GM-CSF. J. Leukoc. Biol. 64:537-545.
48. Sibille, Y., W. W. Merrill, J. A. Cooper Jr., L. Polomski, and J. B. Gee. 1984. Effects of a series of chloromethyl ketone protease inhibitors on superoxide release and the glutathione system in human polymorphonuclear leukocytes and alveolar macrophages. Am. Rev. Respir. Dis. 130: 110-114 [Medline].
49.
Bhaskaran, G.,
A. Nii,
S. Sone, and
T. Ogura.
1992.
Differential effects of
interleukin-4 on superoxide anion production by human alveolar macrophages stimulated with lipopolysaccharide and interferon-
.
J. Leukoc.
Biol.
52:
218-223
[Abstract].
50.
Widmann, C.,
S. Gibson,
M. B. Jarpe, and
G. L. Johnson.
1999.
Mitogen-
activated protein kinase: conservation of a three-kinase module from yeast
to human.
Physiol. Rev.
79:
143-180
51. Guthridge, C. J., D. Eidlen, W. P. Arend, A. Gutierrez-Hartmann, and M. F. Smith Jr.. 1997. Lipopolysaccharide and Raf-1 kinase regulate secretory interleukin-1 receptor antagonist gene expression by mutually antagonistic mechanisms. Mol. Cell. Biol. 17: 1118-1128 [Abstract].
52.
Winston, B. W.,
A. Lange-Carter,
A. M. Gardner,
G. L. Johnson, and
D. W. Riches.
1995.
Tumor necrosis factor
rapidly activates the mitogen-activated protein kinase (MAPK) cascade in a MAPK kinase kinase-independent, c-Raf-1-independent fashion in mouse macrophages.
Proc. Natl.
Acad. Sci. USA
92:
1614-1618
53.
Haystead, C. M.,
P. Gregory,
A. Shirazi,
P. Fadden,
C. Mosse,
P. Dent, and
T. A. Haystead.
1994.
Insulin activates a novel adipocyte mitogen-activated protein kinase kinase that shows rapid phasis kinetics and is distinct
from c-Raf.
J. Biol. Chem.
269:
12804-12808
54.
Monick, M. M.,
A. B. Carter,
G. Gudmundsson,
R. Mallampalli,
L. S. Power, and
G. W. Hunninghake.
1999.
A phosphatidylcholine-specific phospholipase C regulates activation of p42/44 mitogen-activated protein kinases in
lipopolysaccharide-stimulated human alveolar macrophages.
J. Immunol.
162:
3005-3012
55. Monick, M. M., A. B. Carter, G. Gudmundsson, L. J. Geist, and G. W. Hunninghake. 1998. Changes in PKC isoforms in human alveolar macrophages compared with blood monocytes. Am. J. Physiol. 275: 389-397 .
56. El Benna, J., J. Han, J. W. Ark, E. Schmid, R. J. Ulevitch, and B. M. Babior. 1996. Activation of p38 in the stimulated human neutrophils: phosphorylation of the oxidase component p47phox by p38 and ERK but not JNK. Arch. Biochem. Biophys. 15: 395-400 .
57. Fine, S. M., S. B. Maggirwar, P. R. Elliott, L. G. Epstein, H. A. Gelbard, and S. Dewhurst. 1999. Proteasome blockers inhibit TNF-alpha release by lipopolysaccharide stimulated macrophages and microglia: implications for HIV-1 dementia. J. Neuroimmunol. 95: 55-64 [Medline].
58. Schreck, R., K. Albermann, and P. A. Baeuerle. 1992. Nuclear factor kappa B: an oxidative stress-responsive transcription factor of eukaryotic cells. Free Radic. Res. Commun. 17: 221-237 [Medline].
59. Blackwell, T. S., T. R. Blackwell, E. P. Holden, B. W. Christman, and J. W. Christman. 1996. In vivo antioxidant treatment suppresses nuclear factor-kappa B activation and neutrophilic lung inflammation. J. Immunol. 157: 1630-1637 [Abstract].
60. Brennan, P., and L. A. O'Neill. 1995. Effects of oxidants and antioxidants on nuclear factor kappa B activation in three different cell lines: evidence against a universal hypothesis involving oxygen radicals. Biochim. Biophys. Acta 1260: 167-175 [Medline].
61. Bonizzi, G., E. Dejardin, B. Piret, J. Piette, M. P. Merville, and V. Bours. 1996. Interleukin-1 beta induces nuclear factor kappa B in epithelial cells independently of the production of reactive oxygen intermediates. Eur. J. Biochem. 242: 544-549 [Medline].
62.
Kretz-Remy, C.,
P. Mehlen,
M. E. Mirault, and
A. P. Arrigo.
1996.
Inhibition of I kappa B-alpha phosphorylation and degradation and subsequent
NF-kappa B activation by glutathione peroxidase overexpression.
J. Cell
Biol.
133:
1083-1093
63. Schreck, R., P. Rieber, and P. A. Baeuerle. 1991. Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kappaB transcription factor and HIV-1. EMBO J. 10: 2247-2258 [Medline].
64. Israel, N., M. A. Gougerot-Pocidalo, F. Aillet, and J. L. Virelizier. 1992. Redox status of cells influences constitutive or induced NF-kappa B translocation and HIV long terminal repeat activity in human T and monocytic cell lines. J. Immunol. 149: 3386-3393 [Abstract].
65. Rezaul, K., K. Sada, and H. Yamamura. 1998. Involvement of reactive oxygen intermediates in lectin-induced protein-tyrosine phosphorylation of Syk in THP-1 cells. Biochem. Biophys. Res. Commun. 246: 863-867 [Medline].
66. Gosset, P., B. Wallaert, A. B. Tonnel, and C. Fourneau. 1999. Thiol regulation of the production of TNF-alpha, IL-6 and IL-8 by human alveolar macrophages. Eur. Respir. J. 14: 98-105 [Abstract].
67. Das, K. C., Y. L. Molock, and C. W. White. 1995. Activation of NF-kappa B and elevation of MnSOD gene expression by thiol reducing agents in lung adenocarcinoma (A549) cells. Am. J. Physiol. 13: L588-L602 .
68. Means, T. K., R. P. Pavlovich, D. Roca, M. W. Vermeulen, and M. J. Fenton. 2000. Activation of TNF-alpha transcription utilizes distinct MAP kinase pathways in different macrophage populations. J. Leukoc. Biol. 67: 885-893 [Abstract].
69.
Monick, M. M.,
A. B. Carter, and
G. W. Hunninghake.
1999.
Human alveolar macrophages are markedly deficient in REF-1 and AP-1 binding activity.
J. Biol. Chem
274:
18075-18080
70. Vincenti, M. P., T. A. Burrell, and S. M. Taffet. 1992. Regulation of NF-kappa B activity in murine macrophages: effect of bacterial lipopolysaccharide and phorbol ester. J. Cell Physiol 150: 204-213 [Medline].
71. Muroi, M., and T. Suzuki. 1993. Role of protein kinase A in LPS-induced activation of NF-kappa B proteins of a mouse macrophage-like cell line, J774. Cell Signal 5: 289-298 [Medline].
72.
Janssen-Heininger, Y. M.,
I. Macara, and
B. T. Mossman.
1999.
Cooperativity between oxidants and tumor necrosis factor in the activation of nuclear
factor (NF)-kappaB: requirement of Ras/mitogen-activated protein kinases in the activation of NF-kappaB by oxidants.
Am. J. Respir. Cell Mol.
Biol.
20:
942-952
73. Hunninghake, G. W.. 1987. Immunoregulatory functions of human alveolar macrophages. Am. Rev. Respir. Dis. 136: 253-254 [Medline].
74. Chabot, F., J. A. Mitchell, J. M. C. Gutteridge, and T. W. Evans. 1998. Reactive oxygen species in acute lung injury. Eur. Respir. J. 11: 745-747 [Abstract].
This article has been cited by other articles:
![]() |
J. C. K. Leung, S. C. W. Tang, L. Y. Y. Chan, W. L. Chan, and K. N. Lai Synthesis of TNF-{alpha} by mesangial cells cultured with polymeric anionic IgA role of MAPK and NF-{kappa}B Nephrol. Dial. Transplant., January 1, 2008; 23(1): 72 - 81. [Abstract] [Full Text] [PDF] |
||||
![]() |
J. E. Bakema, S. de Haij, C. F. den Hartog-Jager, J. Bakker, G. Vidarsson, M. van Egmond, J. G. J. van de Winkel, and J. H. W. Leusen Signaling through Mutants of the IgA Receptor CD89 and Consequences for Fc Receptor {gamma}-Chain Interaction J. Immunol., March 15, 2006; 176(6): 3603 - 3610. [Abstract] [Full Text] [PDF] |
||||
![]() |
Y. Wu, S. Adam, L. Hamann, H. Heine, A. J. Ulmer, U. Buwitt-Beckmann, and C. Stamme Accumulation of Inhibitory {kappa}B-{alpha} as a Mechanism Contributing to the Anti-Inflammatory Effects of Surfactant Protein-A Am. J. Respir. Cell Mol. Biol., December 1, 2004; 31(6): 587 - 594. [Abstract] [Full Text] [PDF] |
||||
![]() |
S.-M. Lin, C. W. Frevert, O. Kajikawa, M. M. Wurfel, K. Ballman, S. Mongovin, V. A. Wong, A. Selk, and T. R. Martin Differential Regulation of Membrane CD14 Expression and Endotoxin-Tolerance in Alveolar Macrophages Am. J. Respir. Cell Mol. Biol., August 1, 2004; 31(2): 162 - 170. [Abstract] [Full Text] [PDF] |
||||
![]() |
C. Pilette, S. R. Durham, J.-P. Vaerman, and Y. Sibille Mucosal Immunity in Asthma and Chronic Obstructive Pulmonary Disease: A Role for Immunoglobulin A? Proceedings of the ATS, April 1, 2004; 1(2): 125 - 135. [Abstract] [Full Text] [PDF] |
||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Proc. Am. Thorac. Soc. | Am. J. Respir. Crit. Care Med. |