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,
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Departments of Dermatology and Pediatrics,
Herman B. Wells Center for Pediatric Research, and
Howard Hughes Medical Institute, Indiana University School of Medicine, Indianapolis, IN 46202;
§
Department of Pediatrics, National Jewish Center for Immunology and Respiratory Medicine, Denver, CO 80206; and
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Department of Pharmacology, Ohio State University College of Medicine, Columbus, OH 43210
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
IL-1, IL-6, IL-8 (3, 9, 10), and eicosanoids (11).
The perception of the role of the keratinocyte in cutaneous
inflammation has changed from one of a passive bystander to that of an
active participant. This new understanding of the part that the
keratinocyte plays in cutaneous pathophysiology is supported by the
ability of these cells to synthesize numerous proinflammatory
cytokines, chemokines, and growth factors. Human keratinocytes
synthesize TNF-, IL-1, IL-6, IL-8, as well as PGs (12). Recent
evidence suggests that many of these keratinocyte-derived mediators can
traverse the basement membrane and have systemic effects
(13).
Cyclooxygenase (COX) catalyzes the first committed step in prostanoid synthesis (reviewed in 14 . There are two separate COX enzymes, both of which are found in epidermal cells. COX-1 is constitutively expressed and synthesizes PGs in the endoplasmic reticulum. This isozyme is thought to mediate physiologic responses requiring a rapid and/or constant biosynthesis of PGs. The second isozyme, COX-2, is normally absent from cells, but can be induced in response to cytokines, growth factors, and tumor promoters. Reports of phenotypes of transgenic mice deficient in either COX-1 or COX-2 suggest that these isozymes have different functions (15, 16). The role of COX enzymes in keratinocyte biology is unclear, although treatment of murine epidermis with irritants has been reported to induce COX-2 (17). Several reports that COX-2 has been found to be overexpressed in some skin- and colon-derived carcinomas suggest that this enzyme might be involved in epithelial carcinogenesis (18, 19).
The pleotropic cytokines IL-6 and IL-8 have been implicated in
epidermal function and cutaneous inflammation (reviewed in 20 .
Resting keratinocytes do not synthesize appreciable amounts of IL-6 or
IL-8, yet will do so in the presence of IL-1, IL-1ß, TNF-, or
PMA. Keratinocytes express functional receptors for these cytokines,
and both IL-6 and IL-8 treatments have been reported to induce
keratinocyte proliferation in vitro (20, 21). In addition, increased
expression of keratinocyte IL-8 (CXC-2) receptors has been recently
demonstrated in lesions of psoriasis, a hyperproliferative disease with
prominent neutrophil involvement (22).
Human keratinocytes also synthesize PAF (23, 24, 25). In addition to synthesizing PAF and other sn-2 acetyl GPCs, keratinocytes express functional PAF-Rs (26). That human keratinocytes are potential targets of PAF action has significance, since PAF has been found in association with inflammatory skin diseases, including psoriasis (27) and urticaria (28). The functional significance of the keratinocyte PAF-R is not clear. However, the recent report of dermatitis and epidermal hyperplasia as part of the phenotypic abnormalities seen in the PAF-R-overexpressing transgenic mouse (29) suggests that the PAF-R may be involved in cutaneous inflammation. One possible mechanism for PAF-R modulation of cutaneous inflammation could be through PAF-induced keratinocyte cytokine biosynthesis. The ability of keratinocytes to synthesize cytokines in response to PAF-R activation has not been previously examined.
To study the consequences of PAF-R activation in epidermal cells, we have created a model system of the PAF-R by retroviral-mediated gene transduction of the PAF-R-negative human epidermal cell line KB with the human leukocyte PAF-R. The present studies use this novel model system and the human keratinocyte-derived cell line HaCaT (30) to assess the ability of the epidermal PAF-R to stimulate the biosynthesis of COX-2 as well as the cytokines IL-6 and IL-8. Finally, the ability of IL-8 to trigger PAF biosynthesis in epidermal cells was also tested. An understanding of sequelae of the activation of the epidermal PAF-R may provide insights into the role of this lipid mediator in epidermal cell pathophysiology as well as its place in the epidermal cytokine network.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Solvents (HPLC grade ethanol, methanol, chloroform, hexane, and acetic acid) and reagent grade diethyl ether were obtained from Fisher (Fairlawn, NJ). PAF (1-O-hexadecyl-2-acetyl-GPC), 1-hexadecyl-2-N-methyl carbamoyl-GPC (CPAF), PMA, and fatty acid-free BSA were purchased from Sigma (St. Louis, MO). The PAF-R antagonists were provided as follows: WEB 2086 (31) from Boehringer Ingelheim (Ridgefield, CT), and A-85783 (32) from Dr. James Summers, Abbott Pharmaceuticals (Abbott Park, IL). The peptide growth hormone endothelin-1 (ET-1) was purchased from Peptides International (Louisville, KY). Ethanolic solutions were made with CPAF and WEB 2086; PMA and A-85783 were dissolved in DMSO. Appropriate controls used ethanol and DMSO (final concentrations of all solvents were <0.5%).
KB PAF-R model system
KB cells, a human epidermoid carcinoma originally obtained from a patient with an oral squamous cell carcinoma (33), and the PAF-R-positive (26) human keratinocyte-derived epidermal cell line HaCaT (30) were grown as previously described (24, 26). To create a model system of the PAF-R, the entire human leukocyte PAF-R cDNA (provided by Dr. Takao Shimizu, Tokyo, Japan) was cloned into the EcoRI site of the multiple cloning region of the MSCV2.1 retroviral vector (34), and orientation was assessed by restriction endonuclease mapping and sequencing. Infectious amphotropic retroviruses were produced from both MSCV2.1PAF-R and control MSCV2.1 backbone using standard protocols (35). Briefly, the murine ecotropic packaging line GP+ E-86 (E-86) (36) was transfected with the MSCV2.1 construct, and transient supernatants collected 48 h later containing infectious virions were then used to infect the amphotropic packaging line GP+ EnvAm12 (Am-12) (37). Individual colonies of Am-12 were selected with G418 (0.75 mg dry powder/ml; Life Technologies, Grand Island, NY)-containing medium. Viral titers from selected clones were evaluated by infection of NIH-3T3 fibroblasts using dilutions of virus containing supernatants from multiple clones. Clones producing high titer virus and infected and G418-selected NIH-3T3 cells were further analyzed by Southern blot analysis to confirm the integrity of the provirus. Two clones, producing 105 G418-resistant colony-forming units/ml (Am12 MSCV2.1PAF-R virus) and 106 colony-forming units/ml (Am12 MSCV2.1) of virus, were used to infect KB cells, and clones of transduced KB cells resistant to 1 mg/ml G418 were further characterized by Southern and Northern blot analyses. All experimental protocols were duplicated in at least two separate clones of KBPAF-R (KBP) and two KBMSCV2.1 (KBM) cells.
Genomic DNA isolation and Southern blot analysis
Genomic DNA was isolated from the cultured cells by the method of Sambrook and Maniatus (38) with modification. Briefly, after washing with PBS (Life Technologies, Grand Island, NY) cells were lysed with extraction buffer (10 mM Tris-HCl, 0.1 M EDTA, 0.5% SDS, 20 µg/ml pancreatic RNase, and 100 µg/ml proteinase K (Boehringer Mannheim, Indianapolis, IN)) for >1 h. Following extraction with phenol (pH 8.0), genomic DNA was precipitated with 2 vol of ethanol. After centrifugation, the pellet was washed with 70% ethanol, air-dried, and dissolved in autoclaved sterile water. Ten micrograms of DNA was digested overnight with the appropriate restriction endonuclease, run on an 0.8% agarose gel, and transfered to a positively charged nylon membrane (Boehringer Mannheim). The blot was then probed with 32P-labeled human PAF receptor cDNA probe, or NEO, using the random primed labeling method (Boehringer Mannheim). ExpressHyb Hybridization Solution (Clontech, Palo Alto, CA) was used for both prehybridization and hybridization at 50°C. After hybridization, the blot was washed following the manufacturer’s recommendation: 2x SSC-0.05% SDS at room temperature twice for 15 min each time, and 0.2x SSC-0.1% SDS at 50°C twice for 20 min each time. The blot was then exposed to an x-ray film with two intensifying screens at -80°C.
Total RNA extraction and Northern blot analysis
Total RNA was extracted using Tripure (Boehringer Mannheim). After the manufacturer’s procedures of purification, an additional phenol (pH 4.2) extraction was performed, followed by ethanol precipitation. The RNA concentration was accessed by UV spectrophotometer. Twenty micrograms of total RNA was separated on a formaldehyde containing gel following the method reported by Sambrook and Maniatis (38) and transfered to a positively charged nylon membrane (Boehringer Mannheim). The blots were then hybridized with various 32P-labeled cDNA probes using ExpressHyb Hybridization Solution (Clontech) following the manufacturer’s protocol. The blots were stripped of the probe using 0.5% SDS at 100°C and later probed with human GAPDH to determine equal loading. Human IL-6 (no. 68636) and GAPDH (no. 57091) cDNA clones were purchased from American Type Culture Collection (Rockville, MD); human IL-8, COX-1, and COX-2 clones were gifts from Dr. Jana Stankova (University of Sherbrooke, Sherbrooke, Canada) and were used as previously reported (39).
Radioligand binding studies
Radioligand binding studies using the water-soluble PAF-R antagonist [3H]WEB 2086 (New England Nuclear) were conducted that were similar to those previously described (40). Briefly, parental KB or clones of KB cells transduced with the MSCV2.1 control or MSCV2.1PAF-R virus were seeded onto 24-well tissue culture treated dishes (Falcon, Oxnard, CA) at an initial density of 200,000 cells in 1 ml for 24 h. The cells were washed and treated with various concentrations (1–100 nM) of [3H]WEB 2086 in the presence or the absence of an excess of unlabeled WEB 2086 (2 µM) for 24 h at 4°C. After washing, the cells were treated with Triton X detergent (Sigma) and scraped, and radioactivity was measured by scintillation counting. No displaceable [3H]WEB 2086 binding was detected in either parental KB or KBM clones. Saturation binding isotherms of KBP clones revealed displaceable binding that reached saturation; Scatchard analysis (41) of the binding data revealed a single high affinity binding site. Three KBP clones with similar binding characteristics (Kd of 8.5–11.3 nM; binding capacity of 133–289 fmol/106 cells) were used in further studies. Radioligand binding studies of these clones in continuous passage (up to 60 passages) over several months were unchanged, indicating the stability of PAF-R protein expression.
Measurement of COX-2 protein in KB cells
Following incubation with the appropriate experimental treatments, cells were washed with cold PBS (Life Technologies) and lysed with 1 ml of lysis buffer (50 mM Tris-HCl, 1% SDS, 2 mM EDTA, and 2 mM PMSF, pH 7.5). The lysate was scraped and sonicated twice. The protein concentration was determined using the Bio-Rad DC Protein Assay Kit (Bio-Rad, Hercules, CA). Twenty-five micrograms of each protein sample was heat denatured, loaded onto a 10% SDS-PAGE gel (Bio-Rad minigel system), and run at 125 V through the stacking gel and 150 V until bromophenol dye run off. The proteins were then transfered to polyvinylidene difluoride Hybond-P transfer membrane (Amersham, Arlington Heights, IL), using a SD semidry electrophoretic cell (Bio-Rad). The membrane was stained with Ponceau Red (Sigma) to determine the effectiveness of transfer and to assess loading. Detection was performed using ECL-Plus (Amersham). Goat-anti COX-2 Ab (Santa Cruz Biotechnology, Santa Cruz, CA; SC 1745) was used at a dilution of 1/7,500. The secondary anti-goat IgG-horseradish peroxidase (SC 2033) was used at a dilution of 1/75,000. Competition assays were also performed using COX-2 control peptide (SC 1745) to ensure the specificity of the Ab.
Intracellular calcium measurements
Intracellular free calcium concentrations ([Ca2+]i) in suspensions of KB cells were assessed using the calcium-sensitive fluorescent dye indo-1/AM as previously described (24, 26).
Measurement of PAF production by epidermal cells
Cell-associated PAF and PAPC production in clones of KBP or KBM cells in response to CPAF and in HaCaT keratinocytes in response to IL-8 (PeproTek, Rocky Hill, NJ) were assessed by GC/MS exactly as previously described (24, 25).
Measurement of arachidonic acid (AA) release and PGE2 production by KB PAF-R cells
KB cells were grown in 10-cm dishes (Costar, Cambridge, MA) until they were approximately 80 to 90% confluent, then were washed three times with HBSS (Sigma) before addition of 10 ml of prewarmed 0.25% BSA in HBSS containing 100 nM of CPAF. At various times, 1 ml of supernatant was removed to measure AA by GC/MS (25) or PGE2 using a specific RIA (42). No more than 4 ml (total) was removed from an individual plate during an experiment. Similarly treated plates of cells were trypsinized and counted (Coulter, Hialeah, FL).
Measurement of IL-6 and IL-8 protein in KB cells
Cells were plated at a density of 100,000 cells in 1 ml on 24-well plates for 24 h, then exposed to media with or without drugs. In experiments using antagonists, cells were treated 1 h before exposure to agonists. The medium was collected 16 h after drug treatment, and IL-6 and IL-8 were assayed using Quantikine ELISA kits (R&D, Minneapolis, MN). Similarly treated cells were trypsinized and counted (Coulter).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As described in Materials and Methods, PAF-R-negative
KB cells (24, 26) were stably transduced with the human wild-type PAF-R
cDNA using the MSCV2.1 retrovirus (Fig. 1
A). The presence of an intact
provirus in transduced KB clones was confirmed by Southern blot
analysis (Fig. 1B), and expression of the integrated
provirus was demonstrated by Northern blot analysis (Fig. 1C). As outlined in Materials and Methods,
radioligand binding studies using the PAF-R antagonist
[3H]WEB 2086 also revealed PAF-R protein.
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D). Treatment of these cell types with 100 nM
of the peptide growth hormone ET-1 resulted in an intracellular calcium
flux, indicating that these cells have the appropriate cellular
machinery to respond to a G protein receptor-mediated signal (Fig. 1D). Treatment of KBP clones with 100 nM PAF resulted in
changes in [Ca2+]i similar to those described
in other PAF-R-expressing cell types (11, 26, 40). Lyso-PAF (1 µM)
treatment of KBP clones did not affect
[Ca2+]i, indicating that the response did not
result from the physical effects of a phospholipid (data not shown).
Pretreatment of KBP cells with the structurally distinct PAF-R
antagonists WEB 2086 (10 µM) and A-85783 (10 µM) inhibited 100 nM
PAF-induced calcium mobilization, yet had no effect on that induced by
100 nM ET-1 (data not shown). PAF biosynthesis in KB PAF-R cells
Activation of the PAF-R has been reported to induce PAF
biosynthesis in both neutrophils (43) and keratinocytes (24). We tested
the ability of the metabolically stable PAF-R agonist CPAF (43) to
stimulate PAF biosynthesis in our KBPAF-R model system. As shown in
Figure 2, CPAF treatment resulted in PAF
biosynthesis in KBP, but not KBM, cells. CPAF treatment of KBP cells
also induced the biosynthesis of comparable amounts of the PAF-R
agonist PAPC (data not shown).
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As PAF could be generated from AA-enriched 1-alkyl-2-acetyl-GPC
precursors, the ability of PAF-R activation to stimulate the release of
AA was next examined. As shown in Figure 3A, CPAF treatment of KBP
cells resulted in the rapid release of AA, with an initial time course
resembling that of PAF production (Fig. 2). However, unlike PAF, which
was metabolized to baseline levels, the AA content in the supernatant
remained elevated at 60 min. CPAF did not induce AA release in KBM
cells (Fig. 3A) or in parental KB cells (data not shown),
consistent with the lack of a functional PAF-R on these cells.
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B, CPAF treatment resulted in PGE2 accumulation
in KBP, but not KBM, clones. In contrast to CPAF-stimulated AA release,
which was maximal by 10 min, significant PGE2 accumulation
above baseline levels was not seen until after 1 h. COX-2 biosynthesis in KB PAF-R cells
Our finding that the time course of CPAF-induced PGE2
production lagged behind arachidonate release suggested the possibility
that PAF-R activation could induce COX-2. To test this, KBP or KBM
clones were treated with CPAF for various times, and total RNA was
isolated. As shown in Figure 4, CPAF
treatment of KBP cells resulted in an accumulation of COX-2 mRNA. The
increased COX-2 mRNA was first seen at 30 min and persisted at 6 h
after CPAF treatment. These findings contrast with the lack of effect
of CPAF treatment on KBM clones (Fig. 4). Treatment of the
PAF-R-positive (26) human keratinocyte cell line HaCaT with CPAF also
resulted in an increased accumulation of COX-2 mRNA (Fig. 4). CPAF
treatment did not result in an increase in mRNA encoding the COX-1
enzyme in KBP or HaCaT keratinocytes.
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, lane 5). CPAF-induced COX-2
protein was seen by 2 h and remained elevated at 24 h (data
not shown). As shown in Figure 5 (lane 7) for an 8-h
period, pretreatment of KBP cells with 10 µM of the PAF-R antagonist
A-85783 resulted in an inhibition of CPAF-induced COX-2 protein.
Similarly, preincubation of KBP cells with the PAF-R antagonist WEB
2086 (10 µM) inhibited subsequent CPAF-stimulated COX-2 protein (data
not shown). As expected, significant amounts of COX-2 protein were not
seen in KBM cells following CPAF treatment. However, treatment of these
cells with the phorbol ester PMA (10 nM) induced COX-2 protein (Fig. 5). Competition assays using a COX-2 control peptide demonstrated the
specificity of the Ab (data not shown). These studies indicate that
activation of the epidermal PAF-R results in increased COX-2 protein
biosynthesis.
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Keratinocytes have been shown to synthesize both IL-6 and IL-8 in
response to various stimuli, including phorbol esters and TNF- (12, 20, 21). The next experiments assessed the ability of PAF-R activation
to generate increased amounts of these cytokines. As shown in Figure 6, incubation of KBP, but not KBM, cells
with CPAF resulted in an increased accumulation of mRNA encoding both
IL-6 and IL-8. With both cytokines, increased mRNA was first seen at 30
min, was maximal at 1 h, and returned to baseline by 6 h.
Unlike KB cells, significant levels of both IL-6 and IL-8 mRNA were
detected in unstimulated HaCaT keratinocytes. However, CPAF treatment
of HaCaT keratinocytes resulted in an increased accumulation of mRNA
from these two cytokines above baseline levels with a time course
similar to that seen in KBP cells (Fig. 6).
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. CPAF
treatment of KBM cells did not result in an increase in IL-6 or IL-8
protein. However, treatment of KBM cells with PMA resulted in IL-6 and
IL-8 protein release. The PAF-R antagonists A-85783 (Fig. 7) and WEB
2086 (10 µM; not shown) inhibited CPAF-induced cytokine biosynthesis
in KBP cells, yet did not affect cytokine synthesis induced by PMA in
KBP. In addition, higher levels of IL-8 were consistently seen in
baseline (i.e., unstimulated) KBP cells compared with those in KBM
cells.
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Since IL-8Rs are linked to an intracellular calcium mobilization
response, which is a known stimulus for PAF biosynthesis, we next
examined whether IL-8 can act on epidermal cells to stimulate PAF
biosynthesis. As shown in Figure 8,
treatment of HaCaT keratinocytes with IL-8 (100 ng/ml) stimulated PAF
and PAPC production. IL-8-induced PAF and PAPC biosynthesis was seen at
1.5 min, was maximal by 2.5 min, and returned to baseline by 5 min. The
amounts of PAF and PAPC biosynthesis induced in these cells by IL-8
treatment at 2.5 min were 170 and 320 pg/106 cells,
respectively. These studies indicate that PAF and IL-8 can act to
stimulate the biosynthesis of each other in epidermal cells that
express receptors for both lipid mediator and chemokine.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although the majority of PAF effects are thought to be mediated by the PAF-R, PAF is metabolized to potentially biologically active molecules (5, 6, 7). Structurally similar lipids such as lysophosphatidylcholines have also been described as having biologic activity. For example, lysophosphatidylcholine has been reported to stimulate IL-6 release from rat anterior pituitary cells in vitro (44). Thus, PAF-R-independent effects exerted by PAF metabolites and other lipids might result in misleading findings attributable to activation of the PAF-R.
To overcome the potential problem of non PAF-R-induced PAF effects and
to account for the diverse non-PAF PAF-R agonists that can signal
through this receptor (45, 46, 47), our laboratory has developed the KB
PAF-R model system. Although often used as a model for human
keratinocytes because of a similar cytokine production profile, KB
cells differ from human keratinocytes and the epidermal cell lines
HaCaT and A-431 in that they do not express functional PAF-Rs. KB cells
do not express PAF-R mRNA (Fig. 1C) and lack PAF-R protein,
as shown by radioligand binding studies with [3H]PAF and
[3H]WEB 2086 (data not shown) and immunohistochemical
studies using a specific PAF-R polyclonal Ab (26). In addition, KB
cells do not respond to PAF-R agonists by an intracellular calcium
mobilization, AA release, or PAF biosynthesis (24). KB cells were
transduced with the human leukocyte PAF-R using a replication-deficient
retrovirus. To control for possible confounding effects due to the
integration of a retrovirus into genomic DNA, KB cells were also
transduced with the empty retrovirus alone (KBM). With both
PAF-R-negative and -positive cells, this unique KB PAF-R model system
can complement existing pharmacologic tools (i.e., PAF-R antagonists)
to study the effects of PAF-R.
Activation of the KB PAF-R resulted in the biosynthesis of PAF (Fig. 2). Similarly, our laboratory has shown that CPAF treatment of human
HaCaT keratinocytes results in PAF biosynthesis (24). The ability of
PAF-R activation to both induce PAF synthesis as well as increase
transcription of the PAF-R (48) indicates the existence of a positive
feedback loop resulting in enhanced PAF-R-induced effects in a cell
such as a keratinocyte that can both synthesize and respond to this
mediator. Positive feedback loops between PAF and the PAF-R may have
potential clinical significance, especially in populations exhibiting
decreased activity of the major PAF-metabolizing enzyme acetylhydrolase
(49).
Activation of the KB PAF-R resulted in AA release and PGE2
production (Fig. 3). Similarly, CPAF treatment of human keratinocytes
results in AA release (25). PAF treatment of feline tracheal cells (50)
and rabbit corneocytes (51) has previously been shown to increase
eicosanoid biosynthesis. In KBP cells, the lag time in PGE2
biosynthesis compared with that of arachidonate release suggested the
induction of COX-2. Indeed, PAF-R activation of KBP cells resulted in
increased COX-2 mRNA (Fig. 4) and protein (Fig. 5) expression.
Treatment of HaCaT keratinocytes with CPAF also resulted in an increase
in baseline COX-2, but not COX-1, mRNA levels, suggesting that the KB
PAF-R model system resembles the endogenous epidermal PAF-R. The fact
that unstimulated HaCaT keratinocytes were found to express COX-2 mRNA
yet KB cells did not (Fig. 4) is compatible with previous findings that
some carcinomas constitutively express COX-2 (18, 19).
Our studies with KB and HaCaT keratinocytes indicate that PAF-R activation alone is an adequate stimulus for COX-2 biosynthesis in epidermal cells. In contrast, PAF alone does not stimulate rat alveolar macrophage COX-2 biosynthesis, but can act to enhance LPS-stimulated COX-2 biosynthesis through transcriptional activation (39). The recent studies by Marcheselli and Bazan (52) indicate that pretreatment of rat brains with the PAF-R antagonist BN 50730 can inhibit kainic acid- and electroconvulsive shock-induced COX-2 induction. Their findings suggest involvement of the PAF-R in the regulation of this enzyme in vivo.
The significance of PAF-R-mediated PG production in epidermal cells is not clear. Depending upon the experimental conditions, PGE2 has been reported to have mitogenic effects (53) or induce differentiation of human keratinocytes (54). In addition to direct effects on keratinocytes, PGE2 can inhibit monocyte/macrophage function (55). Thus, epidermal cell-derived PGE2 could potentially modulate cutaneous immunity through its effects on Langerhans’ cells. Finally, the reported ability of COX inhibitors to enhance and of exogenous PGE2 to inhibit both macrophage PAF production (56) and PAF-R transcription (48) suggest that this eicosanoid could be an endogenous negative regulator of PAF function.
Activation of the epidermal PAF-R was also found to result in an increased accumulation of the cytokines IL-6 and IL-8. These data suggest that the epidermal PAF-R resembles the PAF-R in human lung fibroblasts that synthesize IL-6 and IL-8 in response to PAF challenge (3). These cell types differ from rat macrophages, as PAF alone does not stimulate IL-6 production but will augment muramyl dipeptide-induced IL-6 biosynthesis (57).
Both IL-6 and IL-8 have been reported to stimulate PAF
biosynthesis in human neutrophils (58, 59). As shown in Figure 8, IL-8
can also induce PAF biosynthesis in human epidermal cells. The amount
of cell-associated PAF produced in response to 100 ng IL-8 was 0.17
ng/106 cells at 2.5 min, which is less than what we have
previously reported for 1 µM A23187 or 250 nM CPAF stimulation (2.2
vs 1.7 ng/106 cells at 2.5 min, respectively) of HaCaT
keratinocytes (24). That IL-8 and PAF can both induce the biosynthesis
of each other indicates the possible presence of a "vicious cycle"
that may result in the propagation of the inflammatory response,
especially in situations characterized by the overexpression of
epidermal IL-8Rs (22) or lack of PAF metabolism (49).
These studies demonstrate that activation of the epidermal PAF-R results in increased biosynthesis of PAF, eicosanoids, COX-2, and the cytokines IL-6 and IL-8. These observations have significance, since both eicosanoids and these ILs have been implicated in keratinocyte biology and cutaneous inflammation. Together, these findings indicate that PAF could be an important member of the epidermal cytokine network in part through its ability to stimulate these other mediators. An understanding of the role of the PAF system in the cytokine network of the skin could have potential therapeutic implications given the availability of PAF-R antagonists.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jeffrey B. Travers, Herman B Wells Center for Pediatric Research, James Whitcomb Riley Hospital for Children, Room 2659, Indiana University School of Medicine, 702 Barnhill Dr., Indianapolis, IN 46202. E-mail address:
3 Abbreviations used in this paper: PAF, platelet-activating factor; COX, cyclooxygenase; GPC, glycerophosphocholine; PAF-R, platelet-activating factor receptor; CPAF, 1-hexadecyl-2-N-methyl carbamoyl-glycerophosphocholine; ET-1, endothelin-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; [Ca2+]i, intracellular Ca2+ concentration; PAPC, 1-palmitoyl-2-acetyl glycerophosphocholine; GC/MS, gas chromatography/mass spectrometry; KBP, KB cells transduced with platelet-activating factor receptor; KBM, KB cells transduced with MSCV2.1 retrovirus alone; AA, arachidonic acid.
Received for publication February 5, 1998. Accepted for publication April 14, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Y. Matsumura, S. N. Byrne, D. X. Nghiem, Y. Miyahara, and S. E. Ullrich A Role for Inflammatory Mediators in the Induction of Immunoregulatory B Cells J. Immunol., October 1, 2006; 177(7): 4810 - 4817. [Abstract] [Full Text] [PDF]
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 P. Wolf, D. X. Nghiem, J. P. Walterscheid, S. Byrne, Y. Matsumura, Y. Matsumura, C. Bucana, H. N. Ananthaswamy, and S. E. Ullrich Platelet-Activating Factor Is Crucial in Psoralen and Ultraviolet A-Induced Immune Suppression, Inflammation, and Apoptosis Am. J. Pathol., September 1, 2006; 169(3): 795 - 805. [Abstract] [Full Text] [PDF]
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 L. Cheng, W. Cao, C. Fiocchi, J. Behar, P. Biancani, and K. M. Harnett HCl-induced inflammatory mediators in cat esophageal mucosa and inflammatory mediators in esophageal circular muscle in an in vitro model of esophagitis Am J Physiol Gastrointest Liver Physiol, June 1, 2006; 290(6): G1307 - G1317. [Abstract] [Full Text] [PDF]
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V. O. Melnikova, A. A. Mourad-Zeidan, D. C. Lev, and M. Bar-Eli Platelet-activating Factor Mediates MMP-2 Expression and Activation via Phosphorylation of cAMP-response Element-binding Protein and Contributes to Melanoma Metastasis J. Biol. Chem., February 3, 2006; 281(5): 2911 - 2922. [Abstract] [Full Text] [PDF]
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G. K. Marathe, C. Johnson, S. D. Billings, M. D. Southall, Y. Pei, D. Spandau, R. C. Murphy, G. A. Zimmerman, T. M. McIntyre, and J. B. Travers Ultraviolet B Radiation Generates Platelet-activating Factor-like Phospholipids underlying Cutaneous Damage J. Biol. Chem., October 21, 2005; 280(42): 35448 - 35457. [Abstract] [Full Text] [PDF]
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M. Darst, M. Al-Hassani, T. Li, Q. Yi, J. M. Travers, D. A. Lewis, and J. B. Travers Augmentation of Chemotherapy-Induced Cytokine Production by Expression of the Platelet-Activating Factor Receptor in a Human Epithelial Carcinoma Cell Line J. Immunol., May 15, 2004; 172(10): 6330 - 6335. [Abstract] [Full Text] [PDF]
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S. D. Billings, M. D. Southall, T. Li, P. W. Cook, L. Baldridge, W. B. Moores, D. F. Spandau, J. G. Foley, and J. B. Travers Amphiregulin Overexpression Results in Rapidly Growing Keratinocytic Tumors: An In Vivo Xenograft Model of Keratoacanthoma Am. J. Pathol., December 1, 2003; 163(6): 2451 - 2458. [Abstract] [Full Text]
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T. Li, M. D. Southall, Q. Yi, Y. Pei, D. Lewis, M. Al-Hassani, D. Spandau, and J. B. Travers The Epidermal Platelet-activating Factor Receptor Augments Chemotherapy-induced Apoptosis in Human Carcinoma Cell Lines J. Biol. Chem., May 2, 2003; 278(19): 16614 - 16621. [Abstract] [Full Text] [PDF]
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 M. D. Southall, T. Li, L. S. Gharibova, Y. Pei, G. D. Nicol, and J. B. Travers Activation of Epidermal Vanilloid Receptor-1 Induces Release of Proinflammatory Mediators in Human Keratinocytes J. Pharmacol. Exp. Ther., January 1, 2003; 304(1): 217 - 222. [Abstract] [Full Text] [PDF]
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 J. E. Moore, T. C. B. McMullen, I. L. Campbell, R. Rohan, Y. Kaji, N. A. Afshari, T. Usui, D. B. Archer, and A. P. Adamis The Inflammatory Milieu Associated with Conjunctivalized Cornea and Its Alteration with IL-1 RA Gene Therapy Invest. Ophthalmol. Vis. Sci., September 1, 2002; 43(9): 2905 - 2915. [Abstract] [Full Text] [PDF]
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S. A. Marques, L. C. Dy, M. D. Southall, Q. Yi, E. Smietana, R. Kapur, M. Marques, J. B. Travers, and D. F. Spandau The Platelet-Activating Factor Receptor Activates the Extracellular Signal-Regulated Kinase Mitogen-Activated Protein Kinase and Induces Proliferation of Epidermal Cells through an Epidermal Growth Factor-Receptor-Dependent Pathway J. Pharmacol. Exp. Ther., March 1, 2002; 300(3): 1026 - 1035. [Abstract] [Full Text] [PDF]
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 J. P. Walterscheid, S. E. Ullrich, and D. X. Nghiem Platelet-activating Factor, a Molecular Sensor for Cellular Damage, Activates Systemic Immune Suppression J. Exp. Med., January 14, 2002; 195(2): 171 - 179. [Abstract] [Full Text] [PDF]
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M. D. Southall, J. S. Isenberg, H. Nakshatri, Q. Yi, Y. Pei, D. F. Spandau, and J. B. Travers The Platelet-activating Factor Receptor Protects Epidermal Cells from Tumor Necrosis Factor (TNF) alpha and TNF-related Apoptosis-inducing Ligand-induced Apoptosis through an NF-kappa B-dependent Process J. Biol. Chem., November 30, 2001; 276(49): 45548 - 45554. [Abstract] [Full Text] [PDF]
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 T. Lahiri, J. D. Laporte, P. E. Moore, R. A. Panettieri Jr., and S. A. Shore Interleukin-6 family cytokines: signaling and effects in human airway smooth muscle cells Am J Physiol Lung Cell Mol Physiol, June 1, 2001; 280(6): L1225 - L1232. [Abstract] [Full Text] [PDF]
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N. Merendino, M. B. Dwinell, N. Varki, L. Eckmann, and M. F. Kagnoff Human intestinal epithelial cells express receptors for platelet-activating factor Am J Physiol Gastrointest Liver Physiol, October 1, 1999; 277(4): G810 - G818. [Abstract] [Full Text] [PDF]
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L. C. Dy, Y. Pei, and J. B. Travers Augmentation of Ultraviolet B Radiation-induced Tumor Necrosis Factor Production by the Epidermal Platelet-activating Factor Receptor J. Biol. Chem., September 17, 1999; 274(38): 26917 - 26921. [Abstract] [Full Text] [PDF]
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