Short exposure of intestinal epithelial cells to TNF-a and
histamine induces Mac-1-mediated neutrophil adhesion
independent of protein synthesis
Ryukou Miyata,*† Kazuhisa Iwabuchi,* Sumio Watanabe,† Nobuhiro Sato,† and Isao Nagaoka*
Departments of *Biochemistry and †Gastroenterology, Juntendo University, School of Medicine, Tokyo, Japan
Abstract: Neutrophils play an important role in
intestinal inflammation by interacting with intestinal epithelial cells. In this study, we evaluated
neutrophil adhesion to intestinal epithelial cells
using intestinal epithelial cell line HT29 stimulated
with tumor necrosis factor a (TNF-a) and histamine for a short time (30 min). The TNF-a and
histamine stimulation markedly increased neutrophil adhesion. The increased adhesion was inhibited by anti-CD11b and anti-CD18 monoclonal
antibodies (mAbs), but not by anti-CD11a and
anti-CD54 (ICAM-1) mAbs. It is interesting that
flow cytometric analysis revealed that ICAM-1 expression on HT29 cells was not changed by TNF-a
and histamine stimulation. Moreover, the increased
adhesion was inhibited by proteinase K treatment
but not cycloheximide treatment of HT29 cells.
Together these observations suggest that short exposure of HT29 cells to TNF-a and histamine induces
CD11b/CD18 (Mac-1)-dependent but CD11a/CD18
(LFA-1)-independent neutrophil adhesion to intestinal epithelial cells, and ICAM-1 is not likely to be
involved in the interactions. Furthermore, epithelial cell ligand(s) for neutrophils is likely protein
molecule(s) that is expressed on the cell by stimulation independent protein synthesis. However, it is
also possible that neutrophil activating factor(s),
which stimulates neutrophils to bind with epithelial
ligands via Mac-1, is expressed by epithelial cells
during stimulation. J. Leukoc. Biol. 66: 437–446;
1999.
Key Words: human · neutrophils · inflammation · adhesion
molecules · inflammatory mediators
INTRODUCTION
Neutrophils play an important role in the pathogenesis of
inflammatory bowel diseases such as ulcerative colitis, Crohn’s
disease, and bacterial enterocolitis [1, 2]. These diseases are
characterized by the formation of crypt abscess, accumulation
of neutrophils adjacent to crypt epithelial cells of the intestinal
mucosa [1, 2]. Recently, neutrophils have been recognized not
only to adhere to intestinal epithelia, but also to influence
epithelial functions. For example, neutrophil adhesion to
intestinal epithelia can modulate epithelial barrier function [3]
and promote Cl2 secretion in secretory diarrhea [4]. During
acute inflammation of intestine, neutrophils emigrate from
bloodstream into mucosal tissue, adhere to the basolateral
surface of intestinal epithelial cells, and transmigrate between
these cells into the luminal space [5]. Transmigrated neutrophils can adhere to the apical surface of epithelial cells, and
may give cytotoxic effects on epithelium with local release of
cytokines, proteases, and products of respiratory burst [6]. In
fact, epithelial dysfunction and symptoms in inflammatory
bowel diseases correlate with the magnitude of neutrophil
accumulation in the intestinal epithelium and mucosa [7].
The process of adhesion and migration of neutrophils across
endothelial cells has been well understood. The initial adhesive
interaction between neutrophil and endothelium is regulated by
interactions with selectins and carbohydrate-containing counter
receptors [8–10]. Selectin-mediated adhesion, which is relatively weak, allows neutrophils to roll on endothelium, and
facilitates their firm adhesion through interactions between
neutrophil b2 integrins [LFA-1 (CD11a/CD18) and Mac-1
(CD11b/CD18)] and endothelial cell counter-receptors such as
intercellular adhesion molecule-1 (ICAM-1, CD54) [9, 10].
Recently, it has been shown that neutrophil adhesion to
basolateral surface of intestinal epithelial cells is dependent on
neutrophil b2 integrin Mac-1 (CD11b/CD18) but not on LFA-1
(CD11a/CD18), unlike neutrophil firm adhesion to endothelial
cells [11]. Moreover, it has been demonstrated that the counterreceptor of basolateral intestinal epithelium is not ICAM-1
[12]. On the other hand, it has been demonstrated that
transepithelial migration is mediated by neutrophil and epithelial CD47 molecules, as observed for transendothelial migration, which is mediated by neutrophil and endothelial CD47
molecules [12, 13]. Thus, the process that applies to neutrophilendothelial interactions does not necessarily apply to neutrophilepithelial interactions. Regardless of the detailed investigation
of basolateral adhesion and transepithelial migration of neutrophils, adhesion of post-transmigrated neutrophils to apical
surface of intestinal epithelial cells has been little studied.
Histamine and tumor necrosis factor a (TNF-a) are known to
be mediators of inflammatory reactions [14, 15]. Histamine
Correspondence: Isao Nagaoka, Department of Biochemistry, Juntendo
University, School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421,
Japan. E-mail: [email protected]
Received August 17, 1998; revised April 19, 1999; accepted April 20, 1999.
Journal of Leukocyte Biology
Volume 66, September 1999
437
content and its secretion have been found to be significantly
increased particularly in affected mucosa of ulcerative colitis
and Crohns disease compared with unaffected tissues [16, 17].
Moreover, TNF-a concentrations are demonstrated to be markedly increased in the affected intestinal mucosa, serum, and
feces of active inflammatory bowel diseases [18–20]. TNF-a
and histamine induce rapid up-regulation of CD11b/CD18 on
neutrophils, and P-selectin on endothelial cells and platelets,
respectively, resulting in increased intercellular adhesion [23,
24]. Thus, it is interesting to know whether short exposure of
intestinal epithelial cells to TNF-a and histamine induces
neutrophil-intestinal epithelial cell interactions. In this study,
we have found that short exposure (30 min) of intestinal
epithelial cells HT29 to TNF-a and histamine rapidly increased neutrophil adhesion to epithelial cells.
MATERIALS AND METHODS
Reagents
Human recombinant TNF-a and human recombinant endothelial interleukin-8
(IL-8) were obtained from Genzyme, Boston, MA; endoglycosidase H (endo-bN-acetylglucosaminidase H), Arthrobacter ureafaciens sialidase, and tunicamycin were obtained from Boehringer Mannheim, GmbH (Mannheim, Germany).
Other reagents were obtained from Sigma Chemical Co. (St. Louis, MO). RGD
peptides (Arg-Gly-Asp-Ser) were obtained from Peptide Institute Inc., Osaka,
Japan. All tissue culture supplies were obtained from GIBCO Laboratories (St.
Lawrence, MA) and Iwaki Glass Co. (Tokyo, Japan).
Cell culture
The human colon epithelial cell line HT29 (ATCC HTB38) [25] was obtained
from American Type Culture Collection (Rockville, MD). HT29 cells were
grown in Macoy 5a medium supplemented with 14 mM NaHCO3, 100 U/mL
penicillin, 100 µg/mL streptomycin, and 10% fetal bovine serum (FBS). HT29
cells were cultured in 150 cm2 dishes, and confluent HT29 cells were harvested
by trypsin/EDTA solution (0.25% trypsin and 0.25% EDTA in Ca21- and
Mg21-free phosphate-buffered saline, pH 7.2). Trypan blue dye exclusion
showed that more than 95% of cells were viable. For adhesion assay, the cells
were seeded in a 96-well tissue culture plate at a density of 5 3 104 cells/cm2 in
0.1 mL per well.
In some experiments, the human colon epithelial cell lines T84 (ATCC
CL248) [26], obtained from American Type Culture Collection, Caco-2
(RCB0988) [27], obtained from Riken Gene Bank (Tsukuba, Japan), and human
umbilical endothelial cells (Clonetics, San Diego, CA) were also used.
Antibodies
The following antibodies were used: anti-CD11a mAb G43-25B (murine
IgG2b,k) from PharMingen (San Diego, CA); anti-CD11b mAb D12 (murine
IgG2a,k) from Becton-Dickinson (San Jose, CA); anti-CD18 mAb 7E4 (murine
IgG1) from Immunotech S.A. (Marseille, France); anti-CD54 mAb RR1/1
(murine IgG1) from Bender MedSystems (Vienna, Austria); anti-CD54 mAb
84H10 (murine IgG1) from Serotec (Oxford, UK); anti-CD4 mAb TH/1 (murine
IgG1), used as a control for nonspecific binding of mAb, from Seikagaku Corp.
(Tokyo, Japan); and anti-CD54 mAb R6.5 (murine IgG1) from Dr. R. Rothlein,
Boehringer Ingelheim (Ridgefield, CT). R6.5 F(ab’)2 fragments were prepared
by pepsin digestion and Protein G affinity chromatography (Pierce, Rockford,
IL). Purity of antibody digests was confirmed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE).
Isolation of neutrophils
Neutrophils were isolated from sodium citrate-anticoagulated peripheral blood
of normal healthy human volunteers with the use of Polymorphprepy (Nycomed
Pharma AS, Oslo, Norway) centrifugation as described previously [28]. Isolated
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Volume 66, September 1999
neutrophils were suspended in buffer A (137 mM NaCl, 2.7 mM KCl, 8.1 mM
Na2HPO4, 1.5 mM KH2PO4, pH 7.4) at a concentration of 1 3 106 cells/mL and
used for subsequent experiments. Differential cell counts with Wright-Giemsa
stain showed that more than 98% of the cells were neutrophils. More than 97%
of the cells were viable as determined by trypan blue dye exclusion.
Adhesion assay
Isolated neutrophils were labeled with 51Cr as described previously [29].
Briefly, neutrophils (1 3 106 cells/mL) were incubated with 2 µCi/mL
Na251CrO4 (200–900 Ci/g Cr, ICN Biomedicals Inc., Costa Mesa, CA) at 30°C
for 30 min with gentle shaking. After washing twice with buffer A, labeled
neutrophils were suspended at 5 3 105 cells/mL in buffer B (137 mM NaCl, 2.7
mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, 1 mM MgSO4, 1 mM CaCl2, pH
7.4) containing 0.1 mg/mL bovine serum albumin (BSA). Flow cytometric
analysis revealed that CD11b and CD62L (L-selectin) expression on neutrophils was not affected by the labeling procedure (data not shown). In vitro
adhesion assay was carried out according to the method of Spertini et al. [30]
with slight modification. Confluent intestinal epithelial cell monolayers in
96-well plates were stimulated at 37°C with 100 U/mL TNF-a for 5 h or 30 min,
0.1 mM histamine for 30 min in medium containing 10% FBS, or 0.1 U/mL
thrombin (Miles Inc., Kankakee, IL) for 30 min in FBS-free medium, and
washed with buffer B. Then the labeled neutrophils (100 µL) were added to the
intestinal epithelial monolayers and incubated at 37°C for 30 min under static
condition. After incubation, the monolayers were washed three times with
pre-warmed buffer B to remove nonadherent neutrophils, and then the cells
were lysed in 0.1 N NaOH/1% Triton X-100 for 60 min at room temperature.
Radioactivities of the lysates were counted using a g-counter (model 1282
CompuGamma, Pharmacia LKB Biotechnology, Uppsala, Sweden). Neutrophil
adhesion (%) was calculated as (cpm in lysates/cpm in applied neutrophils) 3
100 and expressed as the percentage of adherent cells. In some experiments,
HT29 cell monolayers were stimulated with 0.1 mM histamine at 37°C for 30
min in the presence of indicated concentrations of diphenhydramine or
cimetidine. To determine the effect of anti-Mac-1 or anti-LFA-1 mAbs on
neutrophil adhesion to intestinal epithelial cells, neutrophils were preincubated with 10 µg/mL anti-CD11a mAb, anti-CD11b mAb, anti-CD18 mAb, or
anti-CD4 mAb at 4°C for 30 min, and then added to the intestinal epithelial
monolayers. In case of treatment with anti-CD54 (ICAM-1) mAbs, TNF-a- or
histamine-stimulated intestinal epithelial cells were preincubated with 10
µg/mL anti-human CD54 mAbs RR1/1, R6.5, or F(ab)2 fragments of R6.5 at
37°C for 30 min, and then added with labeled neutrophils. To determine the
effects of divalent cations on neutrophil adhesion to intestinal epithelial cells,
HT29 cells and labeled neutrophils were washed once with buffer A containing
1 mM EDTA. After washing twice with buffer A, adhesion assay was performed
in buffer A, buffer A containing 1 mM CaCl2, buffer A containing 1 mM EGTA
and 1 mM MgCl2, or buffer B. Furthermore, to examine the effect of neutrophil
activation, labeled neutrophils were stimulated with 5 3 10-8 M IL-8 or 100
U/mL TNF-a at 37°C for 15 min, and used for adhesion assay. In some
experiments, adhesion assay was performed using human umbilical endothelial
cells and human colon epithelial cell lines (T84 and Caco-2), as described
above.
Treatment of intestinal epithelial cells with
proteinase K, glycosidases, or metabolic
inhibitors
TNF-a (5 h and 30 min)- or histamine (30 min)-stimulated epithelial cells were
incubated with 10 µg/mL proteinase K at 37°C for 1 min, and washed with
buffer A containing 4 mM phenylmethylsulfonyl fluoride and 20% FBS.
Moreover, to determine the effect of protein synthesis inhibitor on neutrophil
adhesion to intestinal epithelial cells, epithelial cells were stimulated with
TNF-a (5 h and 30 min) or histamine (30 min) in the presence of 1 µg/mL
cycloheximide at 37°C, and washed with buffer B. After washing, monolayers
were used for adhesion assay.
To clarify the role of sugar chains of intestinal epithelial cells, HT29 cells
were treated with 15 mU/mL endoglycosidase H or 50 mU/mL sialidase (diluted
in FBS-free media) at 37°C for 24 h. In case of tunicamycin treatment,
epithelial cells were grown in media containing 0.5 mg/mL tunicamycin for 3–4
days. After glycosidase or tunicamycin treatment, intestinal cell monolayers
were washed with buffer B, and adhesion assay was performed. The amounts of
http://www.jleukbio.org
epithelial cell-associated sugar chains were quantitated by immuno-slot blot
analysis (see below).
Furthermore, the involvement of glycosylphosphatidyl inositol (GPI)anchored protein in neutrophil adhesion to epithelial cells was investigated by
treating TNF-a- or histamine-stimulated intestinal epithelial cells with 1 U/mL
phosphatidylinositol-specific phospholipase C (PI-PLC) at 37°C for 30 min.
In addition, to examine the involvement of heparin-like glycosaminoglycans
and extracellular matrix proteins containing RGD sequences in neutrophilHT29 cell interactions, HT29 cells were stimulated with TNF-a (5 h and 30
min) or histamine (30 min) in the presence of 0.01–1 IU/mL of heparinase III in
FBS-free medium, or adhesion assay was performed in the presence of 0.01–1
mg/mL RGD peptide [31–33].
Flow cytometry
Intestinal epithelial cells were seeded in 24-well tissue culture plates at a
density of 5 3 104 cells/cm2 in 1.0 mL per well and cultured to confluence.
After stimulation with TNF-a (5 h and 30 min) or histamine (30 min) at 37°C,
intestinal epithelial monolayers were detached by treatment with trypsin/
EDTA. We confirmed that this treatment did not interfere with the detection of
ICAM-1 (data not shown). Detached cells (5 3 105) were incubated with 1
µg/mL anti-CD54 (ICAM-1) mAb 84H10 or anti-CD4 mAb TH/1 for 30 min at
4°C. After incubation, the cells were washed with 0.25% EDTA in buffer A, and
further incubated with fluorescein isothiocyanate (FITC)-conjugated goat
anti-mouse IgG 1 M (H 1 L) (American Qualex International Inc., La Mirada,
CA) for 30 min at 4°C. After washing with 0.25% EDTA in buffer A, the cells
were analyzed by flow cytometry (FACScan, Becton-Dickinson, Rutherford,
NJ), and data were analyzed using Cell Quest software (Becton-Dickinson). Cell
viability was .95% as assessed by propidium iodide staining. In some
experiments, neutrophils were stimulated with 100 U/mL TNF-a or 5 3 10-8 M
IL-8 at 37°C for 15 min. After washing with buffer A, neutrophils were
incubated with 1 µg/mL anti-CD11a mAb G43-25B or anti-CD11b mAb D12 at
4°C for 30 min, and analyzed by flow cytometry as described above. The
median fluorescence intensities were compared between resting and stimulated
cells.
Immuno-slot-blot assay
After treatment with glycosidase or tunicamycin, the amounts of epithelial
cell-associated sugar chains were quantitated using biotinylated lectins as
described previously [29]. Briefly, intestinal epithelial cells treated with
endoglycosidase H, sialidase, or tunicamycin (as described above), were
washed twice with 0.25% EDTA in buffer A, added with 2 mM phenylmethylsulfonyl fluoride, and sonicated for 10 s at 168 W (Ultrasonic Disruptor, Tomy
Corp., Tokyo, Japan). The sonicates were centrifuged at 10,000 g for 15 min at
4°C, and the supernatants were further centrifuged at 100,000 g for 1 h at 4°C.
The resultant membrane pellets were dissolved in lysis buffer (50 mM sodium
phosphate buffer, pH 7.5, 0.1 M NaCl, 0.5% Nonidet P-40), and protein
concentrations were measured by BCA protein assay reagent (Pierce) using
BSA as a standard. The membrane samples were mixed with sample buffer (125
mM Tris-HCl, pH 6.8, 4% sodium dodecyl sulfate, 0.125% Nonidet P-40, 5 mM
dithiothreitol), boiled for 3 min, and loaded onto PVDF PSQ membrane
equilibrated with blotting buffer (25 mM Tris-HCl, 192 mM glycine, pH 8.9,
containing 5 mM methanol) using a micro-sample filtration manifold Minifold II
(Schleicher and Schuell, Dassel, Germany). After aspiration, the blotted
membranes were washed in blotting buffer and dried. The membranes were
washed with blocking buffer (10 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 0.05%
Tween-20) and incubated with 5 µg/mL biotinylated ConA (Canavalia ensiformis) [34] or SSA (Sambucus sieboldiana) lectin [35] (Seikagaku Corp.) in
blocking buffer for 1 h. Then the membranes were washed with blocking buffer
and incubated with horseradish peroxidase-conjugated streptavidin (Zymed
Laboratories Inc., South San Francisco, CA) for 30 min. The membranes were
washed with blocking buffer, and sugar chains were detected by ECLy Western
blotting detection reagents (Amersham International, Buckinghamshire, UK).
The detected sugar chain bands were analyzed using an interpretive densitometer Master Scany 486 (CSP Inc., Billerica, MA).
Data analysis
Statistical analysis was performed using Student’s t test, and P , 0.05 was
considered to be significant. The results are shown as mean 6 SD.
RESULTS
Effect of TNF-a and histamine stimulation on
neutrophil adhesion to intestinal epithelial cells
As shown in Figure 1, 10.7 6 1.2% of neutrophils adhered to
resting HT29 epithelial cells. When epithelial cells were
stimulated with TNF-a for 5 h, neutrophil adhesion significantly increased (3.3-fold). Pretreatment with TNF-a for 30
min also increased neutrophil adhesion to HT29 cells (2.8fold), and the increased level was almost the same as that of
neutrophil adhesion to TNF-a-stimulated (5 h) HT29 cells. It is
interesting that when HT29 cells were pretreated with histamine for 30 min, neutrophil adhesion was increased to the
levels of TNF-a stimulation (5 h and 30 min). Thrombin, which
had been shown to increase neutrophil adhesion to endothelial
cells [36], did not affect neutrophil adhesion to HT29 cells.
Moreover, the number of adherent neutrophils was counted
in 10 microscopic fields (0.09 mm2/field): 470 6 158 (cells/
mm2; mean 6 SD) for resting HT29 cells, 1657 6 505 for
TNF-a (5 h)-stimulated HT29 cells, 1346 6 395 for TNF-a
(30 min)-stimulated HT29 cells, 1422 6 377 for histamine (30
min)-stimulated HT29 cells, and 547 6 211 for thrombin
(30 min)-stimulated HT29 cells. Thus, neutrophil adhesion
calculated using radioactivities reflects the number of neutrophils that actually come in contact with monolayer.
Effect of histamine receptor antagonists on
neutrophil adhesion to histamine-stimulated
intestinal epithelial cells
Histamine modulates cell functions via its specific receptors
such H1 and H2 receptors [14]. To determine the type of
histamine receptors, the effect of diphenhydramine (H1 antago-
Fig. 1. Effect of TNF-a and histamine stimulation on neutrophil adhesion to
intestinal epithelial cells. Confluently cultured HT29 cell monolayers in
96-well plates were incubated at 37°C without (Resting) or with 100 U/mL
TNF-a for 5 h or 30 min, 0.1 mM histamine for 30 min, or 0.1 U/mL thrombin
for 30 min. After washing, 51Cr-labeled neutrophils were added to the
monolayers and incubated at 37°C for 30 min under static condition. After
incubation, the monolayers were washed to remove nonadherent neutrophils
and then lysed. Radioactivities of adherent neutrophils were counted using a
g-counter, and neutrophil adhesion was expressed as percentage of total
neutrophils. Each bar represents the mean 6 SD of six separate experiments.
Values were compared between without (Resting) and with stimulation (TNF-a,
5 h and 30 min; histamine, 30 min). ****P , 0.001.
Miyata et al.
Neutrophil adhesion to intestinal epithelial cells
439
nist) and cimetidine (H2 antagonist) on neutrophil adhesion to
intestinal epithelial cells was examined. As shown in Figure 2,
diphenhydramine inhibited neutrophil adhesion to histaminestimulated HT29 cells in a dose-dependent manner. In contrast,
cimetidine hardly affected neutrophil adhesion to histaminestimulated HT29. These results suggest that histamine stimulates intestinal epithelial cells via H1 receptors but not H2
receptors.
cells is likely independent of ICAM-1. Next, we examined the
expression of ICAM-1 on intestinal epithelial cells by flow
cytometry. As shown in Figure 4, stimulation with TNF-a for 5
h markedly up-regulated ICAM-1 expression on HT29 cells,
whereas stimulation with TNF-a and histamine for 30 min did
not affect ICAM-1 expression. These results demonstrate that
short-duration stimulation (30 min) of HT29 cells with TNF-a
and histamine does not change ICAM-1 expression.
Effect of mAbs on neutrophil adhesion to
intestinal epithelial cells
Effect of divalent cations on neutrophil adhesion
to intestinal epithelial cells
To identify adhesion molecules involved in neutrophil adhesion
to HT29 cells, we examined the effect of neutralizing mAbs
against b2 integrins and ICAM-1. As shown in Figure 3,
anti-CD11b and anti-CD18 mAbs strikingly inhibited neutrophil adhesion not only to TNF-a-stimulated HT29 cells (5 h
and 30 min) but also to histamine-stimulated HT29 cells (30
min). In contrast, anti-CD11a mAb did not inhibit neutrophil
adhesion to HT29 cells stimulated with TNF-a (5 h and 30 min)
and histamine (30 min). Anti-CD54 mAbs [RR1/1, R6.5 and
F(ab’)2 fragments of R6.5] significantly reduced neutrophil
adhesion to TNF-a (5 h)-stimulated HT29 cells. However, they
did not affect neutrophil adhesion to HT29 cells stimulated
with TNF-a or histamine for 30 min. In separate experiments,
we confirmed that neutrophil adhesion to TNF-a-stimulated
human umbilical endothelial cells was inhibited by antiCD11a, anti-CD11b, anti-CD18, or anti-CD54 mAbs, indicating that mAbs used in this study can interfere with the
interactions between Mac-1/LFA-1 and ICAM-1 molecules
(Table 1). Together these observations suggest that neutrophil
adhesion to TNF-a- or histamine-stimulated (30 min) intestinal
epithelial cells is mediated mainly by Mac-1 (CD11b/CD18)
but not by LFA-1 (CD11a/CD18) and ICAM-1 (CD54).
We next examined the effects of divalent cations on neutrophil
adhesion to intestinal epithelial cells. As shown in Figure 5, in
the absence of added divalent cations, neutrophil adhesion to
TNF-a (5 h and 30 min)- and histamine (30 min)-stimulated
HT29 cells was reduced to almost the resting level. The
presence of 1 mM MgCl2 retained the increased levels of
neutrophil adhesion to stimulated HT29 cells. In contrast,
neutrophil adhesion to stimulated HT29 cells was markedly
decreased in the presence of only CaCl2. These results indicate
that the adhesive interaction between neutrophils and TNF-aor histamine-stimulated HT29 cells is dependent on Mg21 but
not on Ca21.
Flow cytometric analysis of ICAM-1 expression
on intestinal epithelial cells
ICAM-1 is known as a counter-receptor for CD11b/CD18 [9,
10]. However, the above results suggest that CD11b/CD18mediated neutrophil adhesion to stimulated intestinal epithelial
Effect of proteinase K and cycloheximide
treatment on neutrophil adhesion to intestinal
epithelial cells
To characterize the intestinal epithelial cell ligand(s) for
neutrophils, we examined the effect of proteinase K or cycloheximide treatment of HT29 cells on neutrophil adhesion. As shown
in Figure 6, proteinase K treatment of HT29 cells considerably
inhibited neutrophil adhesion to TNF-a (5 h and 30 min)- and
histamine (30 min)-stimulated HT29 cells. In contrast, cycloheximide treatment inhibited neutrophil adhesion to TNF-a (5
h)-stimulated HT29 cells by 51% but did not affect neutrophil
adhesion to TNF-a (30 min)- or histamine (30 min)-stimulated
HT29 cells. Propidium iodide staining showed that proteinase
K and cycloheximide treatment did not affect cell viability of
HT29 cells (.95%). Moreover, flow cytometric analysis re-
Fig. 2. Effect of histamine receptor antagonists on neutrophil adhesion to histamine-stimulated intestinal epithelial cells.
HT29 cell monolayers were incubated
without (Resting) or with 0.1 mM histamine in the absence (None) or presence of
indicated concentrations of H1 receptor
antagonist, diphenhydramine, or H2 receptor antagonist, cimetidine at 37°C for 30
min, followed by adhesion assay. Each bar
represents the mean 6 SD of three separate experiments. Values were compared
between the absence (None) and presence
of diphenhydramine. *P , 0.05, ***P ,
0.005.
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Journal of Leukocyte Biology
Volume 66, September 1999
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Fig. 3. Effect of mAbs on neutrophil adhesion to intestinal epithelial cells. Neutrophil adhesion to unstimulated (Resting) and 100 U/mL TNF-a (5 h and 30 min)or 0.1 mM histamine (30 min)-stimulated HT29 were assayed in the absence (None) or presence of blocking mAbs. For b2 integrin mAbs, neutrophils were incubated
at 4°C for 30 min with anti-CD11a mAb, anti-CD11b mAb, anti-CD18 mAb, or anti-CD4 mAb (a negative control for nonspecific binding of mAb; 10 µg/mL each),
and then used for adhesion assay. For ICAM-1 mAbs, HT29 cells were incubated with anti-CD54 mAb RR1/1, R6.5, or F(ab’)2 fragments of R6.5 (10 µg/mL) at 37°C
for 30 min, followed by adhesion assay. Each bar represents the mean 6 SD of three separate experiments. Values were compared between the absence (None) and
presence of mAbs (anti-CD11b, anti-CD18). *P , 0.05, ***P , 0.005, ****P , 0.001.
vealed that proteinase K or cycloheximide treatment markedly
reduced TNF-a (5 h)-induced ICAM-1 expression on HT29
cells, but hardly affected ICAM-1 expression on TNF-a- and
histamine-stimulated (30 min) HT29 cells (data not shown).
TABLE 1. Effect of mAbs on Nontreated- or IL-8-Stimulated
Neutrophil Adhesion to TNF-a-Stimulated Human Umbilical
Endothelial Cells
Effect of endoglycosidase H, sialidase, and
tunicamycin treatment on neutrophil adhesion to
intestinal epithelial cells
Adhesion (% of total)
None
Anti-CD11a
Anti-CD11b
Anti-CD18
Anti-CD54 RR1/1
Anti-CD54 R6.5
Anti-CD54 F(ab8)2
Anti-CD4
These results suggest that the HT29 cell ligand(s) for neutrophils is likely a protein molecule(s), the expression of which is
dependent on protein synthesis for long-duration stimulation
(5 h) with TNF-a, but independent of protein synthesis for
short-duration stimulation (30 min) with TNF-a and histamine.
Nontreated neutrophils
IL-8-stimulated neutrophils
31.21 6 5.11 (100)
10.96 6 3.67 (35.1)
24.89 6 3.23 (79.8)
16.27 6 5.54 (52.1)
13.29 6 4.78 (42.6)
20.23 6 5.66 (64.8)
19.09 6 6.47 (61.2)
30.24 6 4.58 (96.9)
44.75 6 5.37 (100)
11.97 6 3.87 (26.8)
19.50 6 6.37 (43.6)
14.03 6 3.28 (31.3)
16.40 6 6.87 (36.7)
17.60 6 7.19 (39.3)
18.85 6 5.58 (42.1)
42.77 6 8.32 (95.6)
51Cr-labeled neutrophils were incubated without (Nontreated) or with 5 3
1028 M IL-8 (IL-8-stimulated) at 37°C for 15 min, and adhesion assay was
performed by incubating with TNF-a-stimulated (100 U/ml, 5h) human
umbilical endothelial cells in the absence (None) or presence of blocking
mAbs. In parentheses, relative adhesion is expressed as percentage of adhesion
measured in the absence of mAb (None).
To clarify the role of sugar chains of intestinal epithelial cells,
we treated HT29 cells with endoglycosidase H, sialidase, or
tunicamycin. Endoglycosidase H and sialidase treatment of
HT29 cells did not influence neutrophil adhesion to TNF-a (5 h
and 30 min)- or histamine (30 min)-stimulated HT29 cells. In
addition, tunicamycin, an inhibitor of protein glycosylation, did
not influence neutrophil adhesion to TNF-a- or histaminestimulated HT29 cells (data not shown). Immuno-slot-blot
analysis revealed that 62% of mannose residues recognized by
ConA [34] were removed from HT29 cells by endoglycosidase
H treatment, and 60% of sialic acid residues recognized by SSA
lectin [35] were released by sialidase treatment. Tunicamycin
treatment decreased incorporation of ConA-recognizable sugar
Miyata et al.
Neutrophil adhesion to intestinal epithelial cells
441
Effect of neutrophil stimulation on neutrophil
adhesion to HT29 intestinal epithelial cells
Fig. 4. Flow cytometric analysis of ICAM-1 expression on intestinal epithelial
cells. After incubation without (Resting) or with 100 U/mL TNF-a (5 h and 30
min) or 0.1 mM histamine (30 min), intestinal epithelial cells were detached by
treatment with trypsin/EDTA and recovered. ICAM-1 expression was assessed
using anti-ICAM-1 mAb 84H10 and FITC-labeled secondary antibody. Background (BG) was assessed by incubating cells with anti-CD4 mAb TH/1 (a
negative control for nonspecific binding) and FITC-labeled secondary antibody.
Specifically stained cell numbers (cell counts) were indicated on the ordinate
plotted against fluorescence intensity in a log scale on abscissa. Data represent
one of three separate experiments.
Because inflammatory cytokines such as IL-8 and TNF-a are
detected in the lesions of inflammatory bowel diseases [19, 20,
39], these cytokines are assumed to affect neutrophil adhesion
to intestinal epithelial cells by modulating expression of
neutrophil adhesion molecules. As shown in Figure 7, pretreatment of neutrophils with IL-8 or TNF-a increased neutrophil
adhesion to both resting and TNF-a (5 h and 30 min)- or
histamine (30 min)-stimulated HT29 cells (1.3,2.5-fold).
Anti-CD11b and anti-CD18 mAbs inhibited IL-8- or TNF-ainduced neutrophil adhesion to resting and stimulated (TNF-a,
5 h and 30 min; histamine, 30 min) HT29 cells more than 70%,
whereas anti-CD11a and anti-CD54 mAbs did not affect the
adhesion (data not shown). Flow cytometric analysis demonstrated that IL-8 or TNF-a treatment increased CD11b expression (1.7-fold) but did not affect CD11a expression on neutrophils (data not shown). These results suggest that the increased
neutrophil adhesion to HT29 cells by treatment of neutrophils
with IL-8 or TNF-a is mediated by up-regulated Mac-1 but not
by LFA-1.
chains by 60% (data not shown). In our experimental conditions, viability of HT29 cells assessed by propidium iodide
staining was not affected by these treatments (.95%). These
results suggest that sugar chains removed by endoglycosidase
H and sialidase, or sugar chains inhibited by tunicamycin, may
not be involved in CD11b/CD18-mediated neutrophil adhesion
to HT29 cells.
Effect of PI-PLC treatment on neutrophil
adhesion to intestinal epithelial cells
It has been demonstrated that glycosylphosphatidyl-inositol
(GPI)-anchored adhesion molecules such as CD66c are expressed on epithelial cells [37, 38]. To determine whether
GPI-anchored protein(s) is involved in CD11b/CD18-mediated
neutrophil adhesion to HT29 cells, we treated HT29 cells with
PI-PLC. PI-PLC treatment of HT29 cells did not affect
neutrophil adhesion to TNF-a (5 h and 30 min)- or histamine
(30 min)-stimulated HT29 cells (data not shown). Thus, GPIanchored protein(s), if any present on HT29 cells, are not likely
to be involved in CD11b/CD18-mediated neutrophil adhesion
to HT29 cells.
Moreover, we examined the effect of RGD peptide or
heparinase treatment of HT29 cells on neutrophil adhesion.
Neither RGD peptide (0.01–1 mg/mL) nor heparinase (0.01–1
IU/mL) treatment of HT29 cells affected neutrophil adhesion to
TNF-a (5 h and 30 min)- and histamine (30 min)-stimulated
HT29 cells (data not shown). These results suggest that
heparin-like glycosaminoglycans and extracellular matrix proteins containing RGD sequences that are reported to be
recognized by Mac-1 [31–33] are unlikely to be involved in
neutrophil-HT29 cell interactions.
442
Journal of Leukocyte Biology
Volume 66, September 1999
Fig. 5. Effect of divalent cations on neutrophil adhesion to intestinal
epithelial cells. Neutrophil adhesion to unstimulated (Resting)- or stimulated
(100 U/mL TNF-a, 5 h or 30 min; 0.1 mM histamine, 30 min)-HT29 cells was
analyzed in the absence (No metals) or presence of 1 mM MgCl2 and 1 mM
CaCl2 (Mg21/Ca21), 1 mM MgCl2 and 1 mM EGTA (Mg21), or 1 mM CaCl2
(Ca21). Each bar represents the mean 6 SD of three separate experiments.
Values were compared between the presence of MgCl2 and CaCl2 (Mg21/Ca21),
and absence of metals (No metals) or presence of only CaCl2 (Ca21). **P ,
0.01, ***P , 0.005, ****P , 0.001.
http://www.jleukbio.org
Fig. 6. Effect of proteinase K and cycloheximide treatment on neutrophil
adhesion to intestinal epithelial cells. Unstimulated (Resting) and stimulated
(100 U/mL TNF-a, 5 h or 30 min; 0.1 mM histamine, 30 min) HT29 cells were
incubated with 10 µg/mL proteinase K at 37°C for 1 min, and then adhesion
assay was performed. HT29 cells were stimulated at 37°C with 100 U/mL
TNF-a (5 h and 30 min) or 0.1 mM histamine (30 min) in the presence of 1
µg/mL cycloheximide, followed by adhesion assay. Unstimulated or stimulated
HT29 cells without cycloheximide and proteinase K treatment (None) were also
used for adhesion assay. Each bar represents the mean 6 SD of three separate
experiments. Values were compared between without (None) and with cycloheximide or proteinase K treatment. **P , 0.01.
this study, to evaluate the neutrophil adhesion to intestinal
epithelial cells, we added 51Cr-labeled neutrophils to the
intestinal epithelial cell monolayers that had been confluently
cultured on plastic plates. Thus, our experimental system likely
reflects the interactions between neutrophils and apical surface
of intestinal epithelial cells.
It has been reported that neutrophil adhesion to the basolateral surface of intestinal epithelium is dependent on neutrophil
Mac-1 (CD11b/CD18) but not on LFA-1 (CD11a/CD18), and
that ICAM-1 (CD54), a counter-receptor for Mac-1 and LFA-1,
is little expressed on the basolateral surface of intestinal
epithelium [42]. Furthermore, transepithelial migration is reported to be mediated by neutrophil and epithelial CD47
molecules [12]. The present study has revealed that neutrophil
adhesion to confluently cultured HT29 cells is markedly
increased by stimulation of epithelial cells with TNF-a or
histamine (30 min), and the increased adhesion is inhibited by
anti-CD11b and anti-CD18 mAbs but not by anti-CD11a and
anti-CD54 mAbs. Moreover, flow cytometric analysis has shown
that ICAM-1 expression on HT29 cells is not affected by TNF-a
or histamine stimulation (30 min), which substantially increases neutrophil adhesion to HT29 cells. In preliminary
experiments, we have obtained almost the same results using
other intestinal epithelial cell lines (T84 and Caco-2, data not
shown). Together these observations suggest that neutrophil
Mac-1 but not LFA-1 is possibly involved in the interactions
between neutrophils and apical surface of intestinal epithelium,
DISCUSSION
In this study, we have shown that short exposure of HT29 cells
to TNF-a and histamine induces CD11b/CD18 (Mac-1)dependent but CD11a/CD18 (LFA-1)-independent neutrophil
adhesion to intestinal epithelial cells, and ICAM-1 is unlikely
to be involved in the interactions. Furthermore, epithelial cell
ligand(s) for neutrophils may be protein molecule(s) that is
expressed on the cell by stimulation independent of protein
synthesis.
Intestinal inflammatory diseases such as ulcerative colitis,
Crohn’s disease, and bacterial enterocolitis are characterized
by the accumulation of neutrophils in the crypt epithelial
mucosa [1, 2]. Recently, neutrophils have been recognized not
only to adhere to intestinal epithelia, but also to influence
epithelial functions such as barrier maintenance and electrolyte secretion [3, 4]. During acute intestinal inflammation,
neutrophils emigrate from bloodstream into mucosal tissues,
adhere to the basolateral surface of intestinal epithelial cells,
and transmigrate between these cells into the luminal space [5].
Transmigrated neutrophils further adhere to the apical surface
of intestinal epithelial cells, and may influence epithelial
functions by locally releasing cytokines, proteases, and oxygen
metabolites [6]. Intestinal epithelial cell line HT29 cells have
been shown to adhere to substrate via their basolateral surface,
and grow as columnar epithelium with tight junction, with
polarity both in structure and in response to stimuli [40, 41]. In
Fig. 7. Stimulation of neutrophils with IL-8 and TNF-a. 51Cr-labeled
neutrophils were incubated without (Nontreated) or with 5 3 10-8 M IL-8 or 100
U/mL TNF-a at 37°C for 15 min, and adhesion assay was performed by
incubating with unstimulated (Resting) or stimulated (100 U/mL TNF-a, 5 h
and 30 min; 0.1 mM histamine, 30 min) HT29 cells. Each bar represents the
mean 6 SD of three separate experiments. Values were compared between
without (Nontreated) and with IL-8 or TNF-a treatment of neutrophils. *P ,
0.05.
Miyata et al.
Neutrophil adhesion to intestinal epithelial cells
443
and intestinal ICAM-1 molecule is not likely to be utilized as a
counter-receptor for Mac-1 during short-duration stimulation
(30 min) with TNF-a or histamine, although ICAM-1 is
demonstrated to be selectively expressed on the apical intestinal epithelial membrane [42]. The possibility is further supported by the findings that neutrophil adhesion to intestinal
epithelial cells is increased by stimulation of neutrophils with
IL-8 or TNF-a, which up-regulates neutrophil Mac-1 expression. In this study, ICAM-1 expression on HT29 cells was
increased by TNF-a stimulation (5 h), and anti-ICAM-1 mAbs
R6.5 and its F(ab’)2 fragments inhibited neutrophil adhesion to
TNF-a (5 h)-stimulated HT29 cells by 75%. Thus, it is possible
that intestinal epithelial ICAM-1 molecules are involved in the
interactions with neutrophils during long-duration stimulation
with TNF-a.
Ca21 is required for the interactions between selectins and
their counter-receptors, whereas Mg21 is required for the
binding of b2 integrins to their respective ligands [43, 44].
Consistent with this, Mac-1 (b2 integrin)-mediated neutrophil
adhesion to HT29 cells was found to be dependent on Mg21 but
not on Ca21.
It has been shown that TNF-a induces up-regulation of
ICAM-1 and E-selectin molecules on endothelial cells in a
protein synthesis-dependent manner [10], whereas TNF-a
up-regulates Mac-1 molecules on neutrophils in a protein
synthesis-independent manner [21, 22]. Histamine upregulates P-selectin molecules on endothelial cells and platelets independent of protein synthesis [23, 24]. It has been
demonstrated that histamine acts on target cells via distinct
receptors such as H1 and H2 receptors [14], and induces a
rapid and transient increase of chloride secretion by human
colonic epithelial cells via H1 receptor [45]. In this study,
neutrophil adhesion to histamine-stimulated HT29 cells was
inhibited by H1 but not H2 receptor antagonist, suggesting that
histamine apparently induced expression of HT29 cell ligand(s)
via H1 receptors. Moreover, proteinase K treatment markedly
reduced neutrophil adhesion to TNF-a (5 h and 30 min)- and
histamine (30 min)-stimulated HT29 cells, suggesting that
HT29 cell ligand(s) for neutrophils is likely protein molecule(s). However, experiments using protein synthesis inhibitor demonstrated that expression of HT29 cell ligand(s) was
dependent on protein synthesis for long-duration stimulation
(5 h) with TNF-a, but independent of protein synthesis for
short-duration stimulation (30 min) with TNF-a and histamine.
The ligand(s) might be expressed on the cells by short-time
stimulation via up-regulation from intracellular storage pool
and/or conformational change, as with TNF-a-induced Mac-1
expression on neutrophils and histamine-induced P-selectin
expression on endothelial cells and platelets [21–24].
It has been demonstrated that sugar chains play an important
role in the interactions between adhesion molecules and their
ligand(s) [8–10]. Moreover, CD11b molecule has a lectin site
that recognizes mannose, glucose, and N-acetyl-D-glucosamine
residues present in polysaccharides [46]. To clarify the involvement of sugar chains of HT29 cells in neutrophil-intestinal
epithelial cell interactions, we examined the effect of endoglycosidase H, sialidase, and tunicamycin treatment of HT29 cells
on neutrophil adhesion. Unexpectedly, endoglycosidase H,
444
Journal of Leukocyte Biology
Volume 66, September 1999
sialidase, and tunicamycin treatment did not affect neutrophil
adhesion to HT29 cells. Endoglycosidase H preferentially
hydrolyzes the high-mannose type N-glycans [47], and tunicamycin inhibits the formation of N-glycans [48]. In this study,
when N-glycans were quantitated by the amounts of mannose
residues, more than 60% of N-glycans were removed by
endoglycosidase H treatment, and 60% of N-glycan formation
was inhibited by tunicamycin treatment. Sialidase treatment
also removed 60% of sialic acid residues from HT29 cells.
Thus, it is unclear whether sugar chains such as sialic acids
and N-glycans of intestinal epithelial cells are mainly involved
in Mac-1-mediated neutrophil adhesion to HT29 cells. Furthermore, epithelial counter-receptor for neutrophil Mac-1 does not
seem to be a GPI-anchored type protein, and heparin like
glycosaminoglycans, and extracellular matrix protein containing RGD sequences that are reported to be recognized by Mac-1
[31–33], is unlikely involved in neutrophil-HT29 cell interactions.
HT29 cell ligand(s) for neutrophil Mac-1 is not defined at
this stage. However, almost the same effects were observed on
TNF-a- and histamine (30 min)-stimulated HT29 cells using
neutralizing mAbs, divalent cations, proteinase K, glycosidases, glycosylation inhibitor, PI-PLC, heparinase, and RGD
peptide. Thus, it is tempting to speculate that a similar
molecule(s) is involved in Mac-1-mediated neutrophil adhesion
to TNF-a- and histamine-stimulated HT29 cells. It is interesting that cell surface labeling of HT29 cells using 125I followed
by SDS-PAGE analysis [28] preliminarily revealed that proteins
with molecular masses of 310, 250, 145, 85, and 65 kDa were
up-regulated by stimulation with not only TNF-a (5 h and 30
min) but also histamine (30 min; data not shown); however, it is
not clear which of these epithelial proteins is actually mediating the interaction with Mac-1 molecule. Epithelial ligand(s)
could be identified in the future by techniques such as
cross-linking of intestinal epithelial ligand(s) with Mac-1 using
chemical cross-linkers, and cloning of intestinal epithelial
ligand(s) for Mac-1 from intestinal epithelial cell cDNA library.
In contrast, it is possible that TNF-a and histamine trigger the
production of neutrophil activating factor(s) from HT29 cells,
which stimulates neutrophils to bind with ligands constitutively
expressed on HT29 cells via Mac-1 molecules. We observed
that the culture supernatants recovered from TNF-a- or histamine-pretreated HT29 cells did not affect neutrophil adhesion
to HT29 cells and neutrophil Mac-1 expression (data not
shown). However, the possibility that epithelial cells express
neutrophil activating factor(s) on the surface during stimulation
is not ruled out.
IL-8 is produced by cells, including intestinal epithelial
cells, endothelial cells, monocytes, and T cells [49], whereas
TNF-a is produced by cells including monocytes, macrophages, and mast cells [15]. Histamine is stored in mast cells
and extracellularly released by stimulation [14]. It has been
demonstrated that concentrations of TNF-a, histamine, and
IL-8 are increased in the affected intestinal mucosa of inflammatory bowel diseases [16–20, 39], and symptoms correlate
with the magnitude of neutrophil accumulation in the intestinal
epithelium and mucosa [7]. In this study, we have revealed that
stimulation of intestinal epithelial cells with TNF-a or histahttp://www.jleukbio.org
mine increases neutrophil adhesion to epithelial cells, and that
stimulation of neutrophils with IL-8 or TNF-a up-regulates
surface expression of CD11b/CD18 molecules and increases
neutrophil adhesion to epithelial cells. Thus, TNF-a, histamine, and IL-8, which are produced or released in the local
milieu of intestine, likely play an important role in regulation of
neutrophil adhesion to intestinal epithelial cells by modulating
expression of adhesion molecules on intestinal epithelial cells
or neutrophils. Further studies will be required to elucidate the
neutrophil-intestinal epithelial interactions in vivo. Understanding of interactions between neutrophils and intestinal epithelial
cells will be helpful in the future development of therapies for
inflammatory bowel disorders.
ACKNOWLEDGMENTS
15.
16.
17.
18.
19.
20.
We are grateful to Dr. R. Rothlein (Boehringer Ingelheim,
Ridgefield, CT) for kindly providing anti-CD54 monoclonal
antibody R6.5. This study was supported in part by grants from
Takeda Science Foundation and Atopy (Allergy) Research
Center, Juntendo University, and by a Grant-in-Aid from the
Ministry of Education, Science, Sports and Culture of Japan.
21.
22.
23.
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