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and IL-4 or IL-13
From the Department of Ophthalmology, Yamaguchi University School of Medicine, Japan.
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Abstract |
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 Top Abstract IntroductionMethodsResultsDiscussionReferences |
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,
interleukin (IL)-4, and IL-13 on expression of the chemokine eotaxin by
cultured human keratocytes.
METHODS. Cultured human keratocytes were incubated with various combinations and
concentrations of TNF-, IL-4, and IL-13. The concentration of
eotaxin in the culture supernatant was subsequently measured by
enzyme-linked immunosorbent assay, and the amount of eotaxin mRNA in
cell lysates was determined by reverse transcription–polymerase chain
reaction analysis.
RESULTS. Keratocytes incubated in the absence of cytokines did not release
detectable amounts of eotaxin into the culture medium. Whereas
incubation of keratocytes with TNF-, IL-4, or IL-13 alone or with
the combination of IL-4 and IL-13 had only a small effect on eotaxin
release, exposure of the cells to TNF- in combination with either
IL-4 or IL-13 resulted in a marked increase in eotaxin production that
was both time and dose dependent. The abundance of eotaxin mRNA in
keratocytes was also increased in a synergistic manner by incubation of
cells with TNF- together with either IL-4 or IL-13.
CONCLUSIONS. Stimulation of human keratocytes with the combination of TNF- and
either IL-4 or IL-13 resulted in synergistic increases in both the
abundance of eotaxin mRNA and the release of eotaxin protein. This
cytokine-induced increase in eotaxin production by keratocytes may
contribute to eosinophil infiltration in inflammatory ocular diseases
such as vernal keratoconjunctivitis.
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Extravasation and local accumulation of eosinophils during allergic inflammation are regulated by specific chemokines such as RANTES (regulated on activation, normal T expressed and secreted) and eotaxin, the latter of which is a potent and specific eosinophil chemoattractant.3 Furthermore, the activation of accumulated eosinophils is modulated by stromal cells. Thus, coculture of eosinophils with lung fibroblasts increases the extent of eosinophil degranulation.4 The interaction between eosinophils and fibroblasts is an important regulatory step in the pathogenesis of allergic diseases.
Various types of cytokines are produced by inflammatory cells during
allergic reactions. The proinflammatory cytokines and T helper cell-2
(Th-2)–type cytokines play important roles in the pathogenesis of
allergic diseases.5
The proinflammatory cytokine tumor
necrosis factor (TNF)- is expressed by mucosal mast cells and is
released during acute allergic reactions.6
The Th-2 type
cytokine interleukin (IL)-4 induces immunoglobulin isotype switching in
B cells and maintains the production of IgE.7
Increased
concentrations of IL-48
and TNF-9
have
been detected in the tear fluid of individuals with VKC. IL-13 is
another Th-2–type cytokine that also induces IgE production and
modifies IgE-mediated allergic responses.10
Although the
presence of IL-13 in tear fluid has not been demonstrated, the
concentration of IL-13 in bronchoalveolar lavage fluid is increased in
allergen-challenged individuals with asthma.11
It is thus
possible that cytokines, especially TNF-, IL-4, and IL-13, modify
the functions of inflammatory and structural cells in the cornea of
individuals with VKC.
Previous studies have suggested that corneal keratocytes may release
chemokines and thereby contribute to the local accumulation of
eosinophils.12
13
Understanding the pathogenesis of
corneal epithelial disorders associated with VKC necessitates
characterization of the possible effects of proinflammatory cytokines
and Th-2 cytokines on the release of chemokines from corneal stromal
keratocytes. We therefore examined the effects of TNF-, IL-4, and
IL-13 on eotaxin expression, at both the protein and mRNA levels, in
cultured human keratocytes.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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and IL-4 were obtained from Genzyme (Cambridge,
MA), and human recombinant IL-13 was from Peprotech (Rocky Hill, NJ).
The enzyme-linked immunosorbent assay (ELISA) kit for eotaxin was from
R&D Systems (Minneapolis, MN), and the microplate reader (MPR A4i) was
from Tosoh (Tokyo, Japan). The RNeasy mini kit was from Qiagen (Hilden,
Germany), the PCR kit (TaKaRa One-Step RNA; AMV) was from Takara Shuzo
(Shiga, Japan), the PCR amplification system was from Roche Diagnostic
Systems (GeneAmp 2400-R; Basel, Switzerland), ethidium bromide and DNA
molecular size standards (Marker 11) were from Nippongene (Toyama,
Japan), and agarose (NuSieve 3:1) was from FMC Bioproducts (Rockland,
ME). All reagents used for cell culture were endotoxin minimized.
Isolation, Culture, and Stimulation of Human Keratocytes
Four human corneas were obtained from Mid-America Transplant
Service (St. Louis, MO). The donors were white men and women ranging in
age from 4 to 65 years. After the center of each donor cornea was
punched out for corneal transplantation surgery (performed by TN), the
remaining rim of the cornea was used for the present experiments. The
human tissue was used in strict accordance with the basic principles of
the Declaration of Helsinki. Each cornea was digested separately with
collagenase to provide a suspension of keratocytes.14
The
cells from each cornea were cultured separately in MEM supplemented
with 10% FBS, and they were used for the present studies after four to
six passages. The purity of the keratocyte cultures was judged on the
basis of both cell morphology and reactivities with antibodies to
vimentin and to cytokeratin12
(Fig. 1)
. All the cells were positive for vimentin and negative for
cytokeratin, suggesting the absence of contamination of the cultures by
epithelial cells. No changes in cell morphology or immunoreactivity
were apparent after culture for up to four to six passages.
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, IL-4, or IL-13.
After incubation for the indicated periods of time, the medium was
collected from each culture dish and centrifuged at 120g for
5 minutes. The resultant supernatants were frozen at -80°C for
subsequent assay of eotaxin. The keratocytes remaining in the dish were
exposed to trypsin-EDTA, and their number was determined with a
hemocytometer.
Determination of Eotaxin Concentration by ELISA
The concentration of eotaxin in culture supernatants was
quantified with an ELISA kit, with absorbance at 450 nm being
determined with a microplate reader. The limit of detection was 5
pg/ml. The morphology and number of cells were not affected by
incubation with cytokines for 24 hours. The concentration of eotaxin in
the culture medium was therefore normalized by expression as nanograms
of eotaxin per 106 cells.
RT-PCR Analysis of Eotaxin mRNA
Keratocytes were cultured and stimulated as described, after which
the cells were washed with phosphate-buffered saline, and total RNA was
extracted with the commercial kit. The abundance of eotaxin mRNA was
determined by reverse transcription–polymerase chain reaction (RT-PCR)
analysis with the PCR kit. Transcripts of the constitutively expressed
gene for glyceraldehyde-3-phosphate dehydrogenase (G3PDH)
servedas an internal control to confirm that equal amounts of
RNA were analyzed. The sequences of the PCR primers were as follows:
eotaxin forward, 5'-AAGCTTACGCCAAAGCTCACACCT-3'; eotaxin reverse,
5'-GAATCCTGGCTTTGGAGTTGGAGAT-3'; G3PDH forward,
5'-GCCAAAAGGGTCATCATCTC-3'; and G3PDH reverse,
5'-ACCACCTGGTGCTCAGTGTA-3'. These primers yielded PCR products of the
expected sizes of 322 bp for eotaxin mRNA15
and 500 bp for
G3PDH mRNA.16
17
RT-PCR was performed with the commercial
PCR system. The PCR protocol comprised 25 cycles of 1 minute at 94°C,
2 minutes at 60°C, and 3 minutes at 72°C, and the reaction was
terminated by cooling to 4°C. The amplification products were
subjected to electrophoresis on a 4% agarose gel, which was then
stained with ethidium bromide (1 µg/ml). Densitometry was performed
with a system (Nighthawk; pdi, Huntington Station, NY) comprising a
charge-coupled device camera, an ultraviolet transilluminator, and an
analysis program (Quantity One). The abundance of eotaxin mRNA was
normalized on the basis of the amount of G3PDH mRNA.
Statistical Analysis
Data are expressed as means ± SEM from four separate
experiments unless indicated otherwise. Differences were analyzed by
Student’s t-test or by analysis of variance and Fisher’s
protected least significant difference (PLSD) test. P < 0.01 was considered statistically significant.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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and Either IL-4 or IL-13 on
Eotaxin Release by Human Keratocytes, IL-4, and IL-13 on the
production of eotaxin by human keratocytes isolated from four different
donors. The cells were cultured for 24 hours with each cytokine present
at a concentration of 10 ng/ml, either alone or in various
combinations.
Eotaxin was not detected in the culture medium of cells incubated in
the absence of these agents. For all four keratocyte preparations
examined, the addition of TNF-, IL-4, IL-13, or the combination of
IL-4 and IL-13 resulted in only a small increase in the amount of
eotaxin in the culture medium (Fig. 2)
. However, incubation in the presence of TNF- and either IL-4 or
IL-13 induced a marked increase in the release of eotaxin by all four
cell preparations. No correlation was apparent between the extent of
eotaxin release and the age or sex of the corneal donors. Given that
the responses of the cells from the four different donors were almost
identical, we performed subsequent experiments with keratocytes from
one donor.
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(10 ng/ml) and either IL-4
(10 ng/ml) or IL-13 (10 ng/ml). Whereas incubation of cells with
TNF-, IL-4, or IL-13 alone, or with the combination of IL-4 and
IL-13, had only a small effect on the amount of eotaxin in the culture
medium at any of the time points examined (data not shown), exposure of
keratocytes to the combination of TNF- with either IL-4 or IL-13
resulted in an approximately linear increase in eotaxin release with
incubation time (Fig. 3)
; the increase was statistically significant (P <
0.01) at 12 and 24 hours for both combinations of cytokines, compared
with either the zero time point or with the corresponding times for
cells incubated in the absence of cytokines.
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and
IL-4 on the production of eotaxin by keratocytes was then examined.
Keratocytes were cultured for 24 hours with various concentrations of
TNF- in the absence or presence of IL-4 (10 ng/ml; Fig. 4
), or with various concentrations of IL-4 in the absence or presence of
TNF- (10 ng/ml; Fig. 5
). In the absence of IL-4, TNF- had only a small effect on eotaxin
release at any of the concentrations examined; however, in the presence
of IL-4, TNF- induced a dose-dependent increase in eotaxin release
(Fig. 4) . Similarly, in the absence of TNF-, IL-4 had only a small
effect on eotaxin release at any of the concentrations examined;
however, in the presence of TNF-, IL-4 induced a dose-dependent
increase in eotaxin release (Fig. 5)
.
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and IL-13 on the
production of eotaxin by keratocytes was similarly examined. Whereas
TNF- had only a small effect on eotaxin production at any of the
concentrations examined in the absence of IL-13, in the presence of
IL-13 it induced a dose-dependent increase in eotaxin release that was
maximal at 1 ng/ml (Fig. 6)
. Similarly, in the absence of TNF-, IL-13 had only a small effect
on eotaxin production at any concentration examined; however, in the
presence of TNF-, eotaxin release was increased by IL-13 in a
dose-dependent manner that was maximal at an IL-13 concentration of 10
ng/ml (Fig. 7)
.
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and Either IL-4 or IL-13 on the
Abundance of Eotaxin mRNA in Keratocytes, IL-4, and IL-13
on the abundance of eotaxin mRNA in human keratocytes. Cells were
cultured for 24 hours in the absence or presence of cytokines at
concentrations of 10 ng/ml, after which the amount of eotaxin mRNA in
cell lysates was assayed by RT-PCR (Fig. 8)
and quantified by densitometry. Eotaxin mRNA was not detected in
lysates of unstimulated keratocytes, but was apparent in keratocytes
exposed to either TNF-, IL-4, or IL-13. The abundance of this
transcript was increased further by simultaneous exposure of cells to
TNF- together with either IL-4 or IL-13.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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with either IL-4 or
IL-13 induces a synergistic increase in the expression of eotaxin at
both the mRNA and protein levels in cultured human corneal keratocytes.
Exposure of keratocytes to each of these cytokines individually had
only a small effect on eotaxin production. Our results are thus
consistent with the previous observation that eotaxin production by
skin fibroblasts is increased by costimulation with TNF- and
IL-4.18
Local production of eotaxin by fibroblast-like
cells may therefore be widespread throughout the body. In skin, eotaxin
is released from fibroblasts, which are biologically similar to corneal
keratocytes, but not from keratinocytes, which are similar to corneal
epithelial cells.18
Similarly, we have also shown that
simian virus-40–transformed human corneal epithelial cells do not
produce detectable amounts of eotaxin when exposed to TNF- and
either IL-4 or IL-13 (unpublished observations, June, 1999).
The reason for this difference in the ability to produce eotaxin
between keratocytes and corneal epithelial cells requires further
investigation.
Our results suggest that activation of keratocytes is an important step
in the pathogenesis of eosinophilic infiltration in the ocular region
and that keratocytes and local immune cells communicate through
cytokines and chemokines released into tear fluid. Various cytokines
participate in allergic inflammatory reactions. Whereas proinflammatory
cytokines such as TNF- play important roles in inflammatory
reactions regardless of causative factor, such as injury, infection,
immunologic response, or allergy,19
Th-2 cytokines such as
IL-4 and IL-13 are the principal players in allergic
reactions.10
20
The initial pathobiology of allergic
reactions in the conjunctiva is almost identical in individuals with
allergic conjunctivitis and in those with VKC. However, only
individuals with VKC develop corneal ulceration, shield ulcer, or
corneal plaque. Whereas an increased concentration of IL-4 in tear
fluid has been detected in individuals with either seasonal allergic
conjunctivitis or VKC,8
an increased concentration of
TNF- in tear fluid has been demonstrated in only a few patients with
VKC.9
It is thus possible that the increased
concentrations of both IL-4 and TNF- in tear fluid of individuals
with VKC induce the production of eotaxin by keratocytes. Given that
eotaxin is a potent and specific chemoattractant for
eosinophils,21
activated keratocytes may induce the
infiltration of eosinophils into the corneal stroma. The release from
the infiltrated eosinophils of degradative enzymes that target
extracellular matrix proteins then is likely to result in the corneal
ulceration associated with VKC.
Both IL-4 and IL-13 increased the production of eotaxin in keratocytes
in the presence of TNF-. The actions of IL-4 and IL-13 are similar,
because the corresponding receptor complexes both share the IL-4
receptor chain, although these two cytokines are produced by
different T-cell subsets and dendritic cells.10
However,
recent studies have provided evidence for important differences between
these two cytokines, such as in their effects on airway mucus secretion
and airway fibrosis.22
In the present study, the effect of
IL-4 on eotaxin production was greater than that of IL-13 when
keratocytes were stimulated with low concentrations of these cytokines
in the presence of TNF-. Differences in the roles of IL-4 and IL-13
in ocular allergic reactions require further investigation.
Although the main function of eotaxin in allergic reactions is to promote eosinophil accumulation, other actions of this chemokine, such as induction of the expression of adhesion molecules on vascular endothelial cells23 and on epithelial cells24 as well as enhancement of eosinophil degranulation,25 may contribute to allergic eye diseases.
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Acknowledgements |
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Footnotes |
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Commercial relationships policy: N.
Corresponding author: Naoki Kumagai, Department of Ophthalmology, Yamaguchi University School of Medicine, 1-1-1 Minami-Kogushi, Ube City, Yamaguchi 755-8505, Japan. kgnaoki{at}po.cc.yamaguchi-u.ac.jp
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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is localized to nasal mucosal mast cells and is released in acute allergic rhinitis Clin Exp Allergy 25,406-415[Medline][Order article via Infotrieve]
transcripts in human B cells induced by interleukin 4 to switch to IgE production J Exp Med 172,463-4735ß1 expression by interleukin-6 in rabbit corneal epithelial cells Exp Cell Res 218,418-423[Medline][Order article via Infotrieve]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), after which culture
supernatants were assayed for eotaxin by ELISA. Data are means ±
SEM of values from four separate experiments. *P <
0.01 (Student’s t-test) versus the zero time point;
#P < 0.01 versus the corresponding
time point for cells incubated in the absence of cytokines.
