Role of ERK1/2 Mitogen-Activated Protein Kinases and NF- B
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Abstract |
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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.
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Introduction |
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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.
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Materials and Methods |
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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.
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Results |
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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).
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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).
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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.
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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.
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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.
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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.
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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).
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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.
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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.
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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).
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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).
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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.
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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).
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