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RI Ligation of Atopic Monocytes Identified by Suppression Subtractive Hybridization1
* Department of Dermatology, Friedrich-Wilhelms-University, Bonn, Germany; and
Institut National de la Santé et de la Recherche Médicale Equipe Propre 99-08 and Unité 311, Etablissement Français du Sang-Alsace, Strasbourg, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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RI, on APCs plays an
important role in the initiation and chronicity of inflammatory atopic
diseases. To understand the molecular regulation of FcRI-mediated
processes, differentially expressed genes are of great interest to be
identified. Suppression subtractive cDNA hybridization has been used to
identify genes induced after FcRI stimulation on atopic monocytes.
Overexpression of the identified genes was determined by
semiquantitative RT-PCR analysis of transcripts from the tester
(stimulated) and driver (unstimulated) monocytes. Results were
confirmed and kinetics of the transcripts established using blood cells
from additional atopics at 4 and 24 h of FcRI induction. The
following sequences were identified: monocyte chemoattractant protein
1, macrophage-inflammatory protein 1
, IL-6, A subunit
of inhibin/activin, IFN-stimulated gene of 54 kDa, IL-1R antagonist,
and kynurenine 3-monooxygenase. Chemokines are highly expressed during
the early and late phase after FcRI cross-linking, whereas
proinflammatory and differentiation stimuli rapidly decline after an
initial overexpression. Kynurenine 3-monooxygenase, an enzyme involved
in the degradation of the amino acid tryptophan, is significantly
up-regulated during the late phase after 24 h of FcRI
induction. These results demonstrate that the analysis of the profile
of gene induction following activation of FcRI on atopic monocytes
may reveal how these cells might participate in the regulation of
atopic disorders. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI, is
assumed to be crucially involved in the induction and control of
allergic inflammatory diseases such as the atopic eczema/dermatitis
syndrome (AEDS),4
rhinitis, and asthma (1). This receptor is expressed on
two distinct groups of cells: 1) constitutively on effector cells of
anaphylaxis, i.e., on mast cells, basophils, and rarely on eosinophils;
and 2) variably on professional APCs. Thus, FcRI is expressed at
relatively high levels on Langerhans cells and related dendritic cells
(DCs) in lesional skin of patients with AEDS, whereas it is
absent or present in very low amounts on cells from normal skin of
nonatopic individuals (2). On monocytes, FcRI has been
identified in both atopic donors and clinically healthy individuals
with an atopic family background. There, FcRI has been shown to
mediate efficient IgE-dependent allergen uptake and presentation to T
cells (3). It is known that ligation of FcRI on APCs
triggers the synthesis and release of proinflammatory cytokines,
thereby contributing to the establishment of chronic allergic
inflammation (4).
Recently, new immunomodulatory functions of FcRI on APCs became
apparent: the activation of monocytes by FcRI mediates protection of
these cells against apoptosis induced by serum deprivation or
Fas-ligand, thus regulating their survival (5). In
addition, the production of IL-10 by FcRI ligation on monocytes
prevents their differentiation into DCs in vitro (6).
Therefore, binding and cross-linking of FcRI by specific
IgE-allergen complexes on circulating monocytes may suggest special
functions and/or veto fundamental effector mechanisms of allergy
responses. Histologically, AEDS is an eczematous reaction where T
lymphocytes mediate profound pathophysiological outcome
(7). The aggregation of FcRI on APCs may exert
immunomodulatory signals on T cells, thus regulating T cell
responsiveness. It is largely unknown why patients with atopic
disorders enter phases of remission or aggravation and why some
individuals with high FcRI surface expression on APCs are clinically
healthy. Apparently, there is a wide array of response mechanisms by
the engagement of the receptor, probably depending on the activated
cell type or metabolic environment. To elucidate functional
significances of FcRI, differentially expressed genes were
identified and kinetics thereof established after FcRI ligation in
five atopic individuals.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mAb anti-human FcRI
22E7 (mouse (m) IgG1)
was a kind gift from J. Kochan (Department of Autoimmune Diseases,
Hoffmann-La Roche, Nutley, NJ). The mAb 15-1, an anti-human
FcRI Ab interfering with the IgE binding site, was kindly
provided by J.-P. Kinet (Institute of Allergy and Immunology, Boston,
MA). To detect surface-bound IgE, an anti-human (h) IgE mAb-FITC
was used (Nordic, Tilburg, The Netherlands). Monomeric human myeloma
IgE (hIgE) was obtained from Calbiochem (Bad Soden, Germany) and was
filtered to remove material over 300 kDa (Ultrafree-MC filter unit;
Millipore, Bedford, MA). Rabbit anti-human-IgE (RahIgE) and mouse
anti-CD23 mAb (MHM6, mIgG1) were from DAKO (Glostrup, Denmark).
F(ab')2 of the rabbit anti-human IgE Ab (IgG)
were generated by digestion with immobilized pepsin (Perbio Science,
Bezons, France) according to the manufacturer’s instructions and
undigested IgG was removed on immobilized protein G (Amersham
Biosciences, Orsay, France). FITC-labeled F(ab')2
of goat anti-mouse Ab (GaM/FITC) were obtained from Jackson
ImmunoResearch Laboratories (West Grove, PA). Unlabeled or
FITC/PE-labeled mIgG, anti-CD14-PE mAb, anti-IL-6-PE mAb, and
anti-TNF--PE were obtained from BD Immunocytometry Systems (San
Jose, CA). Saponin (S-7900) was purchased from Sigma-Aldrich (St.
Louis, MO). RPMI 1640-very low endotoxin was purchased from Biochrom
(Berlin, Germany). FCS, L-glutamine,
antibiotics/antimycotics, were purchased from Life Technologies
(Eggenstein, Germany).
Monocyte isolation
For the generation of the FcRI-specific cDNA library, human
peripheral monocytes from an atopic donor were isolated by continuous
flow centrifugation, leukocytapheresis, and counterflow centrifugal
elutriation as previously described (8). For kinetic
studies, monocytes of five additional donors were isolated from
peripheral blood using Nycoprep (Nycomed, Oslo, Norway) according to
the manufacturer’s protocol. Monocytes were determined to be >95%
pure by CD14 surface staining. Basophil contamination was ruled out by
the absence of amplification of tryptase sequences. Atopics were
defined as such if 1) the density of FcRI, expressed as the relative
fluorescence index (rFI, see below) after staining with 22E7 mAb on
peripheral monocytes, was >1.5; 2) donors had a history of
AEDS/allergic rhinitis/allergic asthma and a positive family background
for atopic diseases. Serum IgE levels of atopic donors were 
100 kU/ml
and, in all donors, specific IgE could be detected toward either dust
mites, pollen, or animal dander. All atopic donors had stigmata of
atopy like the infraorbital fold (Dennie-Morgan lines), palmar
hyperlinearity, or xerosis. All donors were volunteers and methods were
approved by the local ethics committee.
Flow cytometry
A FACSCalibur (BD Biosciences, Mountain View, CA) was used to
determine the surface density of the high-affinity receptor for IgE,
FcRI, and the low-affinity IgE receptor, CD23, on monocytes. An IgG1
control mAb was used to assess specific binding of the anti-FcRI
and anti-CD23 mAbs. The cells were incubated at 4°C with the
first Ab (22E7; MHM6 or an isotype control Ab at 0.5 µg/ml) for 30
min. The cells were washed in PBS plus 1% FCS plus 0.1% sodium azide
and incubated at 4°C with GaM/FITC for 30 min. After washing,
GaM/FITC was blocked with normal mouse serum for 15 min, washed, and
the cells were counterstained at 4°C with anti-CD14 mAb and
7-amino-actinomycin D (1 µg/ml) for 30 min. To quantify the receptor
expression, the vital and CD14-positive monocyte population was gated
by a combination of forward and side scatter and
CD14/7-amino-actinomycin D gate sets. The mean fluorescence intensity
(MFI) for each receptor, FcRI and CD23, of this population was
determined. rFIs were assessed as follows: rFI =
(MFI(Receptor) - MFI(Control))/MFI(Control).
To quantify the IgE occupation of FcRI on untreated monocytes or
after in vitro incubation with hIgE, cells were stained with a
FITC-labeled anti-hIgE mAb. For intracellular cytokine
determination, cells were fixed in PBS and 4% formaldehyde for 20 min,
washed in PBS, and permeabilized in PBS, 0.5% saponin, 0.5% BSA, and
0.5% sodium azide (saponin buffer). Anti-IL-6-PE and
anti-TNF--PE Ab and an isotype control were added for 30 min on
ice and the cells were washed twice in saponin buffer. After washing
with PBS, cells were surface stained with anti-CD14-PE or an
isotype control. Finally, the cells were analyzed by flow cytometry.
Results are expressed as the percentage of positive cells compared with
the isotype control.
Receptor ligation
FcRI cross-linking and incubation of monocytes for the
subtractive library and for the kinetic studies were done in 14-ml
polypropylene round-bottom Falcon tubes (BD Biosciences) to avoid gene
activation by adherence. Cells were incubated at a density of 1 x
106 cells/ml. Cross-linking was done by
incubating the monocytes for 1 h with 4 µg/ml hIgE at 37°C.
After washing with culture medium, 20 µg/ml RahIgE (library) or
RahIgE F(ab')2 (kinetic studies) was added for
either 4 or 24 h. As controls, cells were incubated with monomeric
IgE only and with RahIgE F(ab')2 only.
Unstimulated monocyte populations were incubated at the indicated time
points without the addition of hIgE and RahIgE
F(ab')2. For the subtractive library, one time
point at 4 h was chosen.
Subtractive cDNA library
For the generation of a library with specifically
FcRI-induced sequences, monocytes were selectively stimulated by
cross-linking FcRI. The library was generated from a single atopic
donor. Activation of monocytes was confirmed by measuring the induction
of intracellular cytokines such as IL-6 and TNF- in the stimulated
population. Polyadenylated mRNA was purified from 50 x
106 stimulated (tester population) and 150
x 106 unstimulated (driver population) monocytes
using an Oligotex mRNA kit from Qiagen (Courtaboeuf, France).
Then 1 and 6.4 µg of poly(A)+ mRNA from
stimulated and unstimulated monocytes, respectively, were reverse
transcribed and processed using a PCR-Select cDNA Subtraction kit
(Clontech Laboratories, Palo Alto, CA), which is based on the method of
suppression subtractive hybridization (9). The subtraction
step combined the exclusion of common sequences in the target and
driver population and equalized abundant and rare cDNA sequences in the
tester population. The following suppression of the PCR further
enriched for differentially expressed cDNA fragments.
Cloning and analysis of subtracted cDNA
Products from the subtracted cDNA were digested with RsaI restriction enzyme and inserted into the EcoRV restriction site of a pKS cloning vector (Stratagene, Amsterdam, The Netherlands). Plasmid DNAs were prepared using a Flexiprep extraction kit (Amersham Biosciences). DNA sequencing reactions were performed using the Big Dye terminator sequencing method (PE Applied Biosystems, Paris, France). Nucleic acid homology searches were performed using the BLAST program at the National Center for Biotechnology Informations (http://www.ncbi.nlm.nih.gov/).
RT-PCR
To analyze the overexpression of cDNA transcripts in the
FcRI-stimulated cDNA fraction, semiquantitative RT-PCR experiments
were run with specific primers for the sequences found in the library.
Reverse transcriptase reactions were performed with avian
myeloblastosis virus using 1 µg of total RNA from stimulated and
unstimulated monocyte preparations. The linear range of signal strength
for -actin was determined by performing titrations for cDNA.
Denaturation at 94°C for 30 s was followed by annealing the
primers at 55°C for 30 s; the extension was conducted at 72°C
for 30 s. A final extension phase of 5 min was added. Specific
primer sequences for the genes were as follows: human -actin:
forward, 5'-GAGCGGGAAATCGTGCGTGACATT-3' and reverse,
5'-GATGGAGTTGAAGGTAGTTTCGTG-3' (240 bp); human tryptase: forward,
5'-CTCCCTCATCCACCCCCAGT-3' and reverse,
5'-GGATCCAGTCCAAGTAGTAG-3' (616 bp); human MCP-1: forward,
5'-GACCACCTGGACAAGCAAAC-3'and reverse, 5'-CTCAAAACATCCCAGGGGTA-3'
(378 bp); human MIP-1: forward, 5'-CAAGTCTGTGCTGATCCCAG-3' and
reverse, 5'-GGGACACTTATCCTTTGGCT-3' (264 bp); human IL-6: forward,
5'-ACGAATTGACAAA CAAATTCGGTACA-3' and reverse,
5'-CATCTAGATTCTTTGCCTTTTTCTGC-3' (335 bp); A
subunit of human inhibin/activin: forward, 5'-AGCCATATAGCAGGCACGTC-3'
and reverse, 5'-TCACTGCTTCATCTCTGAGC-3' (459 bp); human IFN-stimulated
gene of 54 kDa (ISG-54K): forward, 5'-GAACGCCATTGACCCTCTGA-3' and
reverse, 5'-ACTGATCTGCTAGAGCATGG-3' (473 bp); human IL-1R antagonist
(IL-1RA): forward, 5'-CTTCCATAATCTGGACTCCT-3' and reverse,
5'-ATTCGCTGAGTACCTGCCAA-3' (359 bp); and human
kynurenine-3-monooxygenase: forward, 5'-AGGCTGTGCAGCGTTGGCAT-3' and
reverse, 5'-TGCACCCCCCAACCAGTAAT-3' (400 bp). Amplification was
performed on a PerkinElmer GeneAmp PCR System 9600 thermocycler
(Applied Biosystems). The PCR cycle numbers for the detection of
tryptase was 35, all other amplifications were done with 27 cycles. PCR
fragments were separated on 1% agarose gels and visualized using
ethidium bromide staining. The PCR products were evaluated
semiquantitatively by comparing the ratio of the specific products vs
the -actin band by digital image analysis using the WinCam System
(Cybertech, Berlin, Germany). Identity of RT-PCR products was confirmed
by direct sequencing.
Southern blot
To determine the nature of the two specific gene fragments of the subtracted cDNA, Southern blotting and detection via chemiluminescence was conducted. Briefly, the denatured DNA in the gel was transferred onto a positively charged nylon membrane (Amersham Biosciences). Primers of the identified genes were digoxigenin (DIG) labeled at their 3' end (DIG Oligonucleotide 3'-end labeling kit; Roche Diagnostics, Meylan, France) and hybridized to the target nucleic acids on the membrane. DIG-labeled DNA was detected by enzyme-linked immunoassay using an Ab conjugate (anti-DIG alkaline phosphatase conjugate). The hybrids were detected by the chemiluminescent reagent disodium 3-{4-methoxyspiro(1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3,1,13,7]decan}-4-yl-)phenylphosphate on an x-ray film.
ELISA
Release of MCP-1 and IL-6 from monocytes was quantified by
ELISA. Supernatants of FcRI-stimulated monocytes from atopic donors
used in the kinetic studies were stored at -70°C until cytokine
measurement. Quantification of proteins was conducted in duplicate
using ELISA kits (Quantikine; R&D Systems, Wiesbaden, Germany)
according to the manufacturer‘s instructions. Sensitivity of MCP-1
detection was 31.2 pg/ml and 3.12 pg/ml for IL-6.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A differential cDNA bank of FcRI-activated vs nonactivated
monocytes from one atopic donor was performed. FcRI and CD23 surface
densities on these monocytes were first analyzed by flow cytometry
(Fig. 1
a) and densities for
the receptors were estimated using the rFIs as described in
Materials and Methods. FcRI surface expression was high,
whereas CD23 expression on monocytes was low. The level of pre-existing
IgE on monocyte plasma membranes in vivo was low and receptor-bound IgE
was significantly raised by incubating the cells with hIgE in vitro.
Because, on mast cells, surface FcRI density determines the signal
generation beyond the internalization of IgE-Ag immune complexes
(10) and because the incubation of monocytes with
monomeric IgE stabilizes FcRI on the cell surface, which underlies
otherwise constant recycling processes (11) and is rapidly
down-regulated upon culture (5), monocytes were first
incubated with IgE to ensure high FcRI occupancy with hIgE. In
addition, we determined the increase of intracellular cytokines IL-6
and TNF- 4 h after stimulation of the cells by cross-linking
FcRI (Fig. 1b). The binding of monomeric hIgE and
addition of RahIgE resulted in signaling via FcRI followed by the de
novo synthesis of IL-6 and TNF-.
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RI and CD23 were also determined on monocytes
from five other atopic donors used in subsequent kinetic studies (Fig. 1
c). FcRI density was relatively high for all donors
while CD23 expression was weak. Additionally, in vivo preoccupied
FcRI-IgE was measured by FACS analysis and compared with in vitro
FcRI-bound IgE after incubating the cells for 1 h with hIgE. In
vivo, the amount of IgE which already occupied FcRI was
comparatively low but was raised to significantly higher values after
the addition of hIgE. To avoid activation of CD23 by potential
aggregated IgE, ultrafiltrated IgE was used. A role of CD23 in IgE
binding was further excluded by an inhibition experiment using the mAb
15-1 which blocks selectively the IgE-binding site of FcRI
(12). The mAb 15-1 (50 µg/ml) completely abolished the
binding of monomeric IgE (4 µg/ml) to monocytes, whereas an isotype
control did not interfere with this binding (Fig. 1d).
Therefore, we concluded that CD23 is not involved in IgE binding at the
concentrations used for our studies.
Differential cDNA bank of FcRI-induced sequences
The differential cDNA bank was constructed using mRNA from
FcRI-activated monocytes as tester and unstimulated monocytes as
driver. The subtracted cDNA was separated on a 2% agarose gel and
compared with the unsubtracted fraction (Fig. 2). In the subtracted population, a large
smear with two distinct bands at 650 and 390 bp molecular mass
was seen while numerous distinct amplified fragments were observed in
the unsubtracted tester population. These results confirmed the
equalization and amplification of rare sequences in the tester
population although two genes seemed to be preferentially amplified and
therefore present in abundance. Subsequently, the subtracted cDNAs were
cloned, the DNA sequence of 
100 clones was determined and analyzed
for homology in GenBank and European Molecular Biology Laboratory
databases. The results of the identified sequences are summarized in
Table I. Due to the overrepresentation of
clones corresponding to MCP-1 and MIP-1, only seven different clones
were characterized. The subtracted sequences on the gel were
transferred onto nitrocellulose membranes and separately hybridized
with DIG-labeled primers of the different identified genes. By
chemiluminescence detection, the 650- and 390-bp fragments were
identified as the MCP-1 and MIP-1 cDNAs of Table I, respectively
(Fig. 3). Thus, these in abundance
isolated clones of MCP-1 and MIP-1 (80% of the clones)
corresponded to the two major bands seen on the gel.
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RI-mediated induction of genes identified by
suppression subtractive hybridization
To confirm the overexpression of the transcripts characterized by
cDNA subtraction, PCRs were run in the tester (FcRI-stimulated) and
driver (unstimulated) population using oligonucleotides specific from
the identified genes. Five additional atopic donors were used to
semiquantify and establish kinetics of gene expression (Fig. 4). Since incubation of mast cells with
monomeric IgE alone is sufficient to induce cytokine production
(10), we examined whether similar effects can be observed
in monocytes. Thus, cells were incubated with hIgE alone for the
duration of the culture. In some donors, a small but significant
increase of transcripts could be detected. Activation of FcRI
resulting from contamination with agglutinated IgE is rather unlikely,
since the IgE preparation was ultrafiltered to eliminate agglutinated
IgE. This fact strongly suggests that the activation of FcRI results
from the saturation of FcRI on monocytes with monomeric IgE. In
contrast, the up-regulation of the investigated transcripts by the
addition of RahIgE F(ab')2 alone varied among the
donors. The expression levels somehow mirrored preoccupied FcRI with
IgE in vivo and thus are likely to result from the cross-linking of
FcRI. However, these levels were significantly lower than with
monocytes preincubated with hIgE and treated with RahIgE
F(ab')2. As a result of FcRI cross-linking on
monocytes, MCP-1, MIP-1, and IL-6 were strongly induced within the
first 4 h after stimulation. In the conditions used for
semiquantitative RT-PCR analysis, expression of these transcripts in
untreated monocytes was below the sensitivity of the test, a small
increase was observed after incubation with IgE or RahIgE alone, which
was further increased 4–10 times after cross-linking, compared with
the conditions with IgE alone. For MCP-1 and MIP-1, these relative
induction levels were sustained at least 24 h after stimulation
and were even stronger in the case of MIP-1. In contrast, IL-6
declined to lower expression levels or extinction 24 h after
stimulation. The transcripts of the A subunit
of inhibin/activin, normally not expressed in peripheral monocytes, and
ISG-54K were transiently increased during the first 4 h following
FcRI stimulation. The gene for the A
subunit of inhibin/activin appeared to be more responsive to RahIgE
alone than other genes. Compared with the incubation with IgE alone,
FcRI stimulation of IgE-incubated monocytes induced a similar
10-fold induction of these transcripts in every donor. The
overexpression of both of these transcripts at 4 h was followed by
an extinction at 24 h after FcRI cross-linking. The
IL-1RA transcript was also overexpressed after receptor ligation, with
a 3- to 8-fold increase of transcripts after 4 h, compared with
the treatment with IgE alone. This transcript remained expressed at
least for 24 h but declined to lower levels. Kynurenine
3-monooxygenase transcript expression, detectable 4 h after
stimulation, disclosed, in contrast to the other transcripts induced,
an accumulation within the succeeding 24 h after FcRI ligation.
The kinetics of transcript induction for the genes was comparable among
the different donors, whereas the magnitude of gene induction varied to
some extent.
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RI on atopic monocytes
To demonstrate that FcRI activation on atopic monocytes results
in the secretion of cytokines as suggested by the molecular genetic
analyses, MCP-1 and IL-6 were quantified in the cell supernatants from
three atopic individuals (data shown for only two donors, Fig. 5). Monocytes were stimulated with IgE
alone, IgE+ RahIgE F(ab')2,
RahIgE F(ab')2 alone, or left unstimulated. When
atopic monocytes were incubated with IgE alone, a weak secretion of
MCP-1 and IL-6 could be detected at 4 h and amounts increased up
to 24 h of incubation. This result may point to the physiologic
significance of FcRI activation on monocytes by monomeric IgE.
Incubation of atopic monocytes with anti-IgE alone induced small to
relatively high releases of cytokines, depending on the donor. These
releases correlated with the levels of plasma membrane IgE on the
monocytes (rFI for donor 3, 0.4; rFI for donor 5, 0.9) and therefore
might result from cross-linking of FcRI by anti-IgE. A much
stronger secretion of cytokines was observed when atopic monocytes were
incubated in the presence of IgE and anti-IgE. MCP-1 secretion was
high at 4 h and increased substantially at least to 24 h
after cross-linking of FcRI. The kinetics is consistent with a rapid
and sustained, although weaker, expression of mRNA for MCP-1 until at
least 24 h after FcRI ligation. IL-6 could be detected at high
levels in the culture medium 4 h after induction and its amount
slightly decreased/remained stable until 24 h after stimulation.
The kinetics of this accumulation is consistent with a rapid induction
of synthesis followed by an extinction/weaker expression of genes until
24 h after cross-linking FcRI.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI on basophils,
mast cells, and APC is critical in the control of the immune response
to Ags present at the interface with the environment. FcRI-mediated
release of biological effector molecules by these cells is of
pathogenic significance for delayed-type atopic inflammation
(13, 14, 15). The function of this receptor on circulating
blood monocytes from atopic individuals, however, has not been
sufficiently elucidated. To generate local inflammation, monocytes need
to migrate across the endothelial cell barrier and differentiate into
phagocytic cells or subtypes of DCs. Immunomodulatory signals from
monocytes after ligation of FcRI lead to antiapoptotic mechanisms
and directs these cells into macrophage-like cells, all of which may
explain why atopic inflammatory tissues harbor high amounts of
IgE-bearing macrophages and DCs (16, 17, 18). Taken together,
FcRI on monocytes might play a yet to be defined regulatory role in
atopic inflammation.
On monocytes, the expression of surface FcRI is strongly linked to
an atopic genetic background (19). Until now, it is not
clear why some individuals with high serum IgE levels or high FcRI
expression on monocytes are clinically ill, whereas others with these
parameters, such as members of the same family, can be clinically
healthy. Apparently, there is high expression variability of the
disease among family members but these individuals are inherently more
prone to precipitously developing atopic disease. Moreover,
atopic patients enter phases of remission or aggravation, the nature of
which is complex and implies intra- and interindividual
changes/differences in gene-gene regulatory interactions
(20).
As a first step to understand regulatory mechanisms after cross-linking
of FcRI on atopic monocytes, we determined the kinetics of various
genes expressed after activation of FcRI on these cells of different
atopic donors. The donors presented similar clinical and cellular
appearance, i.e., they were in remission of allergic
rhinitis/conjunctivitis or AEDS; all had a strong family atopic
background, high values of FcRI on the cell surface, and high serum
IgE levels. The investigated genes were identified by cDNA subtraction
techniques using FcRI-activated and nonactivated monocytes of a
single atopic donor. Although MCP-1 and MIP-1 transcripts were
overrepresented, we found five additional genes which were activated
within the first 4 h after FcRI stimulation. Remarkably, all of
these genes can be classified into two groups, according to their
short- or long-term inducibility. Thus, IL-6,
A subunit of inhibin/activin, ISG-54K, and
IL-1RA genes were transiently induced and almost not transcribed
24 h after induction. In contrast, MCP-1, MIP-1, and kynurenine
3-monooxygenase were induced not only shortly after FcRI stimulation
but remained overexpressed for at least 24 h, MIP-1 and
kynurenine 3-monooxygenase being even more strongly expressed at
24 h after induction than at 4 h after induction.
Our work suggests several mechanisms of monocyte function in atopy
resulting from the activation of cells through FcRI. MCP-1 and
MIP-1 are two members of the C-C chemokine family and were strongly
induced upon receptor cross-linking. In addition, high amounts of these
cytokines are secreted from atopic monocytes upon FcRI activation.
It is tempting to speculate that these cytokines are major
monocyte/macrophage attractants and account for the sustained
inflammation seen in atopic tissue.
In individuals with AEDS and allergic asthma, significantly higher IL-6
levels have been found in the serum than in a control group (21, 22). In vivo and in vitro, IL-6 acts as a differentiation factor
for B cells and as an activation factor for T cells. Our study implies
a possible role for FcRI on monocytes for the observed increase of
serum IL-6 since this cytokine was found at high concentrations in the
supernatants of FcRI-activated cells.
Interestingly, as revealed by the kinetic studies, we showed that
kynurenine 3-monooxygenase gene induction increases over a period of at
least 24 h after FcRI cross-linking on monocytes. Kynurenine
3-monooxygenase is an enzyme involved in the tryptophan degrading
pathway where indoleamine 2,3-dioxygenase converts tryptophan into
N-formylkynurenine, the latter being subsequently
deformylated into kynurenine. Kynurenine is further catabolized into
quinolinic acid, a neurotoxin (23). Recently, we were able
to demonstrate the overexpression of 2,3-dioxygenase in
FcRI-stimulated monocytes, which functionally results in the
degradation of tryptophan. In these conditions, FcRI-activated
monocytes acquired the ability to suppress T cell proliferation in
vitro by lowering tryptophan levels in the culture medium 24 h
after stimulation (24). The production of quinolinic acid
as a by-product of FcRI stimulation questions whether this
neurotoxin might have an impact on the interaction between the immune
system and the neurologic system during the development of allergy.
The observations in this study strongly implicate that cross-linking of
FcRI-bound IgE with environmental allergens may activate monocytes
and lead to the induction of several genes with a high impact on the
recruitment of inflammatory cells but also, at least in some
individuals, lead to down-regulatory mechanisms of T cell responses.
This pathway may be relevant to allergens which enter the blood, such
as food allergens (25), but should not apply to allergens
such as dust mites or pollen which are rarely encountered in vivo by
monocytes in the peripheral blood. Other mechanisms involved in FcRI
ligation might also account for our observations. In this regard,
binding of monomeric IgE to FcRI has been shown to activate mast
cells, thus leading to potent production of cytokines
(10). The mechanisms by which ligation of FcRI with
monomeric IgE activates monocytes are not known; however, we cannot
exclude signal transduction of these cells upon binding of IgE. This
would be in line with the finding that secretion, although small, of
cytokines was observed by the addition of monomeric IgE to monocytes
from atopics. Alternatively, the presence of significant amounts of
IgE-anti-IgE complexes has been reported in patients with atopic
disorders (26, 27). Such complexes might lead to chronic
activation of the receptor on circulating monocytes leading to the
observed gene and cytokine/chemokine production. In addition, this
mechanism could also explain the presence of high levels of IL-6 in the
blood of these individuals.
The method used to identify genes after activation of FcRI on
monocytes appears to be efficient in revealing the induction of several
genes with evident physiologic effects (28, 29, 30). It is
advantageous in identifying genes that are strongly up-regulated, thus
involving important effects after ligation of FcRI. The identified
genes were shown to be also induced in monocytes from additional atopic
donors, confirming that the results are representative of and
significant for the function of FcRI. The method may have some
limits which will be resolved using different strategies. First,
although the method used is based on the equalization of the amount of
different cDNA, the presence of overrepresented DNA fragments after the
PCR implies a competition of these fragments with other DNA fragments
in either one or several steps of subtraction, amplification, and/or
cloning. This may result in the potential loss of physiologically
important DNA fragments. Although this disadvantage was compensated by
the analysis of a hundred clones, the method may yield limited
information and some genes known to be overexpressed by FcRI
triggering were not detected (31). Second, repressed genes
as well as very weakly up- or down-regulated genes might be missed;
other methods like microarray techniques could be conducted to be
combined with our strategy (32, 33).
Differences in gene induction could also be elucidated by investigating donors in-season and off-season or in remission or aggravation of disease. Large numbers of individuals may be necessary to validate the results to more general conclusions but the presented strategy clearly demonstrated its successful approach in further defining monocyte function in atopic individuals.
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Acknowledgments |
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Footnotes |
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2 T.B. and H.d.l.S. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Henri de la Salle, Institut National de la Santé et de la Recherche Médicale EP99-08, Etablissement Français du Sang-Alsace 10, rue Spielmann BP 36, 67065 Strasbourg, Cedex, France. E-mail address: henri.delasalle{at}efs-alsace.fr
4 Abbreviations used in this paper: AEDS, atopic eczema/dermatitis syndrome; DIG, digoxigenin; GaM, goat anti-mouse; DC, dendritic cell; MCP-1, monocyte chemoattractant protein 1; MFI, mean fluorescence intensity; MIP-1, macrophage-inflammatory protein 1; ISG-54K, IFN-stimulated gene of 54 kDa; IL-1RA, IL-1R antagonist; RahIgE, rabbit anti-human IgE; rFI, relative fluorescence intensity; h, human; m, mouse.
Received for publication April 30, 2002. Accepted for publication September 27, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI). J. Exp. Med. 175:1285.RI) mediates IgE-dependent allergen presentation. J. Immunol. 154:6285.[Abstract]
RI. J. Immunol. 155:184.
RI) blocks apoptosis in normal human monocytes. J. Clin. Invest. 105:183.[Medline]
RI on human monocytes induces the production of IL-10 and prevents their differentiation in dendritic cells. J. Immunol. 167:797.RI. J. Immunol. 167:1290.RI) on monocytes of atopic individuals. J. Exp. Med. 179:745. and interleukin-6 by alveolar macrophages consecutive to the development of the late asthmatic reaction. J. Allergy Clin. Immunol. 88:561.[Medline]
RI induces the tryptophan degradation pathway involved in regulating T cell responses. J. Immunol. 169:1810.RI on human monocytes and dendritic cells induces NF-
B activation. J. Invest. Dermatol. 118:830.[Medline]
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) whereas in the
unsubtracted cDNA several distinct bands can be seen.
) or
were treated with IgE, RahIgE F(ab')2, or with IgE plus
anti-IgE. Data are presented as relative OD signals to the
