ABBS 2005,38(02): Regulatory Peptides Modulate Adhesion of Polymorphonuclear Leukocytes to Bronchial Epithelial Cells through Regulation of Interleukins, ICAM-1 and NF-kB/IkB

*Corresponding
author: Tel, 86-731-2650341; Fax, 86-731-2650400; E-mail, [email protected]

Abstract        A complex network of regulatory
neuropeptides controls airway inflammation reaction, in which airway epithelial
cells adhering to and activating leukocytes is a critical step. To study the
effect of intrapulmonary regulatory peptides on adhesion of polymorphonuclear
leukocytes (PMNs) to bronchial ­epithelial cells (BECs) and its mechanism,
several regulatory peptides including vasoactive intestinal peptide (VIP),
epidermal growth factor (EGF), endothelin-1 (ET-1) and calcitonin gene-related
peptide (CGRP), were investigated. The results demonstrated that VIP and EGF
showed inhibitory effects both on the secretion of IL-1, IL-8 and the adhesion
of PMNs to BECs, whereas ET-1 and CGRP had the opposite effect. ­­Anti-intercellular
adhesion ­­­molecule-1 (ICAM-1) antibody could block the adhesion of PMNs to
ozone-stressed BECs. Using immunocytochemistry and reverse transcription-polymerase
chain reaction (RT-PCR), it was shown that VIP and EGF down-regulated the
expression of ICAM

Key words        polymorphonuclear leukocytes; bronchial
epithelial cells; interleukins; intercellular adhesion molecule-1 (ICAM-1); NF-kB/IkB

Bronchial epithelial
cells (BECs) are not only an ­important barrier against environmental injury
factors, but also ­active participants in a variety of acute and chronic ­inflammatory
reactions in the airway. BECs stimulated by many factors can release
proinflammatory cytokines that promote ­leukocytes survival and mediate
leukocytes recruitment [1]. Polymorphonuclear leukocytes (PMNs) are major ­subsets
of leukocytes found in significant numbers in the lumen of the airway during
airway hyper-responsiveness (AHR), infection or injury [2]. The recruitment of
PMNs in the airway may largely depend on the adhesion of PMNs to BECs. Many
factors, such as secretion of interleukins (ILs) or other inflammatory media
and expression of adhesion molecules, are involved in the adhesion process.
Interleukin-1 is an important proinflammatory cytokine secreted from BECs,
which stimulates the production and ­accumulation of granulocyte-macrophage ­colony-stimulating
factor, IL-6 and IL-8 from BECs. IL-8 plays a ­crucial role in PMN survival and
recruitment [3]. Adhesion of PMNs to BECs is also correlated with the local
expression level of intercellular adhesion molecule-1 (ICAM-1) [4]. The
expression of adhesion molecules on BECs is ­modulated by many factors
including ILs, ­inflammatory media and nuclear factors [5]. However, in the
process of PMN adhesion to BECs, a cooperative action between ICAM-1 expression
and IL ­secretion from BECs has not been reported, and the signal pathway for
ICAM-1 expression is also not very clear.

Vasoactive intestinal
peptide (VIP), endothelin-1 ­(ET-1), calcitonin
gene-related peptide (CGRP), and epidermal growth factor (EGF) exist widely in
lung and especially in airway epithelial cells [6–10]. Many studies indicated that VIP and EGF attenuated
inflammation, promoted wound repair and protected cells from injury [6,7]. It w­as also reported that ET-1 and CGRP increased
inflammatory adhesion, ­stimulated the development of inflammation and
strengthened the ­injury of cells [8–10]. Although
there are many reports about the function of the four regulatory peptides on
inflammation and AHR, the different regulatory effects and correlative
mechanisms of the regulatory peptides in BEC ­inflammation signal transmission
have not been systemically reported. Previously, we found that in a novel AHR
animal model, VIP and CGRP in lung tissues increased temporally and
procedurally, indicating that multiple kinds of regulatory peptides were
involved in airway inflammation signal ­transmission [11]. However, different
peptides play ­different roles in this process.

To demonstrate the ­different
modulatory effects and the mechanism of different ­regulatory peptides in
airway inflammation, in the present study, we examined the effects of several
regulatory ­peptides on the process of PMN activation, including ­secretion of
IL-1 and IL-8, adhesion of PMNs to BECs, activation of nuclear factor NF-kB and degradation of IkB. Our results indicate that VIP and EGF may inhibit the ­inflammatory
process, while ET-1 and CGRP may evoke inflammation.

Materials and Methods

Materials

Isolation and
cultivation of BECs

Assay of IL-1

Radioimmunoassay of IL-1
was performed according to the protocol. 125I-IL-1b was added to each sample or standard reactant and
incubated for more than 16 h at 4 ºC. Then IL-1b antiserum was added into each tube at room temperature
for 20 min. Each tube was centrifuged at 4 ºC with 3500 rpm for 25 min and
decanted the supernatant. Radiointensity of 125I in sediment was measured using the
g-counter. Concentration
of IL-1 was calculated based on the standard curve of the standard reaction.

Assay of IL-8

The principle of IL-8
activity assay bases on the ­reaction that IL-8 selectively stimulates PMNs to
release peroxidase. PMNs were isolated from blood specimen then ­supernatant
from cultured BECs was added to PMN suspension and incubated at 37 ºC for 30
min. After incubation, the ­suspension was centrifuged and 100 ml of supernatant was taken and reacted with 300 ml PBS containing O-phenylene diamine
dihydrochloride (OPD) and hydrogen peroxide (H2O2) at room temperature for 30 min.
Absorbance was measured at 460 nm every 15 s. Peroxidase active unit ­(1 U) is
equal to absorbance change in unit time at 25 ºC [13].

Preparation of PMNs

Heparin (0.5%) was added
to the blood specimen, ­followed by addition of 6% dextran. Leukocyte-rich
plasma was centrifuged and erythrocyte was dissolved. The ­pellet was diluted
in D-Hanks solution and was carefully layered over lymphocyte separation medium
in

Adhesion study

Rabbit BECs were grown
to confluence in standard ­24-well culture plates. Twenty-four hours before the
­adhesion assay, the complete medium was replaced with fresh ­serum-free
medium. Before addition of leukocyte, the ­medium was removed from the rabbit
BEC culture and the cells were washed once with 0.5 ml RPMI 1640 medium.
Purified PMNs (5´107 cells/well) were added in a final volume of
0.25 ml RPMI 1640. The plates were returned to a 37 ºC incubator for 30 min to
re-equilibrate the temperature and allow the leukocytes to contact the
confluent BEC cells. Nonadherent cells were then ­removed by shaking the plates
for 15 s, then the s­upernatant was decanted. After
washing away the nonadherent cells, 0.5% hexadecyl-trimethyl ammonium bromide
was added to each well to extract cellular peroxidase. 150 ml PBS containing OPD and H2O2 was added to a 50 ml aliquot from each well. Absorbance was measured at 460
nm. The number of adherent cells was determined by comparing peroxidant
activity with that of dilutions of a standard number of purified PMNs, and the
adhesion rate of PMNs was calculated. We also examined the effects of ­a­nti-ICAM-1
antibody, H7 and W7 on adhesion of PMNs to BECs.

Immunocytochemistry
analysis of ICAM-1

After exposing to the
experimental conditions, BECs were centrifuged and sediment on a glass slide
which had been treated with polylysine. BECs were fixed in ­anhydrous alcohol
and treated with 3% H2O2.
Non-specific bindings were blocked by 10% goat serum. Incubation with the
diluted specific primary anti-ICAM-1 antibody was performed at 37 ºC for 1 h,
then overnight at 4 ºC in a moist chamber. After incubation with the
corresponding diluted secondary antibody, sABC was added. The peroxidant
activity was visualized by the DAB reaction. PBS replaced the primary antibody
or secondary antibody as a negative control. The intensity and ­distribution of
the specific immunocytochemical staining reaction was evaluated using a
semiquantity method. Fifty cells in ­every visual field were counted and 3–5 visual fields of every slide were observed. The
average photointensity values were obtained.

RT-PCR analysis of
ICAM-1

Total cellular RNA was
extracted using acid guancidinium thiocyanate/phenol/chloroform. RNA quality,
which ­was assayed by 1% agarose gel electrophoresis, showed three bands: 5S,
18S and 28S RNA. A260/A280 of total RNA was 1.890.12. RT-PCR was carried
out ­using RT-PCR kit according to the manufacturer’s instructions. Briefly,
after an initial reverse transcription at 50 ºC for 30 min, samples were run
for 20 cycles of PCR under the ­following conditions: 1 min denaturation at 94
ºC, 1 min annealing at 56 ºC, and 2 min extension at 72 ºC. Primers for ­amplification
of ICAM-1 were 5‘-AGGTCCACCACTGACACGTT-3‘ and 5‘-CCAATATGATTCCACCCATG-3‘;
and primers for amplification of GAPDH were 5‘-GAGCTGTTTGAGAACACCTC-3‘
and 5‘-TCACACTTCACTGTCACCTC-3‘. Yielded products were 300 bp and
600 bp, respectively. RT-PCR products were assayed on 2% ­agarose gel
electrophoresis and visualized by staining with ethidium bromide. The intensity
of the bands was measured with a microscope graph analysis system.

Extraction of nuclear
protein

After exposing cells to
the experimental conditions, extraction of nuclear protein was performed on ice
with ice-cold reagents. Briefly, cells were washed once with cold PBS then
dissolved with four-pellet volume of buffer A
containing

EMSA for NF-kB

The NF-kB-specific oligonucleotide probe used for EMSA (5‘-GAGATGACGTAGTTTTCGCGCTT-3‘)
­corresponded to the NF-kB binding
site in the ICAM-1 promoter and was synthesized at SBS Genetech (

Western blot for IkB

Following the above
procedure, 30 mg of each
sample was separated with 12% sodium dodecyl sulphate polyacrylamide gel
electrophoresis (SDS-PAGE) gel, and then transferred onto immun-blot PVDF
membrane. The membrane was then blocked and incubated with mouse anti-human IkB antibody at room temperature for 1 h. After
three 5 min washes, the membrane was incubated with anti-mouse IgG-peroxidase
antibody at room temperature for 1 h. After a further three 10 min washes, the
peroxidant activity was visualized by the DAB reaction. A picture was taken and
a microscope graph analysis system was used to analyze the density of each
band.

Statistical analysis

Calculation was
performed using the SPSS program (SPSS Inc.,

Results

Effects of regulatory
peptides on secretion of ILs

As shown in Table 1
and Table 2, the secretion of IL-1 and IL-8 was significantly increased
in ozone-stressed BECs compared with that in resting BECs. Pretreatment with
VIP or EGF attenuated the secretion of IL-1 and IL-8 from ozone-stressed BECs,
while pretreatment with ET-1 or CGRP promoted the secretion of IL-1 and IL-8
from ozone-stressed BECs. The effect of these four regulatory peptides on the
secretion of IL-1 and IL-8 exhibits a dose-dependent manner. This result
indicated that VIP and EGF had an inhibitory effect on the secretion of IL-1
and IL-8 from ozone-stressed BECs, while ET-1 and CGRP increased the secretion
of IL-1 and IL-8. The secretion of IL-1 and activity of IL-8 are highly
correlated with the adhesion rate of PMNs [Fig. 1(A,B)].

Effects of regulatory
peptides on adhesion of PMNs to BECs

In order to study
whether or not pulmonary regulatory peptides modulate the process of PMN
adhesion to BECs, we measured the adhesion of PMNs to BECs after stimulation of
BECs with four regulatory peptides under ozone stress. The result is shown in Table
3. It is evident that ozone stress resulted in a significant increase in
adhesion of PMNs to BECs. The adhesion rate decreased more than 50% with
pretreatment of ozone-stressed BECs with VIP or EGF. In contrast, the adhesion
rate was increased ­significantly by pretreatment with ET-1 and CGRP. The
effect of these four regulatory peptides on the PMN ­adhesion also exhibited a
dose-dependent pattern. To ­i­nvestigate the mechanism affecting the adhesion
of PMNs to BECs, specific calmodulin inhibitor, W7 (10–

Effects of regulatory
peptides on ICAM-1 expression

We next wished to
determine if the effect of the ­pretreatments of ozone-stressed BECs with VIP,
EGF, ­ET-1 or CGRP on PMN adhesion could be attenuated by ­co-treatment with
anti-ICAM-1 antibody (10–

Effects of regulatory
peptides on NF-kB activity

It was of interest to
investigate if ICAM-1 expression is related to the NF-kB pathway. Ozone-stressed BECs were treated with MG132, a
specific NF-kB inhibitor,
and the expression of ICAM-1 was then measured. As shown in Fig. 3, the
expression of ICAM-1 could be inhibited by MG132 (10^–

Effects of regulatory
peptides on IkB activity

The degradation of IkB in ozone-stressed BECs had the consistent
tendency to NF-kB activation
during the 2 h observation [Fig. 5(A)]. VIP and EGF treatment was found
to suppress IkB
degradation, while CGRP and ET-1 had the opposite effect [Fig. 5(B)].

Discussion

During the initiation of
airway inflammation, recruitment of leukocytes in the airway microenvironment is
a critical step. Under stressful conditions, inflammatory signals can be
transmitted from BECs to PMNs through release of chemoattractants or cytokines
such as IL-1 and IL-8 from BECs and synchronously, expression of adhesion
molecules such as ICAM-1 and integrins on BECs. In this study, a correlation
between adhesion of PMNs to stressed BECs and levels of IL-1 and IL-8 released
from ozone-stressed BECs was observed. Our results indicate that VIP and EGF
had an inhibitory effect on the secretion of IL-1 and IL-8 from ozone-stressed
BECs and the ­adhesion of PMNs to ozone-stressed BECs, while ET-1 and CGRP had
the opposite effect. The increased PMN adhesion induced by ET-1 and CGRP could
be inhibited by W7 and H7, indicating that the increased PMN adhesion induced
by ET-1 or CGRP might depend on calmodulin and the PKC pathway.

ICAM-1 is the main
adhesion molecule that may ­mediate PMN adhesion [14]. In our cell injury
model, we also observed that ICAM-1 expression was increased and this increase
could be further boosted by pretreatment with ET-1 and CGRP, but was inhibited
by pretreatment with VIP and EGF. The effect of these regulatory peptides on
PMN adhesion to BECs could be blocked by 50% with anti-ICAM-1 antibody. These
results are consistent with a previous report that adhesion of PMNs to BECs is ­correlated
with the local expression of ICAM-1 [15].

It was also shown that
NF-kB inhibitor MG132
blocked the expression of ICAMIn conclusion, through
release of interleukins, expression of ICAM-1, which depends on the NF-kB/IkB ­transcription
regulatory pathway and adhesion of PMNs, BECs ­transmit the inflammatory or
stress signals to PMNs and initialize the inflammatory reaction. VIP and EGF
have a downregulatory effect to attenuate the inflammation, while ET-1 and CGRP
have an upregulatory effect to enhance the inflammation. The imbalance between
the two kinds of neuropeptides probably explains the occurrence of AHR. The
present study will help develop an optimal treatment for AHR and inflammation.

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