Seediscussions,stats,andauthorprofilesforthispublicationat:https://www.researchgate.net/publication/12122056
Regulatedproductionofinterferon-inducible
T-cellchemoattractantsbyhumanintestinal
epithelialcells
ArticleinGastroenterology·January2001
ImpactFactor:16.72·DOI:10.1053/gast.2001.20914·Source:PubMed
CITATIONS
READS
161
25
4authors,including:
MichaelBDwinell
MedicalCollegeofWisconsin
78PUBLICATIONS2,051CITATIONS
SEEPROFILE
Availablefrom:MichaelBDwinell
Retrievedon:10May2016
GASTROENTEROLOGY 2001;120:49 –59
Regulated Production of Interferon-Inducible T-Cell
Chemoattractants by Human Intestinal Epithelial Cells
MICHAEL B. DWINELL, NORBERT LÜGERING, LARS ECKMANN, and MARTIN F. KAGNOFF
Laboratory of Mucosal Immunology, Department of Medicine, University of California, San Diego, La Jolla, California
See editorial on page 291.
Background & Aims: Human intestinal epithelial cells inducibly express neutrophil and monocyte chemoattractants, yet little is known about the regulated production
of T-cell chemoattractants by the intestinal epithelium.
IP-10, Mig, and I-TAC are 3 CXC chemokines that are
known to act as CD4ⴙ T-cell chemoattractants. Methods:
We studied constitutive chemokine expression in human colon, and defined the regulated expression of
these chemokines by reverse-transcription polymerase
chain reaction, enzyme-linked immunosorbent assay,
and immunohistology using cultured human intestinal
epithelial cell lines and a novel adaptation of an in vivo
human intestinal xenograft model. Results: IP-10 and
Mig were constitutively expressed by normal human
colon epithelium, and their cognate receptor, CXCR3,
was expressed by mucosal mononuclear cells. Interferon (IFN)-␥ stimulation increased mRNA expression
and the polarized basolateral secretion of these chemokines by human colon epithelial cell lines; infection with
enteroinvasive bacteria, or stimulation with the proinflammatory cytokines tumor necrosis factor ␣ and interleukin 1␣, strongly potentiated IFN-␥–induced epithelial
cell IP-10, Mig, and I-TAC production. Epithelial cell
mRNA and protein expression of IP-10, Mig, and I-TAC
were rapidly up-regulated in human intestinal xenografts
in response to stimulation with IFN-␥ alone or in combination with IL-1. Conclusions: The constitutive and regulated production of the IFN-␥–inducible chemokines
IP-10, Mig, and I-TAC by human intestinal epithelium,
and the expression of their cognate receptor, CXCR3, by
mucosal mononuclear cells, suggest that the intestinal
epithelium can play a role in modulating physiologic and
pathologic T cell–mediated mucosal inflammation.
he single layer of intestinal epithelial cells that lines
the intestinal mucosa acts as a physical barrier to
separate the host’s internal milieu from the contents of
the intestinal lumen and plays an important role in
intestinal absorption and secretion. When human intestinal epithelial cells are stimulated with proinflammatory
cytokines or infected with microbial pathogens, they
T
up-regulate a program of proinflammatory genes1–5
whose products chemoattract neutrophils and monocytes
and can signal the onset of an acute mucosal inflammatory response. Each of the components of this epithelial
cell proinflammatory gene program are target genes of
the nuclear transcription factor NF-␬B, which acts as a
central regulator of this response.6,7 In contrast, intestinal epithelial cells do not produce cytokines such as
interferon (IFN)-␥, interleukin (IL)-2, IL-4, and IL-51,3
that play a role in host adaptive immune responses, and
little is known about whether the intestinal epithelium
produces or can be regulated to produce signals that
chemoattract T cells important in physiologic and pathologic mucosal inflammation.4,8
Chemokines are a diverse set of low-molecular-weight
cytokines that selectively direct the migration and activation of specific populations of leukocytes.9,10 Chemokines can be categorized into 4 families based on the
number and spacing of the amino-terminal cysteines
(i.e., CXC, CC, C, and CX3C chemokines). Members of
the CXC family of chemokines have a single amino acid
located between the 2 NH2-terminal cysteines. This
family can be further subdivided into CXC chemokines
that have an NH2-terminal glutamic acid–leucine–arginine (ELR) motif, which are chemotactic for neutrophils,
and those lacking the ELR sequence, various members of
which chemoattract T and B cells.9,10 IFN-␥ up-regulates expression of 3 non-ELR motif CXC chemokines,
IFN-␥–inducible protein 10 (IP-10), monokine induced
by IFN-␥ (Mig), and IFN-inducible T-cell ␣ chemoattractant (I-TAC), which are expressed by monocytes and
various other cell types.11–16 IP-10, Mig, and I-TAC
share ⬃40% amino acid sequence identity and each
binds to the chemokine receptor CXCR3 expressed
Abbreviations used in this paper: ELISA, enzyme-linked immunosorbent assay; IFN, interferon; IL, interleukin; IP-10, interferon ␥–inducible protein 10; I-TAC, interferon-inducible T-cell ␣ chemoattractant;
HRP, horseradish peroxidase; Mig, monokine induced by interferon ␥;
RT-PCR, reverse-transcription polymerase chain reaction.
© 2001 by the American Gastroenterological Association
0016-5085/01/$10.00
doi:10.1053/gast.2001.20914
50
DWINELL ET AL.
mainly by activated CD4⫹ memory (CD45RO⫹) T cells
that produce a T helper (Th) cell 1 pattern of cytokines
(i.e., IFN-␥, IL-2).17,18
Intestinal epithelial cells express receptors for IFN-␥,
and stimulation of these cells with IFN-␥ affects epithelial cell gene expression and functions.19,20 For example,
IFN-␥ stimulation of intestinal epithelial cells up-regulates expression of major histocompatibility complex
(MHC) class I and class II molecules,21 nonclassic MHC
class I molecules (e.g., CD1d),22 and the adhesion molecule ICAM-1.23 In addition, IFN-␥ alters intestinal
epithelial cell chloride secretory responses and decreases
epithelial barrier function.20,24 IFN-␥ stimulation of human intestinal epithelial cells in combination with IL-1
up-regulates the production of nitric oxide (NO)25 and,
in combination with tumor necrosis factor (TNF)-␣,
induces epithelial cell apoptosis.26
IFN-␥ has been shown to be a major inducer of IP-10,
Mig, and I-TAC messenger RNA (mRNA) expression.12–14 Because intestinal epithelial cells have receptors for IFN-␥,19 we hypothesized that the intestinal
epithelium may produce IFN-␥–inducible chemokines.
This was of particular interest because these chemokines
are known to function as chemoattractants for T-cell
populations that have been shown to be important for
mediating physiologic and pathologic inflammation in
the intestinal tract. Our studies expand the role for
IFN-␥ in modulating intestinal epithelial cell responses
and suggest a concept in which the intestinal epithelium
participates in physiologic and pathologic mucosal inflammatory responses through the regulated production
of chemoattractants for subpopulations of CD4⫹ T cells.
Materials and Methods
Reagents
Recombinant human cytokines were obtained as follows: IP-10 and Mig from R&D Systems (Minneapolis, MN);
IL-1␣, IL-4, IL-13, I-TAC, and TNF-␣ from PeproTech
(Rocky Hill, NJ); and IFN-␥ from BioSource International
(Camarillo, CA). Polyclonal biotin-conjugated or unconjugated affinity-purified goat antibodies to IP-10 and Mig and
murine monoclonal antibodies to IP-10 (clone 49106.11) and
Mig (clone 33036.211) were obtained from R&D Systems, and
rabbit anti-I-TAC antibody was from PeproTech. Murine
monoclonal antibody to I-TAC (clone 8G4) was provided by
K. Neote (Pfizer Research Center, Groton, CT). Murine monoclonal antibody to CXCR3 (clone 1C6) was obtained from
PharMingen (San Diego, CA).
Cell Culture and Stimulation Protocols
The human colon adenocarcinoma cell lines HT-29,
HCA-7 colony 29, HCT-8, I-407, Caco-2, and T84 were
GASTROENTEROLOGY Vol. 120, No. 1
cultured as described previously.27 For cytokine stimulation,
intestinal epithelial cells were plated into 6-well plates (Costar,
Cambridge, MA), 6-well collagen-coated Transwell inserts
(24-mm diameter, 0.4-␮m-pore size; Costar), or 60-mm tissue
culture plates (Costar) and grown to confluence. Confluent
epithelial cell cultures were stimulated with TNF-␣ (20 ng/
mL), IL-1␣ (20 ng/mL), and IFN-␥ (40 ng/mL), alone or in
combination. Additional cultures of confluent HT-29 cells
were preincubated for 60 minutes with IL-4 (20 ng/mL) or
IL-13 (20 ng/mL) before adding IFN-␥ or IFN-␥ and TNF-␣.
For bacterial infection, confluent monolayers of HT-29 cells in
6-well plates or 60-mm dishes were incubated with Salmonella
dublin or enteroinvasive Escherichia coli O29:NM2,3 at a multiplicity of infection (MOI) of 100 and 500, respectively. After
45 minutes, medium was removed and the cells washed before
adding fresh gentamicin (50 ␮g/mL)-containing medium to
kill any remaining extracellular bacteria. Some HT-29 cultures
were additionally stimulated with IFN-␥ (40 ng/mL) after
bacterial infection.
Human Fetal Intestinal Xenografts
Human fetal intestine (gestational age, 12–18 weeks)
was transplanted subcutaneously onto the backs of C57BL/6
severe combined immunodeficiency disease (SCID) mice (Jackson Laboratory, Bar Harbor, ME).2,23,28 Human fetal intestinal
xenografts were allowed to grow for 10 weeks after implantation at which time the mucosa and the epithelium, which is
strictly of human origin, is fully differentiated.28 Littermate
control SCID mice were injected subcutaneously with ⬃106
HT-29 cells, and tumors were allowed to develop for 5 weeks.
Mice with mature xenografts or HT-29 tumors were injected
intraperitoneally with 10 ␮g human IFN-␥ alone or together
with 1 ␮g human IL-1␣ in 200 ␮L phosphate-buffered saline
(PBS), whereas controls received 200 ␮L PBS alone. Intestinal
xenografts or HT-29 epithelial tumors were removed 5 hours
later, and adjacent segments were frozen in liquid nitrogen for
RNA isolation, or for immunohistochemical analysis were
embedded in OCT compound (TissueTek, Miles Scientific,
Naperville, IL) and frozen in isopentane/dry ice or fixed in 10%
neutral buffered formalin. Procedures involving the human
intestinal xenograft model were approved by the UCSD Human and Animal Subjects Committees.
RNA Isolation and Detection of mRNA
Expression
Total cellular RNA was extracted using an acid
guanidinium–phenol– chloroform method (TRIzol Reagent;
GIBCO Life Technologies, Grand Island, NY) and treated
with ribonuclease-free deoxyribonuclease (Stratagene, La Jolla,
CA). For reverse-transcription polymerase chain reaction
(RT-PCR), 1 ␮g of total cellular RNA was reverse transcribed,
and complementary DNA was amplified as described previously.27 The primers for I-TAC (sense 5⬘-GCT ATA GCC
TTG GCT GTG ATA TTG TG-3⬘ and antisense 5⬘-CTG
CCA CTT TCA CTG CTT TTA CC-3⬘) and for Mig (sense
5⬘-GTA TCT GAG GCA CAT GTC AG-3⬘ and antisense
January 2001
5⬘-AAA GGC ACT GCA TTG TGG TAG G-3⬘) were designed from available sequences from GenBank (AF030514
and NM_002416, respectively); primers for IP-10 and ␤-actin
were as described previously.3,4 The amplification profile for
IP-10, Mig, and I-TAC was 25–35 cycles of 450-second
denaturation at 95°C and 2.5-minute annealing and extension
at 60°C. Primers for IP-10, Mig, and I-TAC did not amplify
sequences of murine genomic DNA or cDNA. Quantitative
competitive RT-PCR for IP-10 was performed as described
previously3 using the standard plasmid construct pHCQ5.4
Negative control reactions had no added RNA in the RT and
subsequent PCR amplification. Positive control reactions for
IP-10, Mig, and I-TAC used RNA from peripheral blood
mononuclear cells that were stimulated overnight with 10
␮g/mL phytohemagglutinin P and 2 ␮g/mL bacterial lipopolysaccharide (E. coli O111:B4).
Enzyme-Linked Immunosorbent Assay
Polystyrene 96-well plates (Immulon-4; Dynex Technologies Inc., Chantilly, VA) were coated with murine monoclonal antibody to IP-10, Mig, and I-TAC diluted in carbonate
buffer, as a capture antibody. Affinity-purified biotinylated
goat anti–IP-10 and anti-Mig, or unconjugated rabbit anti–
I-TAC, were diluted in PBS, 1.0% bovine serum albumin, and
0.1% Tween 20 and used as detection antibodies. Second-step
reagents were horseradish peroxidase (HRP)-conjugated
streptavidin for the IP-10 and Mig enzyme-linked immunosorbent assay (ELISA) and HRP-conjugated goat anti-rabbit
immunoglobulin (Ig) (BioSource) for the I-TAC ELISA.
Bound HRP was visualized with TMB (3,3⬘5,5,⬘-tetramethylbenzadinedihydrochloride; Sigma Chemical Co., St. Louis,
MO) and H2O2 diluted in sodium acetate buffer, pH 6.0; the
color reaction was stopped by addition of 1.2 mol/L H2SO4;
and absorbance was measured at 450 nm. Chemokine concentrations were calculated from standard curves using recombinant human IP-10, Mig, or I-TAC. IP-10, Mig, and I-TAC
ELISAs were sensitive to 50 pg/mL.
Immunohistochemistry
Human intestinal xenografts were fixed overnight in
10% neutral buffered formalin and embedded in paraffin, and
5-␮m sections were prepared. Serial sections were blocked in
PBS, 1% bovine serum albumin, 2% human serum, and 2%
rabbit serum and incubated overnight at 4°C with either
optimally diluted goat antibody to IP-10, mouse monoclonal
anti-Mig, control goat IgG, or control mouse IgG1 monoclonal antibody. Immunostaining was visualized with Cy3-conjugated rabbit anti-mouse or donkey anti-goat antibodies
(Amersham Pharmacia Biotech, Piscataway, NJ).
Sections of human colon tissue from histologically normalappearing resection margins of surgical specimens from individuals undergoing partial colectomy were embedded in OCT
and snap-frozen in isopentane/dry ice as described previously.27
Serial cryostat sections (5 ␮m) were prepared, fixed in acetone
for 10 minutes, and blocked as described above. Sections were
incubated overnight at 4°C with monoclonal antibodies to
IFN–INDUCIBLE CHEMOKINE SECRETION BY EPITHELIAL CELLS
51
IP-10, Mig, CXCR3 or a mouse monoclonal IgG1 isotype
control antibody after which endogenous biotin was blocked
(Avidin/Biotin Blocking Kit; Vector Laboratories, Burlingame, CA). Sections were subsequently stained with biotinconjugated rabbit anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) followed by
streptavidin-Cy3 (Amersham).
Results
Constitutive Expression of IP-10 and Mig
and Their Cognate Receptor CXCR3 in
Human Colon
We first determined if adult human intestinal
epithelium expresses the IFN-␥–inducible chemokines
IP-10 or Mig. Normal human colon epithelium constitutively expressed both of those chemokines (Figure 1A
and B). To assess whether normal human colon mucosa
also contains potential targets for epithelial IP-10 and
Mig, additional sections of colon from the same individuals were immunostained for CXCR3, the receptor for
these chemokines. Many mononuclear cells in the lamina
propria, but not colon epithelial cells, expressed CXCR3
(Figure 1D). Moreover, CXCR3-bearing cells were abundant in the T-cell zone, but not the B cell–rich germinal
center of mucosal lymphoid follicles in the colon (Figure
1E), consistent with the predominant T-cell expression
of this receptor.
Constitutive and Regulated Expression of
IP-10, Mig, and I-TAC mRNA by Cultured
Human Intestinal Epithelial Cells
To characterize the possible regulated expression
of IP-10, Mig, and I-TAC by human intestinal epithelium, we first used human colon epithelial cell lines.
Several human intestinal epithelial cell lines (i.e., HT29, T84, Caco-2, HCA-7, HCT-8, and I-407) constitutively express mRNA for IP-10 and I-TAC, and several
of these cell lines additionally express Mig mRNA (Figure 2). We next defined the regulated expression of these
chemokines using HT-29 cells and HCA-7 cells, the
latter of which constitutively expressed only low levels of
mRNA for these cytokines. IP-10, Mig, and I-TAC
expression increased by 2–3 hours after IFN-␥ stimulation and reached maximal levels by 8 –16 hours. In
addition, the proinflammatory cytokine TNF-␣ increased IP-10 and I-TAC, but not Mig, mRNA in
HT-29 and HCA-7 cells (Figure 3). Moreover, as assessed
by qualitative RT-PCR, both intestinal epithelial cell
lines appeared to express greater amounts of chemokine
mRNA when stimulated with IFN-␥ in combination
with TNF-␣ (data not shown). To verify this, we used
quantitative RT-PCR to assess IP-10 transcript levels in
52
DWINELL ET AL.
GASTROENTEROLOGY Vol. 120, No. 1
Figure 1. Expression of IP-10 and
Mig and their cognate receptor,
CXCR3, in normal human colon.
Frozen sections of normal human
colon were immunostained for (A )
IP-10 or (B) Mig, and an adjacent
section was stained with (C ) an
isotype control antibody. Sections
are oriented with the lumen to the
right. Sections in D and E were
immunostained for CXCR3, and an
adjacent section in F was stained
with an isotype control antibody.
Colon epithelium expresses IP-10
(A ) and Mig (B). Scattered lamina
propria mononuclear cells also
stain positively for Mig (B). (D) Numerous mononuclear cells in the
lamina propria (arrowheads), but
not epithelial cells lining the crypt
lumen (*) express CXCR3. (E )
CXCR3-staining cells were abundant in the T-cell zone (arrowheads), but not the germinal center (gc) of a lymphoid follicle
contained in the same section as
shown in D. (Original magnification
200⫻ [A–C ] and 400⫻ [D–F ].)
HT-29 cells stimulated with IFN-␥ alone and in combination with TNF-␣. Levels of IP-10 mRNA in unstimulated, IFN-␥–stimulated, and IFN-␥ ⫹ TNF-␣–
stimulated cells were 1.5 ⫻ 105, 6.0 ⫻ 105, and 2.0 ⫻
107 transcripts/␮g cellular RNA, respectively, as assessed
16 hours after stimulation.
Secretion of IP-10, Mig, and I-TAC by
Human Intestinal Epithelial Cell Lines
To determine if IP-10, Mig, and I-TAC mRNA
expression by the cell lines was accompanied by secretion
of these chemokines, chemokine levels in culture supernatants were assayed by ELISA. In the absence of stimulation, HT-29, HCA-7, and Caco-2 cells secreted ⬍50
pg/mL IP-10, Mig, or I-TAC (Figure 4 and data not
shown). IP-10, Mig, and I-TAC secretion increased significantly in HT-29 cells after IFN-␥ stimulation (Figure
4). Consistent with the up-regulation of mRNA expression, TNF-␣ induced IP-10 and I-TAC secretion but did
not stimulate Mig secretion. In contrast to TNF-␣, the
proinflammatory cytokine IL-1␣ stimulated IP-10 but
Figure 2. Constitutive expression of IP-10, Mig, and I-TAC mRNA by
human intestinal epithelial cell lines. Total RNA was isolated from the
indicated cell lines, and the varying chemokine mRNAs were amplified
by RT-PCR using 32 amplification cycles. RNA from peripheral blood
mononuclear cells (PBMC) was a positive control, whereas negative
control reactions had no added RNA.
January 2001
IFN–INDUCIBLE CHEMOKINE SECRETION BY EPITHELIAL CELLS
53
Figure 3. Regulated expression of IP-10, Mig, and I-TAC mRNA in
response to stimulation with inflammatory cytokines. Total RNA was
isolated from HT-29 and HCA-7 cells stimulated for 6 hours with the
indicated cytokines or from control unstimulated HT-29 and HCA-7
cells. IP-10, Mig, I-TAC, and ␤-actin mRNAs were amplified by RT-PCR
using 25 amplification cycles for IP-10 and I-TAC and 32 amplification
cycles for Mig.
not Mig or I-TAC secretion (Figure 4). As shown in
Table 1, TNF-␣ and IL-1␣ potentiated IFN-␥–stimulated secretion of IP-10, Mig, and I-TAC by HT-29
cells. IFN-␥ was also a more potent stimulus than either
TNF-␣ or IL-1␣ alone for IP-10, Mig, and I-TAC secretion in HCA-7 cells, and TNF-␣ and IL-1␣ potentiated IFN-␥–stimulated IP-10, Mig, and I-TAC secretion
by those cells (data not shown).
Intestinal epithelial cells are structurally and functionally polarized into apical and basolateral domains. To
determine if intestinal epithelial cell IP-10, Mig, and
I-TAC secretion was apical or basolateral, Caco-2 cells,
which can be grown as polarized epithelial monolayers on
microporous filter supports (Transwells), were stimulated
with IFN-␥ or costimulated with IFN-␥ and IL-1␣.
IL-1␣, rather than TNF-␣, was used as an agonist in
these studies because we previously noted that Caco-2
cells respond to IL-1␣, but respond poorly, if at all, to
TNF-␣ stimulation in terms of the up-regulated expression of several other chemokine genes.1,6 As shown in
Table 2, IP-10, Mig, and I-TAC were secreted into the
basal rather than the apical chamber of the transwell
epithelial cell cultures, indicating that intestinal epithelial cells can secrete these chemokines basolaterally, in
the direction that is physiologically relevant for the
chemoattraction of T cells in the intestinal mucosa.
Figure 4. Secretion of IP-10, Mig, and I-TAC by HT-29 cells stimulated
with IFN-␥, TNF-␣, or IL-1␣. Confluent HT-29 cells were stimulated with
IFN-␥ (ƒ), TNF-␣ (E), or IL-1␣ () for the indicated times. (A–C) IP-10,
Mig, and I-TAC secretion, respectively. Control unstimulated HT-29
cells (F) secreted ⬍50 pg/mL IP-10, Mig, and I-TAC. Values are
mean ⫾ SEM of 2– 4 repeated experiments.
54
DWINELL ET AL.
GASTROENTEROLOGY Vol. 120, No. 1
Table 1. TNF-␣ and IL-1␣ Potentiate IFN-␥–Stimulated IP-10,
Mig, and I-TAC Secretion
Chemokine secretion (ng/mL)a
Table 3. S. dublin or Enteroinvasive E. coli Infection
Potentiates IFN-␥–Stimulated IP-10, Mig,
and I-TAC Secretion
Chemokine secretion (ng/mL)a
Stimuli added
IP-10
Mig
I-TAC
None
TNF-␣
IL-1␣
IFN-␥
IFN-␥ ⫹ TNF-␣
IFN-␥ ⫹ IL-1-␣
⬍0.05
11.4 ⫾ 2
0.8 ⫾ 0.2
19.3 ⫾ 10
779 ⫾ 60
233 ⫾ 35
⬍0.05
⬍0.05
⬍0.05
5.5 ⫾ 2
237 ⫾ 20
43 ⫾ 13
⬍0.05
0.3 ⫾ 0.01
⬍0.05
5.8 ⫾ 4
44.4 ⫾ 5
14.6 ⫾ 4
NOTE. HT-29 cells were stimulated with the indicated cytokines for 8
hours, and IP-10, Mig, and I-TAC secretion was assessed by ELISA.
Similar potentiation was also noted at 4, 14, and 24 hours after
cytokine stimulation (data not shown).
a Mean ⫾ SEM; n ⫽ 3.
IP-10, Mig, and I-TAC Secretions Are
Increased in Response to Bacterial
Infection of Intestinal Epithelial Cells
To determine if infection of the intestinal epithelium with enteroinvasive bacteria plays a role in the
activation of epithelial T-cell chemoattractants, regulated production of IP-10, Mig, and I-TAC was determined in HT-29 cells in response to infection with S.
dublin or enteroinvasive E. coli O29:NM. Infection with
these enteroinvasive bacteria resulted in a small increase
in IP-10 secretion; little, if any, increase in I-TAC; and
no increase in Mig secretion (Table 3). However, stimulation of bacteria-infected cells with IFN-␥ resulted in
a 4 –5-fold increase in secretion of these 3 CXC chemokines compared with cultures stimulated with IFN-␥
alone (Table 3).
Table 2. Vectorial Secretion of IP-10, Mig, and I-TAC
by Polarized Caco-2 Epithelial Cells
Chemokine secretiona
Chemokine
IP-10
Mig
I-TAC
Stimulus
ng/apical
compartment
ng/basal
compartment
None
IFN-␥
IFN-␥ ⫹ IL-1␣
None
IFN-␥
IFN-␥ ⫹ IL-1␣
None
IFN-␥
IFN-␥ ⫹ IL-1␣
⬍0.05
⬍0.05
0.93
⬍0.05
⬍0.05
⬍0.05
⬍0.05
⬍0.05
⬍0.05
⬍0.05
1.3
12.8
⬍0.05
0.34
1.3
⬍0.05
0.7
3.6
NOTE. Cultures were left unstimulated or were stimulated for 9 hours
with either IFN-␥ or IFN-␥ ⫹ IL-1␣ added to the basal compartment.
Mean transepithelial resistance of the Caco-2 epithelial cell monolayers after cytokine stimulation and in control cultures was 236 ⍀cm2.
a Values are mean of 2–3 replicates.
Bacteria
added
None
S. dublin
E. coli
O29:NM
IFN-␥
addition
⫺
⫹
⫺
⫹
⫺
⫹
IP-10
Mig
⬍0.05
⬍0.05
101 ⫾ 19.5
157 ⫾ 49.9
2.2 ⫾ 0.8
⬍0.05
1515 ⫾ 220 2276 ⫾ 1055
1.5 ⫾ 0.7
934 ⫾ 140
⬍0.05
1434 ⫾ 376
I-TAC
⬍0.05
9.7 ⫾ 1.0
0.07 ⫾ 0.02
40.1 ⫾ 11.3
⬍0.05
40.8 ⫾ 7.3
NOTE. HT-29 cells were either infected with the indicated bacteria for
45 minutes, or remained uninfected, and subsequently stimulated
with IFN-␥ for 24 hours (⫹IFN-␥) or not further stimulated (⫺IFN-␥).
IP-10, Mig, and I-TAC secretion was assayed by ELISA. Similar potentiation was noted 3 and 6 hours after IFN-␥ stimulation of bacteriainfected cultures (data not shown).
a Mean ⫾ SEM; n ⫽ 2–3.
The Th2 Cytokines IL-4 and IL-13 Inhibit
IP-10 and Mig and I-TAC Secretion
IP-10, Mig, and I-TAC are cognate ligands for
CXCR3, a chemokine receptor preferentially expressed
on activated T cells that produce Th1 type cytokines
(i.e., IFN-␥, IL-2).17,18 In addition to IFN-␥ receptors,
intestinal epithelial cells respond to the Th2 cytokines
IL-4 and IL-13 that are known to down-regulate IFN␥–stimulated responses in several cell types.29 IL-4 has
been shown to decrease IP-10 mRNA expression in
human neutrophils but not in bronchial epithelium.15,16
As shown in Table 4, IL-4 and IL-13 partially inhibited
IFN-␥–stimulated IP-10, Mig, and I-TAC secretion by
human colon epithelial cells, suggesting they may function as antagonists of IFN-␥–inducible T-cell chemoattractants produced by the intestinal epithelium. In contrast, IL-4 and IL-13 did not significantly inhibit IP-10,
Mig, and I-TAC production in HT-29 cells costimulated
with IFN-␥ and TNF-␣ (data not shown).
IP-10, Mig, and I-TAC mRNA and Protein
Expression Is Up-regulated in Human
Intestinal Xenografts in Response to
Cytokine Stimulation
To determine if epithelial cell expression of those
chemokines can also be regulated in vivo, we took advantage of a human intestinal xenograft model.2,23,28 To
determine the suitability of the xenograft model for these
studies, we first assessed whether HT-29 colon epithelial
cells, which we have shown in the present study to
respond to cytokine stimulation in vitro, also do so in
vivo when implanted subcutaneously in SCID mice. As
January 2001
IFN–INDUCIBLE CHEMOKINE SECRETION BY EPITHELIAL CELLS
55
Table 4. IL-4 and IL-13 Decrease IP-10, Mig, and I-TAC Secretion by IFN-␥–Stimulated HT-29 Cells
IP-10
Mig
I-TAC
Stimulation
conditions
Addition to IFN-␥–
stimulated cells
Secreted
(ng/mL)a
%
Control
Secreted
(ng/mL)
%
Control
Secreted
(ng/mL)
%
Control
1
None
IL-4
None
IL-13
120.8 ⫾ 15.6
76.2 ⫾ 1.4
104.0 ⫾ 9.3
60.7 ⫾ 11.1
100
63
100
58
19.4 ⫾ 1.3
8.4 ⫾ 1.6
19.7 ⫾ 2.3
9.9 ⫾ 1.6
100
43
100
50
9.6 ⫾ 0.4
8.3 ⫾ 0.5
7.5 ⫾ 1.1
4.5 ⫾ 0.09
100
86
100
60
2
NOTE. The cytokines IL-4 (experiment 1) or IL-13 (experiment 2) were added 1 hour before IFN-␥ stimulation, and chemokine secretion was
assayed 16 hours after IFN-␥ stimulation. Unstimulated control cells and cells stimulated with IL-4 (20 ng/mL) or IL-13 (20 ng/mL) alone did not
secrete detectable levels of IP-10, Mig, or I-TAC.
a Mean ⫾ SEM; n ⫽ 3– 4.
shown in Figure 5, subcutaneous HT-29 tumors responded to intraperitoneal injection of IFN-␥ and IL-1␣
with increased expression of IP-10, Mig, and I-TAC
mRNA, indicating that subcutaneously implanted human intestinal epithelial cells can be stimulated by systemic injection of cytokines in SCID mouse recipients.
Of note, the primers used to amplify IP-10, Mig, and
I-TAC did not amplify murine genomic or cDNA sequences.
We next used human intestinal xenografts implanted
subcutaneously in SCID mice and the same cytokine
treatment protocols to assess regulated chemokine production by normal human intestinal epithelium. This
model allowed us to directly assess early changes in
epithelial chemokine gene expression and regulation in
response to cytokine stimulation in vivo (i.e., under
Figure 5. Expression of IP-10, Mig, and I-TAC mRNA in human intestinal xenografts. Total RNA was isolated from intestinal xenografts
(XG1-3) or HT-29 cells, implanted subcutaneously in SCID mice, 5
hours after intraperitoneal injection of recombinant human IFN-␥ (XG
1) or IFN-␥ and IL-1␣ (XG 2, XG 3). IP-10, Mig, and I-TAC mRNA
expression was detected using RT-PCR with 33 amplification cycles.
Positive and negative control reactions are as in Figure 1.
conditions that would not be readily possible in the
setting of acute or chronic intestinal mucosal inflammation in humans). Because intestinal epithelial cells have
receptors for IFN-␥19,20 and IFN-␥ has been noted to
up-regulate the expression of IP-10, Mig, and I-TAC
mRNA by human monocytes, neutrophils, astrocytes,
and bronchial epithelial cells,14 –16 we first examined the
regulated mRNA expression of these chemokines in the
xenografts in response to IFN-␥ stimulation. Although
IP-10, Mig, and I-TAC levels in control xenografts varied among samples, administration of recombinant human IFN-␥, alone or in combination with human IL-1␣,
stimulated the up-regulated expression of each of those
chemokines in human intestinal xenografts (Figure 5).
Whereas human IFN-␥ is species specific and injection of
this cytokine alone up-regulated human IP-10, Mig, and
I-TAC mRNA in the xenografts, human IL-1␣ might
additionally stimulate murine mediators that potentially
act on human cells in the xenograft.
To show that chemokine mRNA expression by the
xenografts was accompanied by epithelial cell protein
production, adjacent segments of the intestinal xenografts were immunostained for IP-10 and Mig. The
epithelium of human intestinal xenografts weakly expressed IP-10. However, IP-10 immunostaining was increased in xenografts from mice injected with human
IFN-␥ in combination with IL-1␣ (Figure 6). Similar
findings were obtained in xenografts from mice injected
with IFN-␥ alone (data not shown). Further, constitutive
epithelial production of Mig was noted in some of the
xenografts, and increased epithelial Mig immunostaining
was seen in xenografts from mice injected with IFN-␥
and IL-1␣ (Figure 6).
Discussion
Intestinal epithelial cells are known to up-regulate a program of inflammatory genes whose products can
chemoattract neutrophils and monocytes in response to
56
DWINELL ET AL.
GASTROENTEROLOGY Vol. 120, No. 1
Figure 6. Epithelial cell expression of IP-10 and Mig in response
to cytokine stimulation of human
intestinal xenografts. Sections of
human intestinal xenografts from
SCID mice injected with human
IFN-␥ and IL-1␣, or sections of
xenografts from unstimulated SCID
mice, were immunostained for
IP-10 and Mig. (A and B) Sections
stained with anti-IP-10; (D and E )
sections stained with anti-Mig; (C
and F ) adjacent sections stained
with the respective isotype control
antibodies. Sections in A and D
were obtained from unstimulated
control xenografts; sections in B,
C, E, and F were from xenografts
stimulated with IFN-␥ and IL-1␣.
IP-10 and Mig immunostaining
was predominately epithelial with
a perinuclear and basal localization and immunostaining was
markedly increased after cytokine
stimulation. (Original magnification
400⫻.)
TNF-␣ and IL-1␣ stimulation or infection with enteroinvasive bacteria.1,3,5,6,30 The likely importance of components of that epithelial cell gene program for host
survival was recently shown in an animal model of enteroinvasive Shigella infection.31 Our findings of high
levels of IP-10, Mig, and I-TAC production by human
intestinal epithelial cells in vitro, and of their production
in vivo using a novel adaptation of the human intestinal
xenograft model and normal adult human colon, indicate
intestinal epithelial cells have the potential to participate
in physiologic and pathologic mucosal T-cell responses.
By chemoattracting IFN-␥–producing CD4⫹ T cells in
close proximity to the epithelium, epithelial-derived IP10, Mig, and I-TAC, which themselves are up-regulated
by IFN-␥, may activate a positive feedback loop. The net
effect of this would be amplification of the mucosal Th1
response important in physiologic mucosal inflammation
and inflammatory disease of the intestinal mucosa.
The healthy human intestinal mucosa can be regarded
as physiologically inflamed in that it normally contains
abundant populations of B and T cells. The majority of
T cells within the lamina propria adjacent to the epithelium are CD4⫹ activated/ memory (CD45RO⫹) T cells
that produce a Th1 pattern of cytokines (e.g. IFN-␥,
IL-2).32 However, the mediators that are important for
the predominance of this T-cell subset in the mucosa are
not known. We and others have shown an abundance of
CXCR3-expressing mononuclear cells within the mucosa
of normal colon. Further, we found constitutive expression of IP-10, Mig, and I-TAC by intestinal epithelial
cells in vitro and of IP-10 and Mig by intestinal epithelium in intestinal xenografts and normal adult human
colon in vivo. It is tempting to speculate that IP-10,
Mig, and/or I-TAC, constitutively produced by intestinal
epithelial cells, chemoattract activated/memory CD4⫹ T
cells that express the CXCR3 receptor and produce Th1
cytokines17,18 in the vicinity of the epithelium. Consistent with this, gut-homing CD45RO⫹␤7 integrin– expressing intraepithelial and lamina propria lymphocytes
were shown to express functional CXCR3 and to migrate
in response to I-TAC stimulation in vitro.33 Although
intestinal epithelial cells do not produce IFN-␥,1,3 intraepithelial T cells, which are among the earliest cells to
populate the intestinal tract during ontogeny, do produce IFN-␥.34 Thus, they and/or IFN-␥–producing mucosal dendritic cells present in close proximity to the
epithelium may provide a regulatory signal to epithelial
cells and thereby act as an initial stimulus for epithelial
cell IP-10, Mig, and I-TAC production.
January 2001
Physiologic inflammation normally present in the intestinal mucosa can become dysregulated in diseases such
as Crohn’s disease or ulcerative colitis or after infection
with enteric pathogens. In this regard, IP-10, Mig, and
I-TAC production by IFN-␥–stimulated intestinal epithelial cells was strongly potentiated in an environment
in which epithelial cells concurrently encountered the
proinflammatory mediators TNF-␣ or IL-1␣, or were
infected with enteroinvasive bacterial pathogens. Thus,
TNF-␣ and IL-1, produced by monocytes and T cells in
the intestinal mucosa, and enteroinvasive bacterial
pathogens, can markedly amplify IFN-␥–stimulated intestinal epithelial cell signals that are known to chemoattract CXCR3 bearing CD4⫹ T cells. Like TNF-␣ or IL-1
stimulation, enteroinvasive bacteria activate the transcription factor NF-␬B in intestinal epithelial cells6 and,
in addition, can up-regulate epithelial cell TNF-␣ production.3 The latter could, in an autocrine or paracrine
manner, further amplify IFN-␥–stimulated epithelial IP10, Mig, and I-TAC secretion by bacteria-infected cells.
Of note, our data with human colon epithelial cells are
consistent with a prior report of synergy in the transcriptional activation of the murine IP-10 gene by IFN-␥ and
TNF-␣ in fibroblasts35 and data in human respiratory
epithelial cell lines,16 but differ from studies in normal
human bronchial epithelium where IFN-␥ alone maximally induced IP-10, Mig, and I-TAC mRNA expression.16
Many of the currently characterized chemokine receptors are promiscuous in that they frequently bind more
than one chemokine ligand.9,10 The parallel expression
and time course of production by intestinal epithelial
cells of 3 IFN-␥–inducible CXC chemokines that utilize
a single receptor (i.e., CXCR3) might reflect redundancy
of the chemokine signaling system. Such redundancy
would provide a relatively “fail safe” mechanism to ensure the preservation of chemotactic responses that are
necessary for host survival. Alternatively, quantitative
differences in the amount of IP-10, Mig, and I-TAC
produced may be paralleled by differences in downstream
signaling by those chemokines. In this regard, epithelial
cell secretion of IP-10 and Mig in response to cytokine
stimulation was greater than that of I-TAC. Furthermore, IP-10 and I-TAC, but not Mig, were up-regulated
in response to TNF-␣, whereas only IP-10 was upregulated in response to IL-1. It is also possible that
receptor occupancy with one of these chemokines may
attenuate the delivery of functional signals to target cells
by the others because IP-10, Mig, and I-TAC have
different receptor-binding affinities and desensitization
profiles for CXCR3.14,36 Alternatively, these chemokines
IFN–INDUCIBLE CHEMOKINE SECRETION BY EPITHELIAL CELLS
57
may mediate different or complementary functions if
they ultimately are shown to bind to additional receptors
or to be induced by additional stimuli that have yet to be
identified.
Human intestinal xenografts in SCID mice contain an
epithelium that is strictly of human origin and have
provided a useful model in our prior studies to investigate early epithelial cell responses to infection by luminal
microbial pathogens.2,23,37 We have now successfully
adapted the intestinal xenograft model to study early in
vivo intestinal epithelial cell responses to stimulation
with one or more human cytokines. For example, the
ability to directly stimulate human intestinal epithelium
in xenograft-bearing SCID mice with human IFN-␥
provides a powerful tool to quantitatively and qualitatively assess regulated epithelial cell gene expression in
an in vivo setting. This is particularly the case because
similar controlled in vivo assessments of epithelial responses are not possible in human subjects due to the
production of unknown quantities of multiple cytokines
during the course of acute and chronic intestinal mucosal
inflammation.
Positive feedback between CD4⫹ T cells that produce
IFN-␥, and the intestinal epithelium that, in response to
IFN-␥ stimulation, produces IP-10, Mig, and I-TAC
that chemoattract IFN-␥–producing T cells would predictably be subject to counterregulation. Intestinal epithelial cells respond to the cytokines IL-4 and IL-13 that
are secreted by Th2 type CD4⫹ T cells, and these cytokines are known to down-regulate cellular responses to
IFN-␥ in several other cell types, including IFN-␥–
stimulated NO production by intestinal epithelial
cells.38 Consistent with this, IL-4 and IL-13 attenuated
IFN-␥–stimulated IP-10, Mig, and I-TAC secretion by
intestinal epithelial cell lines. Although IL-4 down-regulated IP-10 production in IFN-␥–stimulated human
neutrophils and mouse macrophages,15,39 this was not the
case for human bronchial epithelial cells,16 suggesting
cell type–specific differences in the down-regulation of
this IFN-␥–inducible chemokine.
Our findings suggest a role for the intestinal epithelium and epithelial IP-10, Mig, and I-TAC production
in the pathogenesis of intestinal inflammatory responses
in which IFN-␥–producing T cells predominate, by chemoattracting CXCR3-expressing activated/memory T
cells into the vicinity of the epithelium. Increased numbers of IFN-␥–producing Th1 cells in the vicinity of the
epithelium are a central feature of several chronic inflammatory diseases of the human intestinal tract (e.g.,
Crohn’s disease and celiac disease)40,41 and enteric bacterial infections. In other chronic inflammatory diseases of
58
DWINELL ET AL.
the intestine such as ulcerative colitis, Th2 cytokine–
producing cells predominate in the intestinal mucosa.40
Although patients with ulcerative colitis were reported
to contain increased numbers of IP-10 –staining cells in
the lamina propria,42,43 epithelial IP-10 did not appear to
be increased, which may simply reflect the fixation and
immunostaining methods used in those studies or, less
likely, down-regulation of epithelial IP-10 production
by Th2 cytokines. Taken together, our data are consistent with a role for IFN-␥ in regulating and modulating
the intestinal epithelial cell proinflammatory gene program and suggest that the epithelium may participate in
the regulation of adaptive immune responses in the
inflamed mucosa by chemoattracting a specific population of activated T cells to the proximity of the epithelial
barrier.
References
1. Eckmann L, Jung HC, Schurer-Maly C, Panja A, Morzycka-Wroblewska E, Kagnoff MF. Differential cytokine expression by human
intestinal epithelial cell lines: regulated expression of interleukin
8. Gastroenterology 1993;105:1689 –1697.
2. Eckmann L, Stenson WF, Savidge TC, Lowe DC, Barrett KE, Fierer
J, Smith JR, Kagnoff MF. Role of intestinal epithelial cells in the
host secretory response to infection by invasive bacteria. Bacterial entry induces epithelial prostaglandin H synthase-2 expression and prostaglandin E2 and F2␣ production. J Clin Invest 1997;
100:296 –309.
3. Jung HC, Eckmann L, Yang SK, Panja A, Fierer J, MorzyckaWroblewska E, Kagnoff MF. A distinct array of proinflammatory
cytokines is expressed in human colon epithelial cells in response to bacterial invasion. J Clin Invest 1995;95:55– 65.
4. Yang SK, Eckmann L, Panja A, Kagnoff MF. Differential and
regulated expression of C-X-C, C-C, and C-chemokines by human
colon epithelial cells. Gastroenterology 1997;113:1214 –1223.
5. Kagnoff MF, Eckmann L. Epithelial cells as sensors for microbial
infection. J Clin Invest 1997;100:6 –10.
6. Elewaut D, DiDonato JA, Kim JM, Truong F, Eckmann L, Kagnoff
MF. NF-␬B is a central regulator of the intestinal epithelial cell
innate immune response induced by infection with enteroinvasive
bacteria. J Immunol 1999;163:1457–1466.
7. Jobin C, Panja A, Hellerbrand C, Iimuro Y, DiDonato J, Brenner DA,
Sartor RB. Inhibition of proinflammatory molecule production by
adenovirus-mediated expression of a nuclear factor–␬B superrepressor in human intestinal epithelial cells. J Immunol 1998;
160:410 – 418.
8. Tanaka Y, Imai T, Baba T, Ishikawa I, Uehira M, Nomiyama H,
Yoshie, O. Selective expression of liver and activation-regulated
chemokine (LARC) in intestinal epithelium in mice and humans.
Eur J Immunol 1999;29:633– 642.
9. Rollins BJ. Chemokines. Blood 1997;90:909 –928.
10. Locati M, Murphy PM. Chemokines and chemokine receptors:
biology and clinical relevance in inflammation and AIDS. Annu
Rev Med 1999;50:425– 440.
11. Luster AD, Unkeles JC, Ravetch JV. Gamma-interferon transcriptionally regulates an early-response gene containing homology to
platelet proteins. Nature 1985;315:672– 676.
12. Taub DD, Lloyd AR, Conlon K, Wang JM, Ortaldo JR, Harada A,
Matsushima K, Kelvin DJ, Oppenheim JJ. Recombinant human
interferon-inducible protein 10 is a chemoattractant for human
monocytes and T lymphocytes and promotes T cell adhesion to
endothelial cells. J Exp Med 1993;177:1809 –1814.
GASTROENTEROLOGY Vol. 120, No. 1
13. Liao F, Rabin RL, Yannelli JR, Koniaris LG, Vanguri P, Farber JM.
Human Mig chemokine: biochemical and functional characterization. J Exp Med 1995;182:1301–1314.
14. Cole KE, Strick CA, Paradis TJ, Ogborne KT, Loetscher M, Gladue
RP, Lin W, Boyd JG, Moser B, Wood DE, Sahagan BG, Neote K.
Interferon-inducible T cell ␣ chemoattractant (I-TAC): a novel nonELR CXC chemokine with potent activity on activated T cells
through selective high affinity binding to CXCR3. J Exp Med
1998;187:2009 –2021.
15. Gasperini S, Marchi M, Calzetti F, Laudanna C, Vicentini L, Olsen
H, Murphy M, Liao F, Farber J, Cassatella MA. Gene expression
and production of the monokine induced by IFN-␥ (MIG), IFNinducible T cell ␣ chemoattractant (I-TAC), and IFN-␥–inducible
protein-10 (IP-10) chemokines by human neutrophils. J Immunol
1999;162:4928 – 4937.
16. Sauty A, Dziejman M, Taha RA, Iarossi AS, Neote K, GarciaZepeda EA, Hamid Q, Luster AD. The T cell–specific CXC chemokines IP-10, Mig, and I-TAC are expressed by activated human
bronchial epithelial cells. J Immunol 1999;162:3549 –3558.
17. Qin S, Rottman JB, Myers P, Kassam N, Weinblatt M, Loetscher
M, Koch AE, Moser B, Mackay CR. The chemokine receptors
CXCR3 and CCR5 mark subsets of T cells associated with certain
inflammatory reactions. J Clin Invest 1998;101:746 –754.
18. Loetscher M, Gerber B, Loetscher P, Jones SA, Piali L, ClarkLewis I, Baggiolini M, Moser B. Chemokine receptor specific for
IP10 and Mig: structure, function, and expression in activated
T-lymphocytes. J Exp Med 1996;184:963–969.
19. Valente G, Ozmen L, Novelli F, Geuna M, Palestro G, Forni G,
Garotta G. Distribution of interferon-␥ receptor in human tissues.
Eur J Immunol 1992;22:2403–2412.
20. Colgan SP, Parkos CA, Matthews JB, D’Andrea L, Awtrey CS,
Lichtman AH, Delp-Archer C, Madara JL. Interferon-␥ induces a
cell surface phenotype switch on T84 intestinal epithelial cells.
Am J Physiol 1994;267:C402–C410.
21. Mayer L, Eisenhardt D, Salomon P, Bauer W, Plous R, Piccinini L.
Expression of class II molecules on intestinal epithelial cells in
humans. Differences between normal and inflammatory bowel
disease. Gastroenterology 1991;100:3–12.
22. Colgan SP, Morales VM, Madara JL, Polischuk JE, Balk SP,
Blumberg RS. IFN-␥ modulates CD1d surface expression on intestinal epithelia. Am J Physiol 1996;271:C276 –C283.
23. Huang GT, Eckmann L, Savidge TC, Kagnoff MF. Infection of
human intestinal epithelial cells with invasive bacteria upregulates apical intercellular adhesion molecule-1 (ICAM)-1 expression and neutrophil adhesion. J Clin Invest 1996;98:572–583.
24. Madara JL, Stafford J. Interferon-␥ directly affects barrier function
of cultured intestinal epithelial monolayers. J Clin Invest 1989;
83:724 –727.
25. Witthoft T, Eckmann L, Kim JM, Kagnoff MF. Enteroinvasive bacteria directly activate expression of iNOS and NO production in
human colon epithelial cells. Am J Physiol 1998;275:G564 –
G571.
26. Abreu-Martin MT, Vidrich A, Lynch DH, Targan SR. Divergent
induction of apoptosis and IL-8 secretion in HT-29 cells in response to TNF-␣ and ligation of Fas antigen. J Immunol 1995;
155:4147– 4154.
27. Dwinell MB, Eckmann L, Leopard JD, Varki NM, Kagnoff MF.
Chemokine receptor expression by human intestinal epithelial
cells. Gastroenterology 1999;117:359 –367.
28. Savidge TC, Morey AL, Ferguson DJ, Fleming KA, Shmakov AN,
Phillips AD. Human intestinal development in a severe-combined
immunodeficient xenograft model. Differentiation 1995;58:361–
371.
29. Mosmann TR, Coffman RL. TH1 and TH2 cells: different patterns
of lymphokine secretion lead to different functional properties.
Annu Rev Immunol 1989;7:145–173.
30. McCormick BA, Colgan SP, Delp-Archer C, Miller SI, Madara JL.
January 2001
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
Salmonella typhimurium attachment to human intestinal epithelial monolayers: transcellular signalling to subepithelial neutrophils. J Cell Biol 1993;123:895–907.
Sansonetti PJ, Arondel J, Huerre M, Harada A, Matsushima K.
Interleukin-8 controls bacterial transepithelial translocation at
the cost of epithelial destruction in experimental shigellosis.
Infect Immun 1999;67:1471–1480.
Kanof ME, James SP, Strober W. The phenotype and function of
T cells in the lamina propria of the human intestine. Reg Immunol
1998;1:190 –195.
Agace WW, Roberts AI, Wu L, Greineder C, Ebert EC, Parker CM.
Human intestinal lamina propria and intraepithelial lymphocytes
express receptors specific for chemokines induced by inflammation. Eur J Immunol 2000:30:819 – 826.
Lundqvist C, Melgar S, Yeung MM, Hammarstrom S, Hammarstrom ML. Intraepithelial lymphocytes in human gut have lytic
potential and a cytokine profile that suggest T helper 1 and
cytotoxic functions. J Immunol 1996;157:1926 –1934.
Ohmori Y, Hamilton TA. The interferon-stimulated response element and a ␬B site mediate synergistic induction of murine IP-10
gene transcription by IFN-␥ and TNF-␣. J Immunol 1995;154:
5235–5244.
Loetscher M, Loetscher P, Brass N, Meese E, Moser B. Lymphocyte-specific chemokine receptor CXCR3: regulation, chemokine
binding and gene localization. Eur J Immunol 1998;28:3696 –
3705.
Laurent F, Eckmann L, Savidge TC, Morgan G, Theodos C, Naciri
M, Kagnoff MF. Cryptosporidium parvum infection of human intestinal epithelial cells induces the polarized secretion of C-X-C
chemokines. Infect Immun 1997;65:5067–5073.
Kolios G, Rooney N, Murphy CT, Robertson DA, Westwick J.
Expression of inducible nitric oxide synthase activity in human
colon epithelial cells: modulation by T lymphocyte derived cytokines. Gut 1998;43:56 – 63.
Deng W, Ohmori Y, Hamilton TA. Mechanisms of IL-4 –mediated
suppression of IP-10 gene expression in murine macrophages.
J Immunol 1994;153:2130 –2136.
Fuss IJ, Neurath M, Boirivant M, Klein JS, de la Motte C, Strong
SA, Fiocchi C, Strober W. Disparate CD4⫹ lamina propria (LP)
IFN–INDUCIBLE CHEMOKINE SECRETION BY EPITHELIAL CELLS
59
lymphokine secretion profiles in inflammatory bowel disease.
Crohn’s disease LP cells manifest increased secretion of IFN-␥,
whereas ulcerative colitis LP cells manifest increased secretion
of IL-5. J Immunol 1996;157:1261–1270.
41. Nilsen EM, Jahnsen FL, Lundin KE, Johansen FE, Fausa O, Sollid
LM, Jahnsen J, Scott H, Brandtzaeg P. Gluten induces an intestinal cytokine response strongly dominated by interferon-␥ in
patients with celiac disease. Gastroenterology 1998;115:551–
563.
42. Uguccioni M, Gionchetti P, Robbiani DF, Rizzello F, Peruzzo S,
Campieri M, Baggiolini M. Increased expression of IP-10, IL-8,
MCP-1, and MCP-3 in ulcerative colitis. Am J Pathol 1999;155:
331–336.
43. Grimm M, Doe W. Chemokines in inflammatory bowel disease
mucosa: expression of RANTES, macrophage inflammatory protein (MIP)-1␣, MIP-1␤ and ␥-interferon–inducible protein 10 by
macrophages, lymphocytes, endothelial cells and granulomas.
Inflamm Bow Dis 1996;2:88 –96.
Received April 13, 2000. Accepted August 2, 2000.
Address requests for reprints to: Martin F. Kagnoff, M.D., Laboratory
of Mucosal Immunology, Department of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 920930623. e-mail: [email protected].
Supported by National Institutes of Health (NIH) grant DK35108; by
NIH Training Grant T32 DK7202 and a Career Development award
from the Crohn’s and Colitis Foundation of America (to M.B.D); by the
Deutsche Forschungsgemeinschaft (LU732/1-1) and the Deutsche Gesellschaft für Verdauungs-und Stoffwechselerkrankungen (to N.L.);
and by a Research Grant from the Crohn’s and Colitis Foundation of
America (to L.E.)
The current affiliation for Dr. Lügering is Medizinische Klinik und
Poliklinik B, Universität Münster, Münster, Germany.
The authors thank J. R. Smith, M. P. Housley, and J. Leopard for
expert technical support; Dr. N. Varki for helpful advice on the immunostaining studies; and Dr. K. Neote for providing the monoclonal
antibody to I-TAC.