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From the Laboratory of Immunology, Istituto Dermopatico dell’Immacolata, Istituto di Ricovero e Cura a Carottere Scientifico, Rome, Italy
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Abstract |
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and interferon 
. These cytokines also promote de novo synthesis of numerous chemokines, including CCL2/MCP-1, CCL5/RANTES, CXCL10/IP-10, and CXCL8/IL-8, in turn responsible for the recruitment of different leukocyte populations. This study demonstrates that stimulation of EGFR down-regulates CCL2, CCL5, and CXCL10, while it increases CXCL8 expression in keratinocytes. Conversely, EGFR signaling blockade produces opposite effects, with increased CCL2, CCL5, and CXCL10, and reduced CXCL8 expression. In a mouse model of contact hypersensitivity, a single topical administration of a selective EGFR kinase blocker before antigen challenge results in a markedly enhanced immune response with increased chemokine expression and heavier inflammatory cell infiltrate. Targeting EGFR on epithelial cells may thus have profound impact on inflammatory and immune responses.
(TGF-), amphiregulin, heparin-binding EGF-like growth factor, and epiregulin. These factors derive from proteolytic cleavage of integral membrane precursors by metalloproteinases.1
Although keratinocytes express multiple EGF receptors (c-erbB1–3), c-erbB1 (also called EGFR) is responsible for the bulk of the autocrine activities of EGF family members in keratinocytes.2,3
Indeed, EGFR activation serves essential functions in skin development, wound healing as well as carcinogenesis, maintaining keratinocytes in an active proliferative state,4,5
enhancing their motility,6
and supporting survival and protection from apoptosis.7
EGFR and its ligand TGF- are overexpressed in a variety of benign and malignant hyperproliferative skin disorders, including psoriasis,8-10
and the role of TGF- in the genesis of epidermal hyperplasia has been confirmed in transgenic mouse models.11,12
However, no consistent information exists on the contribution of EGFR signaling to skin inflammation.
A common feature of chronic inflammatory skin disorders such as psoriasis, atopic dermatitis, and allergic contact dermatitis, is epidermal hyperplasia and thickening, a phenomenon attributed to leukocyte-derived cytokines such as tumor necrosis factor (TNF)- and interferon (IFN)-, which are potent inducers of EGF family growth factors and EGFR.13,14
In the course of T cell-driven skin inflammatory diseases, activated Th1 lymphocytes infiltrating the dermis and the epidermis are the major source of IFN- and TNF-. Among the various leukocyte subsets, Th1 lymphocytes dominate psoriatic and allergic contact dermatitis lesions, but they are present also in chronic atopic dermatitis.15,16
These cytokines initiate a program of increased keratinocyte expression of inflammatory mediators, including adhesion molecules, cytokines, and chemokines. In particular, prominent keratinocyte expression of CCL2 (monocyte chemoattractant protein 1, MCP-1), CCL5 (RANTES), CXCL8 (IL-8), and CXCL10 (IFN--induced protein of 10 kd, IP-10) is a common finding in T cell-mediated skin diseases, and mediates the recruitment of T cells and other leukocyte populations in the skin.17-20
Here we demonstrated that EGFR signaling activation was an early event in keratinocyte response to TNF- or IFN-, and that this mechanism was part of an autocrine loop with regulatory effects on CCL2, CCL5, and CXCL10 expression. In a mouse model of contact hypersensitivity, a T cell-mediated immune response where the contribution of keratinocytes to skin inflammation is well established,20
administration of a selective EGFR kinase blocker before antigen challenge induced enhanced immune response with increased chemokine expression and heavier inflammatory infiltrate. Thus, EGFR appears to play a relevant role in the control of skin inflammation.
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Materials and Methods |
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Four-mm punch biopsies were taken from lesional skin of adult patients with chronic plaque psoriasis (n = 5; three females and two males; age 30 to 48), chronic atopic dermatitis (n = 5; three females and two males; age 25 to 42), chronic allergic contact dermatitis (n = 3; two females and one male; age 30 to 40), and normal skin of healthy subjects (n = 4; two females and two males; age 24 to 39). Epidermal sheets for keratinocyte cultures and full-thickness punch skin explants for organ cultures were obtained from healthy individuals undergoing plastic surgery (mammoplasty or abdominoplasty) (n = 6, three females and three males; age 25 to 40). All subjects were not receiving any systemic or topical therapy before sampling. Informed consent was obtained from all subjects and the study design was approved by the local ethical committee.
Cytokines, Reagents, and Antibodies
Recombinant human TGF-, TNF-, IFN-, and anti-TGF- neutralizing Ab were purchased from R&D Systems (Abingdon, United Kingdom). PD168393, PD153035, and AG1296 were from Calbiochem (La Jolla, CA). Ilomastat came from BIOMOL Research Laboratories (Plymouth Meeting, PA).
Immunohistochemistry
Four-µm cryostatic sections were fixed with 4% paraformaldehyde for 10 minutes, treated with 0.3% hydrogen peroxide, permeabilized with 0.05% Triton X-100 and then incubated for 1 hour at room temperature with the appropriate dilution of each Ab. For the detection of EGFR in human tissues and of TGF-, CCL5, CCL2, and CXCL10 in the mouse, we used Abs from Santa Cruz Biotech (Santa Cruz, CA). TGF- in human tissues was detected with a mAb from Oncogene (Darmstadt, Germany). Abs against human CXCL8 and CXCL10 were from R&D Systems. Anti-mouse CD4, CD8, and CD11b Abs were from BD PharMingen (San Diego, CA). Phospho-ERK1/2 was detected in mouse skin sections with an Ab from Cell Signaling Technologies (Beverly, MA). Secondary biotinylated Abs and staining kits were from Vector Laboratories (Burlingame, CA). Immunoreactivity was revealed using avidin-biotin-peroxidase system and 3-amino-9-ethylcarbazole as chromogen. Sections were counterstained with Mayer’s hematoxylin. As negative controls, primary Abs were omitted or replaced with isotype-matched Ig. Infiltrating cells positive for CD4, CD8, or CD11b were counted in high power fields at x1000 and expressed as cells per area unit ± SD (n [microscopic fields per section] = 6).
Keratinocyte Cultures
Primary cultures of normal human keratinocytes were obtained as described previously.21 Keratinocytes were routinely grown in serum-free Keratinocyte Growth Medium (Clonetics, San Diego, CA), prepared from the essential nutrient solution Keratinocyte Basal Medium supplemented with 10 ng/ml EGF, 0.4 µg/ml hydrocortisone, 2 ml bovine pituitary extract and antibiotics. In the 24 hours preceding the experiments, 80% confluent keratinocyte cultures were switched to EGF-depleted medium.
Organ Cultures of Normal Human Skin
Five mm3 punches of normal human skin from three healthy donors were placed in Keratinocye Basal Medium with 0.1% normal human serum in a humidified incubator at 37°C, with enough medium to just cover the explants. Cultures were treated or not with IFN- (1000 units/ml, 24 hours) following 30 minutes of pre-incubation with PD168393 (2 µmol/L) or its vehicle (0.025% dimethyl sulfoxide (DMSO)). After experiments, the explants were snap-frozen.
TUNEL Staining
TUNEL staining was done on paraformaldehyde-fixed keratinocyte monolayers or paraffin-embedded sections of human and mouse skin using the In Situ Cell Death Detection Kit AP (Boehringer Mannheim, Mannheim, Germany), following the manufacturer’s instructions. For the evaluation of apoptosis in confluent keratinocyte monolayers, TUNEL+ cells were counted in high power fields at x1000 and expressed per 103 cells ± SD (n [microscopic fields in a chamber slide] = 6). In human whole-skin explants and in mouse skin sections, apoptosis was quantified as TUNEL+ cells/500 basal epidermal cells ± SD (n [microscopic fields per section] = 6). The data were confirmed in three independent experiments.
Flow Cytometry Analysis
Keratinocytes were treated with TNF-, IFN-, or medium alone for 15 minutes at 37°C. Cells were then incubated with anti-TGF- ectodomain-specific Ab (R&D Systems) or isotype-matched control Ab for 30 minutes on ice, followed by FITC-conjugated rabbit anti-goat Ab (BD PharMingen) for 20 minutes and finally analyzed with FACSCalibur (BD PharMingen).
Enzyme-Linked Immunosorbent Assay (ELISA)
TGF- was measured in cell-free supernatants using a kit from Oncogene. Chemokines were measured with dedicated kits from BD PharMingen.17
Data are expressed as nanograms per 106 cells ± SD from six independent experiments.
Immunoprecipitation and Western Blot Analysis
Keratinocytes were lysed by adding 1 ml per 10-cm dish of lysis buffer (20 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 1% Triton X-100, 1 mmol/L EDTA, 1 mmol/L phenylmethylsulphonyl fluoride, 0.15 units/ml aprotinin, 1% leupeptin, 1% pepstatin, 1 mmol/L NaF, and 1 mmol/L Na3VO4). EGFR was immunoprecipitated using anti-EGFR Ab from Santa Cruz Biotech, and phospho-EGFR was immunodetected using anti-phospho-tyrosine (PY) Ab from Transduction Laboratories (BD PharMingen). Anti-ERK and phospho-ERK Abs were from Cell Signaling Technologies. Anti-IFN-R1, phospho-IFN-R1, TNFR1, and TRAF2 Abs were from Santa Cruz Biotech.
RNase Protection Assay
Total RNA was extracted from cultured keratinocytes using the Trizol reagent (Invitrogen, Carlsbad, CA). The templates of the human chemokines under investigation and of the housekeeping molecule L32, and the kit for RNase protection assay were purchased from BD PharMingen. Total RNA was hybridized overnight with -32ATP-labeled cDNA templates, and reactions were performed as described.17
RNase protection assay bands were quantified by using laser densitometry supported by the Quantity One software (Bio-Rad, Hercules, CA). The densitometry value of each chemokine mRNA signal was normalized to the value of the corresponding L32 mRNA signal, and expressed as multifold increase (or decrease) as compared to its respective control, or as relative densitometric units ± SD from four independent experiments.
Contact Hypersensitivity Assay
BALB/c mice (Charles River Italia, Calco, Italy) were sensitized by application of 30 µl of 0.5% 2,4-dinitrofluorobenzene (DNFB) (Sigma-Aldrich, Milan, Italy) in acetone/olive oil (4/1) on a 2-cm2 area of the shaved abdomen.19 Five days later, sensitized and unsensitized animals received 10 µl of 0.15% DNFB on each side of each ear. In selected groups, 10 µl of 4 mmol/L PD168393 dissolved in DMSO/absolute ethanol (1/10 v/v), or 10 µl of the vehicle alone were painted on each side of each ear of sensitized and unsensitized mice 30 minutes before challenge. At least five mice per group were used in each experiment. Ear thickness was measured before challenge and in the following 3 days. Data are expressed as the change (from pre-challenge levels) in ear thickness x10-3 inches and represent the mean increase ± SEM. In each experimental group, some mice were sacrificed 48 hours after challenge, and the ears were cut and either paraffin-embedded or snap-frozen for hematoxylin & eosin staining or immunohistochemistry, respectively.
Statistics
The Wilkoxon signed-rank test was applied to compare differences in chemokine gene expression and protein release by cultured keratinocytes. In the statistical evaluation of apoptosis in cultured keratinocytes and in mouse experiments, unpaired Student’s t-test was used to compare the differences between means. Significance was assumed at a P value of 0.05 or less.
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Are Overexpressed in Chronic Inflammatory Skin Disorders
EGFR immunoreactivity could be localized throughout the whole epidermis of normal skin from healthy controls as previously reported,22
although it was more accentuated at the basal cell layer (Figure 1A)
. In parallel with the increased epidermal thickness, we observed a prominent EGFR expression extended to suprabasal keratinocyte layers, not only in psoriasis as previously documented,8
but also in lesional skin of atopic dermatitis and allergic contact dermatitis (Figure 1, B–D)
. Sparse EGFR-positive cells could be detected in the dermis (Figure 1, A–D)
. In normal skin, TGF- stained basal keratinocytes quite faintly (Figure 1E)
. In contrast, accentuated TGF- immunoreactivity was detected throughout basal and suprabasal keratinocyte layers in psoriasis, atopic dermatitis, and allergic contact dermatitis (Figure 1, F–H)
. Moreover, the inflammatory infiltrate displayed intense TGF- staining (Figure 1, F–H)
, suggesting that it represents a relevant source of this growth factor in the inflamed skin.
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and IFN-
Both TNF- and IFN- induce TGF- gene expression in keratinocytes.13,14
By FACS analysis, we observed that both cytokines also induced a rapid TGF- shedding from keratinocyte membranes. Compared to untreated cells, TNF- (100 ng/ml, 15 minutes) induced the loss of 55 ± 9.50% cell-associated fluorescence, whereas a 70 ± 15.25% decrease was caused by IFN- (100 units/ml, 15 minutes) (n = 6; Figure 2A
). Cytokine-induced shedding of TGF- was efficiently prevented by cell pre-incubation with the broad-range metalloproteinase inhibitor Ilomastat23
(25 µmol/L, 30 minutes) (Figure 2B)
, and was paralleled by over 10-fold increase in soluble TGF- (Figure 2C)
. Both cytokines (30 minutes) also increased the levels of EGFR tyrosine phosphorylation, which was efficiently prevented by cell pre-incubation (30 minutes) with PD168393 (2 µmol/L), a selective inhibitor of EGFR/c-erbB2 tyrosine kinase24
(Figure 3A)
. EGFR phosphorylation promoted by TNF- or IFN- could be attenuated by pre-incubation with neutralizing anti-TGF- Ab (10 µg/ml) or Ilomastat, but not by the PDGF receptor-specific tyrphostin AG129625
(2 µmol/L) (Figure 3A)
. Finally, PD168393 totally prevented both TNF-- and IFN--induced ERK1/2 phosphorylation at the time point of their maximal effect on ERK activation (15 minutes; data not shown) as well as at 30 minutes (Figure 3B)
, suggesting that EGFR transactivation occurred during keratinocyte response to these cytokines. Treatment with PD168393 did not interfere with IFN--induced phosphorylation of IFN-R1 or with TRAF2 recruitment by TNFR1 following TNF- (Figure 3B)
. Finally, presence of 0.025% DMSO in the medium, used as vehicle for the three pharmacological inhibitors, did not affect signals compared to medium alone (not shown).
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Modulates Chemokine Expression in Keratinocytes
Keratinocytes are usually cultured in the presence of 10 ng/ml EGF. To test whether EGFR activation was involved in the control of chemokine expression by keratinocytes, they were starved of EGF for 24 hours before treatment with TNF- (100 ng/ml) or IFN- (100 units/ml) alone and together with TGF- (50 ng/ml). When combined to TNF- (Figure 4A
, left panels), TGF- invariably induced a prominent down-regulation of CCL2, CCL5, and CXCL10 mRNA at the time points examined. We also registered a twofold increase in the levels of CXCL8 mRNA at 2 hours and 4 hours, but no detectable influence at 8 hours. In contrast, the effects of TGF- on IFN--driven CCL2, CCL5, and CXCL10 gene expression could be reproducibly observed only after 8 hours co-stimulation (Figure 4A
, right panels). At this time point, TGF- induced a three- to fivefold decrease of IFN--induced expression of CCL2 and CCL5, whereas CXCL10 signal underwent a twofold decrease (from 55.05 ± 7.5 to 23.50 ± 8.25 relative densitometric units, n = 4, P < 0.05). Finally, in contrast to what was observed with TNF-, TGF- induced a persistent up-regulation on IFN--induced CXCL8-specific signal, with an increase from 0.50 ± 0.55 to 2.50 ± 0.50 relative densitometric units (n = 4, P < 0.05) at 8 hours co-stimulation. In association to either TNF- (4 hours) or IFN- (8 hours), TGF- displayed a dose-dependent activity at the mRNA (Figure 4B)
and at the protein level, as measured by ELISA in the supernatants following 24 hours of stimulation (Figure 5)
. TGF- also dose-dependently reduced and augmented basal CCL5 and CXCL8 expression, respectively (Figure 4B
and Figure 5
) The effects of TGF- on cytokine-driven chemokine protein release were significant (P < 0.05) at concentrations higher than 5 ng/ml.
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We observed that pharmacological impairment of EGFR signaling perturbed keratinocyte response to both cytokines with opposite effects compared to TGF- (Figure 6)
. In particular, CCL2, CCL5, and CXCL10 mRNA levels were three to five times higher when stimulation with TNF- (2 hours) was performed following 30 minutes pre-incubation with TGF- neutralizing Ab (10 µg/ml) or Ilomastat (25 µmol/L). Inhibition of EGFR kinase activity by PD168393 (2 µmol/L) induced a four- to sevenfold increase in CCL2 and CXCL10 mRNA levels. In contrast, CXCL8 mRNA was significantly reduced by EGFR blockade (Figure 6
, left panels). Similar effects could be observed with IFN- stimulation (8 hours). Indeed, PD168393 (or Ilomastat) pre-treatment induced three to four times higher CCL2 and CCL5, two times higher CXCL10 (130.05 ± 5.75 vs. 54.50 ± 8.55 relative densitometric units; n = 4, P < 0.05), but down-regulated CXCL8 mRNA levels when compared to IFN- alone (Figure 6
, right panels). ELISA on 24 hours supernatants showed that PD168393 significantly affected constitutive and cytokine-induced chemokine release, both at 0.2 and 2 µmol/L concentration (Figure 7
; P < 0.05). Of note, the profile of TNF-- or IFN--induced chemokine release was similarly perturbed by 0.2 µmol/L PD168393 or 25 µmol/L Ilomastat (data not shown). Moreover, the effects of PD168393 were closely reproduced by a distinct EGFR tyrosine kinase inhibitor, PD153035,2
both at the mRNA and protein level (data not shown). Neither the unrelated tyrphostin AG1296 (Figure 6
and Figure 7
) nor 0.025% DMSO (not shown) could affect chemokine expression as compared to culture medium. Experiments performed on organ cultures of normal human skin confirmed that PD168393 (2 µmol/L) increased CXCL10 and reduced CXCL8 immunoreactivity in the epidermis after a 24-hour stimulation with IFN- (1000 units/ml) (Figure 8)
. Also TGF- expression was impaired by PD168393, in agreement with previous evidence that TGF- de novo synthesis is principally governed by TGF- itself through autocrine EGFR stimulation.26
In the intact epidermis of whole-skin explants, TUNEL+ keratinocytes were not detected either with or without PD168393 treatment, whereas the mean number of TUNEL+ keratinocytes in confluent chamber slides was 4.25 cells ± 3.50 in PD168393-treated cultures, as compared to 4.05 cells ± 2.50 in vehicle-treated controls (per 102 cells ± SD; n [microscopic fields per chamber slide] = 6), with no significant increase in keratinocyte apoptosis index (P = 0.5). Similar results were obtained in three independent experiments.
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We next investigated the impact of EGFR-linked signaling impairment in the expression of a prototypic T cell-mediated skin inflammation in vivo. Sensitized mice treated with a single application of PD168393 (10 µl of 4 mmol/L solution on each side of each ear) 30 minutes before hapten challenge exhibited a marked increase in contact hypersensitivity response to DNFB compared to vehicle-treated groups at all time points (P < 0.02) (Figure 9A)
. Also, PD168393 significantly augmented the modest edema evoked by DNFB in unsensitized mice at 24 hours and 48 hours (P < 0.02) (Figure 9A)
, although painting of PD168393 alone did not affect ear swelling or histological features in respect to vehicle (Figure 9, B and C)
. Increased contact hypersensitivity following PD168393 treatment was associated with stronger edema and heavier inflammatory infiltrate as compared to vehicle-treated ears (Figure 9, D and E)
, as well as enhanced epidermal immunoreactivity for CXCL10, CCL2, and CCL5, but fainter TGF- (Figure 10A)
. In the mouse skin treated with PD168393 we could observe a prominent reduction of epidermis-associated phospho-ERK1/2, which intensely stained keratinocyte nuclei in vehicle-treated sensitized skin (Figure 10A)
. Finally, CXCL8 was hardly detectable in the epidermis in all experimental conditions, in keeping with previous reports.18,19
To identify the cell types more numerously recruited in PD168393-treated skin, we counted the number of infiltrating CD8+, CD4+, and CD11b+ cells. We measured a marked increased number of CD4+ T lymphocytes and CD11b+ leukocytes, but not of CD8+ T cells at the site of PD168393 application (P < 0.02 vs. vehicle-treated skin) (Figure 10B)
. Finally, no evidence of increased keratinocyte apoptosis could be observed after administration of the inhibitor. In PD168393-treated skin, 11.50 ± 2.35 TUNEL+ cells were found in the epidermis compared to 10.25 ± 4.90 TUNEL+ cells in the vehicle-treated skin (per 500 basal cells, n [microscopic fields per section] = 6, P = 0.5). Three independent experiments provided similar values.
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Discussion |
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were overexpressed not only in psoriasis,8-10
but also in chronic atopic dermatitis and allergic contact dermatitis. Moreover, leukocytes showed strong TGF- immunoreactivity in chronically inflamed skin. TNF- and IFN- are released by T cells and other inflammatory cells in these disorders, and are potent inducers of EGFR and TGF- gene expression in keratinocytes.13,14
Of note, we found that both TNF- and IFN- also promoted a rapid metalloproteinase-mediated TGF- shedding from keratinocyte membrane, indicating that a "triple-membrane-passing signal mechanism"31
contributed to early cytokine-induced EGFR transactivation. A variety of proinflammatory stimulants are now known to affect epithelial cell functions through this mechanism.23,31,32
In our system, TGF- neutralization prevented cytokine-induced EGFR phosphorylation only partially, suggesting that other EGFR ligands could possibly participate in this process. Indeed, we collected some evidence that both TNF- and IFN- triggered the early release of the mature forms of other EGFR ligands, including heparin binding EGF-like growth factor and amphiregulin, although at lower levels than TGF- (data not shown). However, the observation that the broad-range metalloproteinase inhibitor Ilomastat was not able to prevent EGFR phoshorylation as effectively as the specific inhibitor of its receptor tyrosine kinase PD168393, possibly indicates that both cytokines may act on EGFR activation state also via EGFR ligand-independent, intracellular pathways. The contribution of a ligand-independent, redox-sensitive EGFR transactivation mechanism in the process of NF
B activation in response to TNF- has been documented in a number of cell types, including the skin carcinoma A431 cells.33
In keratinocytes, the early activation of EGFR-driven signaling cascades by both TNF- and IFN- appeared involved in the down-regulation of CCL2, CCL5, and CXCL10, suggesting that this loop is physiologically switched on to modulate the expression of critical proinflammatory mediators, and eventually to dampen skin inflammation. At the mRNA level, EGFR-mediated down-regulation of CCL2, CCL5, and CXCL10 expression varied substantially in its kinetics according to the cytokine used, appearing early (2 hours) with TNF-, and more delayed (8 hours) with IFN-. CXCL10 expression is maximally induced by IFN-,34
which causes the release of very high levels of this chemokine. Of note, TGF- significantly impaired CXCL10 expression in response to IFN- both at the mRNA and protein level, although with less efficiency in comparison to what was observed with TNF-. In contrast, an early EGFR-dependent potentiation of CXCL8 mRNA expression was evident with both cytokines. The increased CXCL8 expression following EGFR activation can be interpreted as a further mechanism of tissue repair, since CXCL8 is a well-recognized autocrine growth factor for epithelial cells.35
Conversely, impairment of EGFR signaling led to opposite chemokine expression profile, with enhanced CCL2, CCL5, and CXCL10, but reduced CXCL8. Consistent with our in vitro findings, skin application of the EGFR blocker PD168393 before antigen challenge led to exaggerated contact hypersensitivity in the mouse, with increased expression of keratinocyte-associated CCL2, CCL5, and CXCL10 and higher numbers of CD4+ T cells and CD11b+ leukocytes. The latter comprise cells of the monocyte/macrophage lineage and activated polymorphonucleates, and are strongly represented in the lesional skin at the time point of maximal contact hypersensitivity response.18-20
EGFR activation encompasses the involvement of a number of signaling cascades implicated in immediate-early gene expression.36,37
In particular, EGFR control on cell cycling, motility, and rescue from apoptosis is largely mediated by the potent MEK1/2-ERK1/2 activation induced by EGFR ligands.38-40
Indeed, we have observed that the selective abrogation of ERK1/2 performed by two chemically unrelated inhibitors of MEK1/2 activity closely reproduced the effects of the EGFR blocker PD168393 on chemokine expression in keratinocytes (Mascia F, Dattilo C, Girolomani G, Pastore S, manuscript in preparation). In keratinocyte cultures, PD168393 completely prevented the increase of ERK1/2 phosphorylation due to either TNF- or IFN- stimulation. More importantly, we could observe a strong PD168393-associated reduction of phospho-ERK1/2 immunostaining in the epidermis of contact sensitized mice, 2 days after local painting of this EGFR receptor tyrosine kinase inhibitor. The mechanisms through which abrogation of MEK1/2-ERK1/2 signaling may induce increased CCL2, CCL5, and CXCL10 synthesis are unclear at present. Independent reports have proposed an inhibitory cross-talk between ERK1/2 and p38 MAPK, which could explain an increase in p38 MAPK-dependent gene expression following MEK1/2 blockade.41,42
Experiments are now underway to evaluate the reciprocal role of the distinct MAPKs in chemokine expression.
EGFR appears to be strongly involved in the control of epithelial cell-triggered inflammation by down-regulating the expression of CCL2, CCL5, and CXCL10, which attract diverse leukocyte subsets. On the other hand, inhibition of EGFR signaling may be exploited to augment the inflammatory response initiated by immunological mechanisms. EGFR blockade by using either anti-EGFR antibodies or EGFR-specific tyrphostins has emerged as a very efficacious strategy against epithelial cell tumors,43 an activity that has been attributed mainly to neutralization of the proliferative functions mediated by EGFR.4-7 In addition, prolonged exposure to EGFR-specific tyrphostins has been shown to induce apoptosis in keratinocytes not only in culture systems7 but also in the skin of patients undergoing anti-EGFR anti-cancer therapy.43 Our results indicate that inhibition of EGFR signaling might exert anti-cancer activity by favoring the recruitment of inflammatory cells and thus a more pronounced anti-tumor immune response, along with down-regulation of CXCL8, which is an important growth factor for malignant epithelial cells.
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Footnotes |
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Supported by the Italian Ministry of Health.
Accepted for publication April 10, 2003.
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References |
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converting enzyme (TACE) regulates epidermal growth factor receptor ligand availability. J Biol Chem 2002, 277:12838-12845 in psoriatic epidermis. Science 1989, 243:811-814 is widely expressed in differentiated as well as hyperproliferative skin epithelium. J Invest Dermatol 1991, 96:328-332[Medline]
during epidermal development and differentiation. Genes Dev 1991, 5:714-727 in the epidermis of transgenic mice elicits hyperplasia, hyperkeratosis, and spontaneous, squamous papillomas. Cell Growth Differ 1993, 4:1071-1082[Abstract]
and the epidermal growth factor receptor in neonatal skin explants. J Invest Dermatol 1992, 99:350-356[Medline]
and transforming growth factor in normal human epidermal keratinocytes and squamous carcinoma cells. J Biol Chem 1999, 274:29138-29148 producing T cells in psoriatic lesions: localization to both CD4+ and CD8+ subsets. J Invest Dermatol 1998, 111:1072-1078[Medline]
cleavage. J Biol Chem 1996, 271:3279-3284. Am J Respir Cell Mol Biol 2002, 26:430-437B activation. J Biol Chem 2001, 28:25953-25958
B (NF-B)-inducing kinase to activate NF-B. J Biol Chem 2001, 276:8865-8874This article has been cited by other articles:
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S. Blanchet, K. Ramgolam, A. Baulig, F. Marano, and A. Baeza-Squiban Fine Particulate Matter Induces Amphiregulin Secretion by Bronchial Epithelial Cells Am. J. Respir. Cell Mol. Biol., April 1, 2004; 30(4): 421 - 427. [Abstract] [Full Text] [PDF]
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) or IFN-
) or vehicle alone (10% DMSO in absolute ethanol) (•). DNFB challenge solution was also applied to non-sensitized mice pre-treated with PD168393 (
) or vehicle alone (
) (A). Data represent the mean of the mean changes in ear thickness at each data point from five different experiments. *, P < 0.02 vs. the respective vehicle-treated group. Histological features of ear skin samples collected 48 hours after painting unsensitized mice with vehicle alone (B) or PD168393 (C), or 48 hours after DNFB challenge of sensitized mice pre-treated with vehicle alone (D) or PD168393 (E). Ear sections were stained with hematoxylin & eosin. Magnification, x100.