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*
Department of Dermatology and Allergy, University of Kiel, Kiel, Germany, and
St. John’s Institute of Dermatology, University of London, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterial and MethodsResultsDiscussionReferences |
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were not detected as Eo chemotaxins under these
conditions. Using reverse transcriptase-PCR techniques, we found that
IL-4 dose and time dependently induces eotaxin mRNA in dermal
fibroblasts. Stimulation with IL-4 and TNF- caused a 10- to 20-fold
increase of the release of three biochemically different eotaxin forms,
each consisting of a mixture of N-terminally truncated and
O-glycosylated variants having the same backbone amino acid
sequence but different specific activities. Our findings support the
hypothesis that eosinophil recruitment seen in IL-4-mediated skin
reactions, at least in part, may be due to Th2 cytokine-mediated
induction of eotaxin in dermal fibroblasts. |
Introduction |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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For unknown reasons Eos infiltrate the dermis of patients with atopic dermatitis after patch test reactions with aeroallergens (4). In lesional atopic dermatitis skin, dermal deposits of the Eo major basic protein have been demonstrated, suggesting that Eos degranulate in the affected skin (5).
Because Eos predominantly infiltrate the dermis rather than epidermis in all the above-mentioned skin diseases, including parasite infection, it is intriguing to speculate that dermal cells may contribute to Eo infiltration through the production of Eo attractants.
Elevated IgE level, activation of mast cells, basophils, Th2-type CD4+ T lymphocytes, and Eo tissue infiltration are known to be strongly associated with helminth parasite infections (6, 7). Key mediators produced by Th2 lymphocytes for defense against helminths are IL-5 and IL-4.
Whereas IL-5 mainly promotes blood eosinophilia (8), IL-4 induces IgE synthesis (9) and endothelial vascular cell adhesion molecule-1 expression (10), which, together with ß1 integrin very late antigen-4 expression on eosinophils (11), may increase Eo adherence to the vessels. It does not, however, explain Eo tissue accumulation. Therefore we speculate that, in addition, an Eo-specific chemoattractant such as eotaxin (12, 13, 14) is involved.
This raises the possibility that fibroblasts, which represent the major cellular component of the dermis, are capable of producing Eo chemotactic factors after treatment with a Th2 cytokine. In this study we investigated whether cultured human foreskin-derived fibroblasts have the capability to produce Eo attractants when stimulated with the Th2 cytokine IL-4.
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Material and Methods |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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Human surgically removed skin (foreskin) was incubated with a mixture of 0.05% (w/v) trypsin and 0.02% (w/v) EDTA over night at 4°C, and epidermis was removed. Thereafter, dermis was cut into pieces of about 2 mm in diameter. These were transferred into 25-mm2 flasks and supplied with MEM (Life Technologies-AMIMED, Allschwil, Switzerland) supplemented with penicillin (10 U/ml), streptomycin (100 µg/ml), glutamine (2 mM), and 10% FCS. When dermal fibroblasts had started growth, dermal pieces were removed. Medium was changed once a week. Passage was performed by the use of a mixture of 0.05% (w/v) trypsin and 0.02% EDTA for 5 min at 37°C. Fibroblasts from culture of the 5th to 10th passages were transferred either into six-well plates (9 cm2/well) or, for preparative purposes, into 175-cm2 or 500-cm2 flasks (Nunc, Wiesbaden, Germany) and were used at confluence.
Isolation of Eos
Eosinophils were isolated from human peripheral blood of normal donors having mild eosinophilia using Percoll density gradient centrifugation as previously described (15). Eo preparations used for experiments contained more than 90% Eos.
Eo chemotaxis assay
Screening of cell culture supernatants and HPLC fractions for
eosinophil chemotactic activity was assayed as previously described
(15) using blind well Boyden chambers (Bio-Rad, Munich, Germany), which
were filled with samples at appropriate dilutions and covered with a
polyvinylpyrrolidone-containing filter (pore size: 3 µm) (Costar,
Tübingen, Germany). Chemotactic activity was expressed as
chemotactic index:
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In some experiments, Eos were determined microscopically using a modification of Boyden’s method, as previously described in detail (15).
Production of Eo chemotactic proteins by human fibroblasts
Confluent growing cultures of dermal fibroblasts (
5 x
107 cells/flask) in 500-cm2 flasks were washed
twice with MEM and subsequently stimulated for 48 h with 20 ng/ml
human rIL-4 (Pepro Tech, London, UK) dissolved in 50 ml MEM/flask
containing the supplements but not FCS. Thereafter supernatants were
harvested and centrifuged and stored below -70°C until further
use.
High performance liquid chromatography
HPLC was performed at room temperature with a Spectra Physics liquid chromatography system equipped with a pump, a SP 8700 solvent delivery system, a Kratos spectroflow 783 spectrophotometer (Kratos, Westwood, NJ), and a SP 4270 integrator.
Purification of Eo chemotactic proteins
Pooled supernatants (500 ml/experiment, supernatants of 10 500-cm2 flasks) of IL-4 (20 ng/ml)-stimulated dermal fibroblasts (corresponding to approximately 5 x 108 cells) were concentrated and diafiltered against Tris/citrate buffer (10 mM/10 mM), pH 8.0, by using an Amicon YM-3 filter (cut off: 3kDa), and clarified by centrifugation.
Concentrated supernatants were then applied to a prepacked heparin-Sepharose column (Hi Trap, Pharmacia, Uppsala, Sweden) (1 ml volume) equilibrated with 10 mM Tris/10 mM citrate buffer, pH 8.0, using a HPLC pump and a flow rate of 1 ml/min. Thereafter the column was washed with equilibration buffer until no UV (280 nm)-absorbing material appeared in the effluent.
Bound material was eluted using 1 M NaCl in equilibration buffer and the pooled fractions were concentrated and diafiltered against 0.1% TFA in water until pH 2 was reached.
Heparin-bound proteins of stimulated fibroblasts were then applied to a preparative wide pore RP-8-HPLC column (300-7 C8 Nucleosil, 250 x 12.6 mm, Macherey and Nagel, Düren, Germany), and proteins were eluted from the column using a gradient of increasing concentrations of acetonitrile in 0.1% aqueous TFA (flow: 3 ml/min). During the HPLC separation of proteins, absorbance was monitored at 215 nm. Integration values obtained by the peak integrator were used to determine the amounts of proteins eluting in a given peak. To convert integration units to protein concentrations, known amounts of ubiquitin (Sigma, St. Louis, MO) were used for calibration. Nearly 106 integration units corresponded to 1 µg ubiquitin.
Fractions collected from preparative RP-8-HPLC, containing Eo chemotactic activity, were concentrated when necessary using YM-3 filters or were directly applied to a micro-Mono S HPLC column attached to a Smart Micro-HPLC apparatus (Pharmacia) previously equilibrated with 50 mmol/L ammonium formate, pH 4.0, containing 25% (v/v) acetonitrile. Proteins were eluted with a gradient of increasing concentrations of NaCl (maximum: 1 mol/L) in equilibration buffer using a flow rate of 100 µl/min. During the HPLC runs, absorbance at 215 nm, 254 nm, and 280 nm, as well as conductivity, were monitored.
Fractions containing Eo chemotactic activity were directly applied to a micro-RP-18-HPLC column (Pharmacia) previously equilibrated with 0.1% aqueous TFA. Proteins were eluted with a gradient of increasing concentrations of acetonitrile as shown in the chromatograms.
SDS-PAGE analyses
SDS-PAGE for polypeptides was performed with commercially available Tricin-buffer containing discontinuous gels ("high density gels," Pharmacia) and a "Phast" electrophoresis system (Pharmacia).
Proteins were fixed with 0.5% (v/v) glutaraldehyde containing 0.1% (w/v) sodium thiosulfate in 30% aqueous (v/v) ethanol containing 0.4 mol/L sodium acetate at pH 6.0 and were visualized by the use of a silver-staining kit (Sigma).
Peptide mapping experiments
From 2 to 4 µg of purified eotaxin variants or recombinant eotaxin (Pepro Tech) were treated with 100 ng of different endoproteinases (Lys C, EC 3.4.21.50; Asp N, EC 3.4.24.33; Glu C, EC 3.4.21.19) at appropriate conditions according to the instructions of the manufacturer (Boehringer Mannheim, Mannheim, Germany) in a volume of 40 to 100 µl. Thereafter samples were separated by micro RP-18 HPLC (Smart System, Pharmacia) and peaks were analyzed for UV absorbance at 215, 254, and 280 nm or sequenced and investigated for molecular masses or for presence of carbohydrates.
Amino acid sequence determinations
Amino acid sequences were determined by Edman degradation in an Applied Biosystems 476 A-pulsed liquid protein sequencer using reversed-phase (RP)-HPLC for phenylthiohydantoin-derivative detection.
Cysteine residues were confirmed after on filter reduction with tributylphosphine and alkylation with 4-vinylpyridine.
Matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) mass spectrometry (MS)
A MALDI instrument (TOF SPER, V6-Fison Instruments, Manchester,
UK) was used for mass determinations. A 10-mg/ml solution of
-cyano-4-hydroxy-cinnamic acid (Sigma) in aqueous 70% acetonitrile
containing 0.1% TFA was used as matrix. For ESI MS analysis, a Trio-2
quadruple (VG Biotech-Fisons Instruments, Manchester, UK) was used.
Samples were preconcentrated using a reverse-phase trapping system
(16).
Glycan ELISA
For determination of carbohydrate chains in eotaxin preparations, a digoxigenin glycan ELISA kit (Boehringer Mannheim) was used. Briefly, different amounts (0 to 100 ng) eotaxin were bound to ELISA plates and, by use of the kit, investigated for the presence of carbohydrate chains, as described by the manufacturer. The detection limit was near 2 to 5 ng glycoprotein. In other experiments, instead of eotaxin HPLC fractions, off peptide-mapping experiments were used to localize peptide fragments containing carbohydrate linkage.
Semiquantitative duplex RT-PCR
Total RNA from dermal fibroblast was isolated by acidic guanidinium thiocyanate-phenol-chloroform extraction (17). One microgram of total RNA was reverse transcripted using an oligo T18 primer and standard reagents (Life Technologies, Eggenstein, Germany). Intron spanning sets of primers specific for GAPDH and eotaxin (18) were used to differentiate between genomic and cDNA templates. cDNA corresponding to 50 ng RNA served as template in a duplex PCR reaction containing 0.8 µM eotaxin-specific primers (forward primer: 5'-CCCAACCACCTGCTGCTTTAACCTG-3'; reverse primer: 5'-TGGCTTTGGAGTTGGAGATTTTTGG-3') and (as internal control for equal amounts of cDNA before PCR) 0.1 µM GAPDH-specific primer pair (18). Amplification and analysis of the PCR products were performed as described (18).
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Results |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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In initial experiments, we found that stimulation of cultivated dermal fibroblasts with 20 ng/ml IL-4 for 48 h resulted in the release of Eo chemotactic activity, whereas nonstimulated cells did not contain this activity. Biochemical characterization experiments revealed that Eo attractants completely bound to a heparin-Sepharose column. The nonbinding proteins, as well as lipids containing effluent, did not show any strong Eo chemotactic activity (data not shown).
Further purification of the heparin-binding Eo chemotactic proteins by
preparative RP HPLC revealed early eluting fractions containing EO
chemotactic activity (Fig. 1
). Other
leukocytes, i.e., neutrophils or monocytes, did not respond to these
HPLC fractions. This Eo chemotaxin was then further purified by the use
of micro cation exchange HPLC followed by micro-RP HPLC (Fig. 2). The purified material we termed
eotaxin ß showed a single band upon Tricine (Sigma) SDS-gel
electrophoresis corresponding to a molecular mass of 13 kDa.
Recombinant eotaxin as control protein gave a single band near 10 kDa.
(Fig. 2).
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In order to obtain information about the structure of this chemotaxin, we performed N-terminal amino acid sequence analyses of the purified attractant and peptide-mapping experiments using different endoproteinases.
N-terminal sequence analyses revealed the presence of three N termini starting with Ala-Ser-Val (50%), Gly-Pro-Ala-Ser-Val (40%), and Ser-Val (10%), which correspond to the predicted N terminus of human eotaxin.
MALDI MS analyses, however, indicated the presence of a complex mixture
of different proteins (Fig. 3). Masses of
the principal components were 9322 and 9020. Further analyses of the
purified Eo attractant by peptide mapping with the endoproteinase Lys C
and comparison of the pattern with that of recombinant eotaxin show
some similarities as well as marked differences: natural eotaxin is
missing the peaks 1 and 4 present in the recombinant material. Instead,
four other peaks (numbers 1, 2, 4, and 5) can be seen in natural
eotaxin (Fig. 4). Sequence analyses of
the peaks different in the patterns of natural and recombinant material
revealed identical sequences for peaks having identical numbers and, in
addition, peaks 2 and 5 in natural material have identical sequences as
1 and 4, respectively. The complete amino acid sequence of natural
eotaxin produced by IL-4-stimulated dermal fibroblasts could be
evaluated by comparing and aligning sequences obtained from different
peptide-mapping experiments (Fig. 5).
Interestingly, in all experiments we were unable to determine residue
71. Peak 1 in recombinant eotaxin clearly represents a
carboxyl-terminal fragment starting with serine, which contains the
nonidentified residue 71 predicted to be threonine (Fig. 5).
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) were positive, whereas none of the fractions of
recombinant material showed any glycan-ELISA activity (data not
shown). MALDI MS analyses of peaks 1 and 2 revealed molecular masses of 649 and 1068 for both peaks. The calculated molecular mass of the peptide fragment predicted is 529.
Dose dependency and kinetics of IL-4-induced eotaxin mRNA expression
In order to analyze the conditions of eotaxin induction after stimulation with IL-4, we analyzed in semiquantitative reverse transcriptase (RT)-PCR eotaxin mRNA production in cultivated dermal fibroblasts.
Half-maximum eotaxin mRNA expression occurred after stimulation with
0.1 ng/ml IL-4, and maximum mRNA expression was seen at concentrations
higher than 1 ng/ml (Fig. 6A).
Eotaxin mRNA was up-regulated within 1 h and showed maximum
responses after 18 h (Fig. 6B).
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C, a
combination of IL-4 and TNF- seems to represent a very potent
stimulus. We therefore analyzed whether release of bioactive eotaxin is
also enhanced.
IL-4 in combination with TNF- strongly increases release of
different eotaxin variants
Heparin-bound proteins that were secreted from fibroblasts
stimulated with IL-4 (20 ng/ml) together with TNF- (20 ng/ml) were
separated by preparative RP HPLC (Fig. 7A). Two peaks of Eo-activity
were identified in HPLC fractions. The later eluting Eo attractant is
identical with RANTES (Fig. 7A) as could be revealed by
further purification and N-terminal sequencing (data not shown).
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, ß, and 
) that eluted together as a single
and one of the major at 215-nm absorbing protein peaks (Fig. 7A). SDS-PAGE analyses of this eotaxin preparation (Fig. 7A, inset) revealed a single band of 13-kDa size.
The amount of eotaxin reproducibly (n > 14) appeared
to be 10- to 20-fold higher when compared with IL-4 alone as revealed
by comparison of the peak integration units. Upon micro-Mono S-HPLC
(Fig. 7B), however, two Eo chemotactic 13-kDa proteins were
separated. Whereas two isoforms (eotaxin ß and eotaxin ) (Fig. 7C, left panel) were separated by micro-RP HPLC
of the earlier eluting protein peak I (Fig. 7B), the
stronger and most abundant cationic protein peak II (termed eotaxin
) appeared to be homogeneous giving a single peak (Fig. 7D). Comparison of the peptide-mapping patterns seen after
digestion of eotaxins , ß, and , respectively, with Lys C, with
the pattern of the eotaxin we isolated from IL-4-stimulated
fibroblasts, (Fig. 4) showed identity with eotaxin ß. N-terminal sequences of all eotaxins were found to be identical giving in each case a mixture of three N-terminal variants in a similar ratio as shown before. Furthermore, all variants gave positive results in a glycan assay indicating glycosylation. MALDI MS analyses revealed a size heterogeneity in all eotaxin variants with principal masses at 9320, 9020, and 8475. The intensities of some masses varied; eotaxin ß also contains a component having a mass of 7882.
Natural eotaxin variants express different biologic activity
When the different purified variants of natural eotaxin were
studied for eosinophil chemotactic activity, we found the major eotaxin
, which represents 60% of total eotaxin, showing similar potency
and efficacy (percentage of input migrating cells) in an in vitro Eo
chemotaxis assay as seen for recombinant eotaxin (Fig. 8). Both eotaxin ß and eotaxin were
found to be slightly less active regarding potency, but showing
identical efficacy as recombinant eotaxin.
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Discussion |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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Th2 cells are known to favor activation of mast cells and Eos and stimulation of B cell growth and isotype switching to cells producing IgE and thus are believed to have a protective role in helminth infection (7).
A body of evidence supports a role of Eos as effector cells in killing helminthic parasites, especially in the larval stage (19). Thus it is intriguing to speculate that there is a link between Th2 cells and tissue recruitment of Eos. Initial experiments in our laboratory revealed that blood- and tissue-derived Th2 cell clones produced almost no Eo-attracting RANTES or other Eo attractants (our unpublished results). This unexpected finding was supported by a recent report that Th1 cells, but not Th2 cells, produce RANTES (20). Furthermore, experiments failed to show that T lymphocyte preparations express eotaxin mRNA—even after stimulation (Ref. 12 and our unpublished results); however, eotaxin mRNA expression in Ag-induced pulmonary eosinophilia in rodents is T cell dependent (21, 22), which favored the hypothesis that Th2 cytokines may indirectly induce tissue infiltration of Eos.
In this study we could identify dermal fibroblasts as the first
cellular source of secreted and biologically active eotaxin, which is
released as a post-translational modified isoform of eotaxin—termed
eotaxin ß—when fibroblasts were stimulated with IL-4. This finding
is of particular importance because it would explain why Eos usually
appear in the dermis rather than epidermis in eosinophilic skin
diseases (5). Originally RANTES appeared to be of high relevance as
attractant involved in Eo tissue accumulation (23). Indeed dermal
fibroblasts produce high amounts of biologically active RANTES when
stimulated with TNA- (24). However, in allergic lung inflammation of
mice, eosinophilic infiltrates appeared to depend upon the presence of
T lymphocytes but not RANTES (25). In this study, we did not find any
RANTES as well as MCP-3 bioactive and immunoreactive protein after
stimulation of dermal fibroblasts with IL-4 when solid phase ELISAs
with specific mAbs were used (our unpublished observations).
Furthermore, the discovery of only minute amounts of RANTES in lesional
scales of atopic patients (26) and lack of RANTES mRNA correlation with
Eo tissue infiltration in allergen-induced cutaneous late phase
responses (27) point toward a comparatively minor role of RANTES as
well as possibly MCP-3 in Th2 cytokine-mediated skin inflammation.
Although there are reports showing that eotaxin mRNA is constitutively
expressed in the gut and skin or rodents (28) as well as the gut of
humans (12, 13), our knowledge about regulation of human eotaxin gene
expression and especially protein release is scarce. As yet there is no
information whether bioactive eotaxin is released by cultured normal
human cells although a recent report indicates the release of minute
amounts of immunoreactive eotaxin in a lung epithelial cell line (29).
Umbilical cord endothelial cells have been reported to express eotaxin
mRNA, when stimulated with TNF-, but not with IL-4 (13). In our
experiments we were unable to detect any Eo chemotactic cytokine in
culture supernatants of TNF-- or IL-4-stimulated umbilical cord
endothelial cells (our unpublished observations).
Furthermore, in our hands, cultured keratinocytes also failed to express eotaxin mRNA and to release Eo attractants (our unpublished observations). In skin, the property to respond toward IL-4 stimulation with a strong release of eotaxin appears to be restricted to dermal fibroblasts. Dermal fibroblasts express IL-4R (30) and produce collagen I upon stimulation with IL-4 (31), thus representing important cells mediating Th2-dependent effector mechanisms in skin.
An association between tissue eosinophilia and IL-4 production
has been presumed because transgenic mice expressing IL-4 in the lung
showed eosinophilic inflammation (32). Furthermore, in IL-4-deficient
mice, tissue eosinophilia is strongly inhibited upon allergen-induced
airway inflammation (33) and upon Onchocerca
volvulus-mediated corneal inflammation (34), but not blood
eosinophilia, which depends upon IL-5. The failure to completely
abolish tissue eosinophilia by the use of anti-IL-4 points toward a
role of other factors. A likely candidate would be IL-13, which binds
to the -chain of the IL-4R (35). This receptor is known to be
expressed in fibroblasts (30) and thus IL-13 could act similarly
as IL-4.
A rather indirect role of IL-4 together with IL-5 in mediating effector mechanisms is likely from experiments showing that CD4+ T lymphocytes are involved in Eo accumulation in airways of Ag-challenged allergic mice: depletion of T cells resulted in a decrease of lung eosinophilia together with a decrease of IL-4 and IL-5 mRNA expression in the lung (36).
Apart from T lymphocytes (mainly CD4+NK1.1+ T cells) (37) other cells such as mast cells (38) and basophils (39) are known to be major cellular sources of stored IL-4, which is released after stimulation.
It is well documented that mast cell and basophil activation in vivo is linked with tissue eosinophilia (40). As mechanism for this association the release of Eo attractants by mast cells and/or basophils has been discussed (41). Our studies would add another likely mechanism whereby activated mast cells and/or basophils release IL-4, which would activate in vivo fibroblasts for eotaxin production.
Our finding that a combination of IL-4 and TNF- induces the release
of large amounts of a mixture of biologically active eotaxin isoforms
(termed eotaxins , ß, and ) is of importance because mast cells
and basophils contain both preformed IL-4 and TNF- (42), which are
released when stimulated through the IgE receptor. Thus it is
conceivable that eosinophil tissue infiltration seen after mast cell
activation is the result of strong eotaxin release in neighboring
fibroblasts, which needs to be proved in in vivo experiments.
Eotaxin is the only Eo attractant that is secreted by IL-4-stimulated dermal fibroblasts. Thus other chemokines including MCP-4 (43, 44) apparently are not released as bioactive molecules or in sufficient amounts and thus appear to be of less importance for recruitment of Eos into human skin in IL-4-mediated reactions. It is interesting to note that MCP-3, which also expresses Eo chemotactic activities (45, 46), is not detected in supernatants of cultured dermal fibroblasts, despite strong mRNA expression (our unpublished observations). This may point toward a differential post-translational regulation of some chemokines and a careful interpretation of gene expression vs protein release.
The natural eotaxin isoform we have isolated from IL-4 stimulated
fibroblasts (eotaxin ß) in principle shows the predicted backbone
protein sequence deduced from the eotaxin cDNA (12, 14, 18). There
are, however, marked differences that include mobility upon
SDS-PAGE analysis and peptide-mapping profiles. With respect to the
previous identification of different eotaxin cDNAs (18), it was
interesting to clarify whether the eotaxin variants are really
released. In the case of the chemokine gro, different forms (gro ,
gro ß, and gro ) are encoded by different genes (47). MIP-1 on
the other hand shows a gene polymorphism with various, slightly
different forms (48). To identify differences in the primary structure,
we therefore performed peptide mapping and sequencing experiments with
natural eotaxin purified from IL-4-stimulated fibroblasts (eotaxin ß)
as well as recombinant eotaxin.
The data from these experiments indicate that the eotaxin variant
encoded by clone 53 (Lys58 is substituted by Arg) (18)
represent 10% of the natural eotaxin (, ß, and ) preparations.
Furthermore, residue 71, predicted to be a Thr, is glycosylated as
shown by the shift to early retention time upon RP HPLC of the
peptide-mapping fragment when compared with recombinant material as
well as positive glycan ELISA results. The polar shift, however, and
duplication of the HPLC peak representing the most prominent
UV-absorbing peak in the peptide-mapping experiment of recombinant
eotaxin using the endoproteinase Lys C (see Fig. 4) indicate
derivatization in the area between residues 58 and 65 (see Fig. 5). The
lack of carbohydrate in these modified fragments (data not shown) and
the clear evidence for a Ser as residue 63 upon Edman degradation
indicate derivatization other than glycosylation, which needs further
investigation.
There is clear evidence that residue 71, which is predicted to be a threonine (12, 13, 14) and which could not be determined by Edman degradation, is O-glycosylated. Instead, HPLC fraction variants containing C-terminal fragments of this natural eotaxin contained carbohydrates as revealed by a glycan-ELISA.
Different eotaxins have also been demonstrated in guinea pig
bronchoalveolar lavage fluid. These differences were ascribed to
differences in O-glycosylation and accompanied by an
unidentified residue (49) also predicted to be a threonine (50).
Additional biochemical variants of natural human eotaxin (termed
eotaxin and eotaxin ) could also be purified from fibroblast
supernatants previously stimulated with IL-4 plus TNF-.
Peptide-mapping patterns of eotaxin and were different from
that of eotaxin ß (data not shown) and again these eotaxin forms
seemed to be O-glycosylated at residue 71.
Glycosylation can affect biologic activity or cytokines (51).
Therefore, it was interesting to investigate specific Eo
chemoattractant activities of the three biochemically distinct forms of
natural eotaxin. Indeed, eotaxin ß, which is released upon
stimulation of fibroblasts with IL-4, shows a slightly decreased
potency in vitro, whereas the eotaxin , which is released as major
variant upon fibroblast stimulation with a mixture of IL-4 and TNF-,
reveals identical activity as seen with recombinant material.
Thus, eotaxin bioactivity, unlike that of other chemokines known to be N- or O-glycosylated (52), can be affected by glycosylation. Because both eotaxin variants are O-glycosylated at residue 71 (threonine), derivatization, possibly at residue 63 (serine), might be responsible for the decreased potency seen for eotaxin ß.
In summary, we have shown that dermal fibroblasts represent a major
cellular source of bioactive natural O-glycosylated eotaxin
when stimulated with the Th2 cytokine IL-4 or—more efficiently—in
combination with TNF-. Our findings would, at least in part, explain
selective recruitment of Eos into affected skin seen in Th2-mediated
skin inflammation.
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Acknowledgments |
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Footnotes |
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2 Both authors contributed equally to this study.
3 Address correspondence and reprint requests to Dr. J.-M. Schröder, Dept. of Dermatology, University of Kiel, D-24105 Kiel, Germany. E-mail address:
4 Abbreviations used in this paper: Eos, eosinophils; MALDI, matrix-associated laser desorption ionization; MS, mass spectrometry; PAF, platelet-activating factor; RP, reversed phase; TFA, trifluoroacetic acid; GAPDH, glyceraldehyde phosphate dehydrogenase; ESI, electrospray ionization.
Received for publication June 2, 1997. Accepted for publication September 16, 1997.
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P. Ogilvie, S. Paoletti, I. Clark-Lewis, and M. Uguccioni Eotaxin-3 is a natural antagonist for CCR2 and exerts a repulsive effect on human monocytes Blood, August 1, 2003; 102(3): 789 - 794. [Abstract] [Full Text] [PDF]
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K. Chibana, Y. Ishii, T. Asakura, and T. Fukuda Up-Regulation of Cysteinyl Leukotriene 1 Receptor by IL-13 Enables Human Lung Fibroblasts to Respond to Leukotriene C4 and Produce Eotaxin J. Immunol., April 15, 2003; 170(8): 4290 - 4295. [Abstract] [Full Text] [PDF]
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K. Raman, M. H. Kaplan, C. M. Hogaboam, A. Berlin, and N. W. Lukacs STAT4 Signal Pathways Regulate Inflammation and Airway Physiology Changes in Allergic Airway Inflammation Locally Via Alteration of Chemokines J. Immunol., April 1, 2003; 170(7): 3859 - 3865. [Abstract] [Full Text] [PDF]
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C. Langdon, C. Kerr, L. Tong, and C. D. Richards Oncostatin M Regulates Eotaxin Expression in Fibroblasts and Eosinophilic Inflammation in C57BL/6 Mice J. Immunol., January 1, 2003; 170(1): 548 - 555. [Abstract] [Full Text] [PDF]
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H. Asakura, Y. Kashio, K. Nakamura, M. Seki, S. Dai, Y. Shirato, M. J. Abedin, N. Yoshida, N. Nishi, T. Imaizumi, et al. Selective Eosinophil Adhesion to Fibroblast Via IFN-{gamma}-Induced Galectin-9 J. Immunol., November 15, 2002; 169(10): 5912 - 5918. [Abstract] [Full Text] [PDF]
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J. D. Campbell, M. J. Stinson, F. E. R. Simons, and K. T. HayGlass Systemic chemokine and chemokine receptor responses are divergent in allergic versus non-allergic humans Int. Immunol., November 1, 2002; 14(11): 1255 - 1262. [Abstract] [Full Text] [PDF]
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M. T. Borchers, T. Ansay, R. DeSalle, B. L. Daugherty, H. Shen, M. Metzger, N. A. Lee, and J. J. Lee In vitro assessment of chemokine receptor-ligand interactions mediating mouse eosinophil migration J. Leukoc. Biol., June 1, 2002; 71(6): 1033 - 1041. [Abstract] [Full Text] [PDF]
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M. Relloso, A. Puig-Kroger, O. M. Pello, J. L. Rodriguez-Fernandez, G. de la Rosa, N. Longo, J. Navarro, M. A. Munoz-Fernandez, P. Sanchez-Mateos, and A. L. Corbi DC-SIGN (CD209) Expression Is IL-4 Dependent and Is Negatively Regulated by IFN, TGF-{beta}, and Anti-Inflammatory Agents J. Immunol., March 15, 2002; 168(6): 2634 - 2643. [Abstract] [Full Text] [PDF]
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J. W. Verbsky, D. A. Randolph, L. P. Shornick, and D. D. Chaplin Nonhematopoietic Expression of Janus Kinase 3 Is Required for Efficient Recruitment of Th2 Lymphocytes and Eosinophils in OVA-Induced Airway Inflammation J. Immunol., March 1, 2002; 168(5): 2475 - 2482. [Abstract] [Full Text] [PDF]
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E. F. Foxman, M. Zhang, S. D. Hurst, T. Muchamuel, D. Shen, E. F. Wawrousek, C.-C. Chan, and I. Gery Inflammatory Mediators in Uveitis: Differential Induction of Cytokines and Chemokines in Th1- Versus Th2-Mediated Ocular Inflammation J. Immunol., March 1, 2002; 168(5): 2483 - 2492. [Abstract] [Full Text] [PDF]
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K. Watanabe, P. J. Jose, and S. M. Rankin Eotaxin-2 Generation Is Differentially Regulated by Lipopolysaccharide and IL-4 in Monocytes and Macrophages J. Immunol., February 15, 2002; 168(4): 1911 - 1918. [Abstract] [Full Text] [PDF]
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F. H. Falcone, P'n. Loke, X. Zang, A. S. MacDonald, R. M. Maizels, and J. E. Allen A Brugia malayi Homolog of Macrophage Migration Inhibitory Factor Reveals an Important Link Between Macrophages and Eosinophil Recruitment During Nematode Infection J. Immunol., November 1, 2001; 167(9): 5348 - 5354. [Abstract] [Full Text] [PDF]
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P. Loetscher and I. Clark-Lewis Agonistic and antagonistic activities of chemokines J. Leukoc. Biol., June 1, 2001; 69(6): 881 - 884. [Abstract] [Full Text] [PDF]
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A. Mathew, J. A. MacLean, E. DeHaan, A. M. Tager, F. H.Y. Green, and A. D. Luster Signal Transducer and Activator of Transcription 6 Controls Chemokine Production and T Helper Cell Type 2 Cell Trafficking in Allergic Pulmonary Inflammation J. Exp. Med., May 7, 2001; 193(9): 1087 - 1096. [Abstract] [Full Text] [PDF]
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C. Bandeira-Melo, K. Sugiyama, L. J. Woods, and P. F. Weller Cutting Edge: Eotaxin Elicits Rapid Vesicular Transport-Mediated Release of Preformed IL-4 from Human Eosinophils J. Immunol., April 15, 2001; 166(8): 4813 - 4817. [Abstract] [Full Text] [PDF]
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J. Hoeck and M. Woisetschlager STAT6 Mediates Eotaxin-1 Expression in IL-4 or TNF-{{alpha}}-Induced Fibroblasts J. Immunol., April 1, 2001; 166(7): 4507 - 4515. [Abstract] [Full Text] [PDF]
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P. Ogilvie, G. Bardi, I. Clark-Lewis, M. Baggiolini, and M. Uguccioni Eotaxin is a natural antagonist for CCR2 and an agonist for CCR5 Blood, April 1, 2001; 97(7): 1920 - 1924. [Abstract] [Full Text] [PDF]
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M. M. Teixeira, A. Talvani, W. L. Tafuri, N. W. Lukacs, and P. G. Hellewell Eosinophil recruitment into sites of delayed-type hypersensitivity reactions in mice J. Leukoc. Biol., March 1, 2001; 69(3): 353 - 360. [Abstract] [Full Text]
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L. Cohn, C. Herrick, N. Niu, R. J. Homer, and K. Bottomly IL-4 Promotes Airway Eosinophilia by Suppressing IFN-{{gamma}} Production: Defining a Novel Role for IFN-{{gamma}} in the Regulation of Allergic Airway Inflammation J. Immunol., February 15, 2001; 166(4): 2760 - 2767. [Abstract] [Full Text] [PDF]
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J. C. Keen, L. Sholl, M. Wills-Karp, and S. N. Georas Preferential Activation of Nuclear Factor of Activated T Cells c Correlates with Mouse Strain Susceptibility to Allergic Responses and Interleukin-4 Gene Expression Am. J. Respir. Cell Mol. Biol., January 1, 2001; 24(1): 58 - 65. [Abstract] [Full Text]
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F. H. Falcone, H. Haas, and B. F. Gibbs The human basophil: a new appreciation of its role in immune responses Blood, December 15, 2000; 96(13): 4028 - 4038. [Full Text] [PDF]
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N. Zimmermann, S. P. Hogan, A. Mishra, E. B. Brandt, T. R. Bodette, S. M. Pope, F. D. Finkelman, and M. E. Rothenberg Murine Eotaxin-2: A Constitutive Eosinophil Chemokine Induced by Allergen Challenge and IL-4 Overexpression J. Immunol., November 15, 2000; 165(10): 5839 - 5846. [Abstract] [Full Text] [PDF]
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T. Sekiya, M. Miyamasu, M. Imanishi, H. Yamada, T. Nakajima, M. Yamaguchi, T. Fujisawa, R. Pawankar, Y. Sano, K. Ohta, et al. Inducible Expression of a Th2-Type CC Chemokine Thymus- and Activation-Regulated Chemokine by Human Bronchial Epithelial Cells J. Immunol., August 15, 2000; 165(4): 2205 - 2213. [Abstract] [Full Text] [PDF]
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D. C. Webb, A. N. J. McKenzie, A. M. L. Koskinen, M. Yang, J. Mattes, and P. S. Foster Integrated Signals Between IL-13, IL-4, and IL-5 Regulate Airways Hyperreactivity J. Immunol., July 1, 2000; 165(1): 108 - 113. [Abstract] [Full Text] [PDF]
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J. Harder, U. Meyer-Hoffert, L. M. Teran, L. Schwichtenberg, J. Bartels, S. Maune, and J.-M. Schröder Mucoid Pseudomonas aeruginosa, TNF-alpha , and IL-1beta , but Not IL-6, Induce Human beta -Defensin-2 in Respiratory Epithelia Am. J. Respir. Cell Mol. Biol., June 1, 2000; 22(6): 714 - 721. [Abstract] [Full Text]
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H. Nagase, M. Miyamasu, M. Yamaguchi, T. Fujisawa, K. Ohta, K. Yamamoto, Y. Morita, and K. Hirai Expression of CXCR4 in Eosinophils: Functional Analyses and Cytokine-Mediated Regulation J. Immunol., June 1, 2000; 164(11): 5935 - 5943. [Abstract] [Full Text] [PDF]
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 N. Kumagai, K. Fukuda, Y. Ishimura, and T. Nishida Synergistic Induction of Eotaxin Expression in Human Keratocytes by TNF-{alpha} and IL-4 or IL-13 Invest. Ophthalmol. Vis. Sci., May 1, 2000; 41(6): 1448 - 1453. [Abstract] [Full Text]
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 J. Zhang, L. J. Lathbury, and L. A. Salamonsen Expression of the Chemokine Eotaxin and Its Receptor, CCR3, in Human Endometrium Biol Reprod, February 1, 2000; 62(2): 404 - 411. [Abstract] [Full Text]
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S. Matsukura, C. Stellato, J. R. Plitt, C. Bickel, K. Miura, S. N. Georas, V. Casolaro, and R. P. Schleimer Activation of Eotaxin Gene Transcription by NF-{kappa}B and STAT6 in Human Airway Epithelial Cells J. Immunol., December 15, 1999; 163(12): 6876 - 6883. [Abstract] [Full Text] [PDF]
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S. Ying, Q. Meng, K. Zeibecoglou, D. S. Robinson, A. Macfarlane, M. Humbert, and A. B. Kay Eosinophil Chemotactic Chemokines (Eotaxin, Eotaxin-2, RANTES, Monocyte Chemoattractant Protein-3 (MCP-3), and MCP-4), and C-C Chemokine Receptor 3 Expression in Bronchial Biopsies from Atopic and Nonatopic (Intrinsic) Asthmatics J. Immunol., December 1, 1999; 163(11): 6321 - 6329. [Abstract] [Full Text] [PDF]
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C. Stellato, S. Matsukura, A. Fal, J. White, L. A. Beck, D. Proud, and R. P. Schleimer Differential Regulation of Epithelial-Derived C-C Chemokine Expression by IL-4 and the Glucocorticoid Budesonide J. Immunol., November 15, 1999; 163(10): 5624 - 5632. [Abstract] [Full Text] [PDF]
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 T. R. Johnson and B. S. Graham Secreted Respiratory Syncytial Virus G Glycoprotein Induces Interleukin-5 (IL-5), IL-13, and Eosinophilia by an IL-4-Independent Mechanism J. Virol., October 1, 1999; 73(10): 8485 - 8495. [Abstract] [Full Text] [PDF]
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S. Ying, D. S. Robinson, Q. Meng, L. T. Barata, A. R. McEuen, M. G. Buckley, A. F. Walls, P. W. Askenase, and A. B. Kay C-C Chemokines in Allergen-Induced Late-Phase Cutaneous Responses in Atopic Subjects: Association of Eotaxin with Early 6-Hour Eosinophils, and of Eotaxin-2 and Monocyte Chemoattractant Protein-4 with the Later 24-Hour Tissue Eosinophilia, and Relationship to Basophils and Other C-C Chemokines (Monocyte Chemoattractant Protein-3 and RANTES) J. Immunol., October 1, 1999; 163(7): 3976 - 3984. [Abstract] [Full Text] [PDF]
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M. E. Rothenberg Eotaxin . An Essential Mediator of Eosinophil Trafficking into Mucosal Tissues Am. J. Respir. Cell Mol. Biol., September 1, 1999; 21(3): 291 - 295. [Full Text]
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G. Devouassoux, D. D. Metcalfe, and C. Prussin Eotaxin Potentiates Antigen-Dependent Basophil IL-4 Production J. Immunol., September 1, 1999; 163(5): 2877 - 2882. [Abstract] [Full Text] [PDF]
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A. Shinkai, H. Yoshisue, M. Koike, E. Shoji, S. Nakagawa, A. Saito, T. Takeda, S. Imabeppu, Y. Kato, N. Hanai, et al. A Novel Human CC Chemokine, Eotaxin-3, Which Is Expressed in IL-4-Stimulated Vascular Endothelial Cells, Exhibits Potent Activity Toward Eosinophils J. Immunol., August 1, 1999; 163(3): 1602 - 1610. [Abstract] [Full Text] [PDF]
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M. Miyamasu, M. Yamaguchi, T. Nakajima, Y. Misaki, Y. Morita, K. Matsushima, K. Yamamoto, and K. Hirai Th1-derived cytokine IFN-{gamma} is a potent inhibitor of eotaxin synthesis in vitro Int. Immunol., June 1, 1999; 11(6): 1001 - 1004. [Abstract] [Full Text]
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J. T. Wise, T. J. Baginski, and J. L. Mobley An Adoptive Transfer Model of Allergic Lung Inflammation in Mice Is Mediated by CD4+CD62LlowCD25+ T Cells J. Immunol., May 1, 1999; 162(9): 5592 - 5600. [Abstract] [Full Text] [PDF]
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S. Struyf, P. Proost, D. Schols, E. De Clercq, G. Opdenakker, J.-P. Lenaerts, M. Detheux, M. Parmentier, I. De Meester, S. Scharpe, et al. CD26/Dipeptidyl-Peptidase IV Down-Regulates the Eosinophil Chemotactic Potency, But Not the Anti-HIV Activity of Human Eotaxin by Affecting Its Interaction with CC Chemokine Receptor 3 J. Immunol., April 15, 1999; 162(8): 4903 - 4909. [Abstract] [Full Text] [PDF]
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J. Teruya-Feldstein, E. S. Jaffe, P. R. Burd, D. W. Kingma, J. E. Setsuda, and G. Tosato Differential Chemokine Expression in Tissues Involved by Hodgkin's Disease: Direct Correlation of Eotaxin Expression and Tissue Eosinophilia Blood, April 15, 1999; 93(8): 2463 - 2470. [Abstract] [Full Text] [PDF]
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L. M. Teran, M. Mochizuki, J. Bartels, E. L. Valencia, T. Nakajima, K. Hirai, and J.-M. Schröder Th1- and Th2-Type Cytokines Regulate the Expression and Production of Eotaxin and RANTES by Human Lung Fibroblasts Am. J. Respir. Cell Mol. Biol., April 1, 1999; 20(4): 777 - 786. [Abstract] [Full Text]
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L. Li, Y. Xia, A. Nguyen, Y. H. Lai, L. Feng, T. R. Mosmann, and D. Lo Effects of Th2 Cytokines on Chemokine Expression in the Lung: IL-13 Potently Induces Eotaxin Expression by Airway Epithelial Cells J. Immunol., March 1, 1999; 162(5): 2477 - 2487. [Abstract] [Full Text] [PDF]
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P. Loetscher, A. Pellegrino, J.-H. Gong, I. Mattioli, M. Loetscher, G. Bardi, M. Baggiolini, and I. Clark-Lewis The Ligands of CXC Chemokine Receptor 3, I-TAC, Mig, and IP10, Are Natural Antagonists for CCR3 J. Biol. Chem., January 26, 2001; 276(5): 2986 - 2991. [Abstract] [Full Text] [PDF]
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A. M. Stutz, J. Hoeck, F. Natt, B. Cuenoud, and M. Woisetschlager Inhibition of Interleukin-4- and CD40-induced IgE Germline Gene Promoter Activity by 2'-Aminoethoxy-modified Triplex-forming Oligonucleotides J. Biol. Chem., April 6, 2001; 276(15): 11759 - 11765. [Abstract] [Full Text] [PDF]
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M. A. Huber, A. Denk, R. U. Peter, L. Weber, N. Kraut, and T. Wirth The IKK-2/Ikappa Balpha /NF-kappa B Pathway Plays a Key Role in the Regulation of CCR3 and eotaxin-1 in Fibroblasts. A CRITICAL LINK TO DERMATITIS IN Ikappa Balpha -DEFICIENT MICE J. Biol. Chem., January 4, 2002; 277(2): 1268 - 1275. [Abstract] [Full Text] [PDF]
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P. E. Moore, T. L. Church, D. D. Chism, R. A. Panettieri Jr., and S. A. Shore IL-13 and IL-4 cause eotaxin release in human airway smooth muscle cells: a role for ERK Am J Physiol Lung Cell Mol Physiol, April 1, 2002; 282(4): L847 - L853. [Abstract] [Full Text] [PDF]
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; eotaxin 
). For comparison, the dose-response curve of recombinant human
eotaxin purification of commercially available material (after
micro-RP-HPLC) is included (
). PAF (10-7 M) served as a
positive stimulus, whereas medium alone (-) served as a negative
control. We estimated an ED50 ± SD of 18 ± 13,
50 ± 11, and 48 ± 42 ng/ml for eotaxin