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αAccumulation Are Mediated by TNF-
of Human Lung Endothelial Cells and IL-8
Staphylococcal Enterotoxin A-Induced Injury
James M. Noble, Keiko Naitoh and Edmund J. Miller
Nobumitsu Fujisawa, Shinichiro Hayashi, Anna Kurdowska,
http://www.jimmunol.org/content/161/10/5627
1998; 161:5627-5632; ;J Immunol
References
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, 10 of which you can access for free at: cites 39 articlesThis article
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Immunologists All rights reserved.
Copyright © 1998 by The American Association of
9650 Rockville Pike, Bethesda, MD 20814-3994.
The American Association of Immunologists, Inc.,
is published twice each month byThe Journal of Immunology
at NSTL China Trial on June 18, 2013http://www.jimmunol.org/Downloaded from
Staphylococcal Enterotoxin A-Induced Injury of Human Lung
Endothelial Cells and IL-8 Accumulation Are Mediated
by TNF-
a
1
Nobumitsu Fujisawa,* Shinichiro Hayashi,
†
Anna Kurdowska,* James M. Noble,*
Keiko Naitoh,
†
and Edmund J. Miller
2
*
Staphylococcal enterotoxin A (SEA), a superantigen produced by some strains of Staphylococcus aureus, causes a variety of clinical
manifestations ranging from food poisoning to shock. S. aureus can also be associated with the development of acute respiratory
distress syndrome, and SEA has been shown to cause an inflammatory reaction in the lung. Therefore, we examined possible
interactions between SEA, PBMCs, polymorphonuclear cells (PMNs), and normal human lung microvascular endothelial cells
(HMVEC-L), as well as the role of these interactions on the secretion of IL-8. Injury to HMVEC-L, as measured by the release
of
51
Cr, increased significantly when HMVEC-L were incubated with SEA and PBMCs. IL-8 was secreted by both PBMCs and
HMVEC-L. The accumulation of IL-8 in the culture medium of HMVEC-L was increased by SEA in a dose-dependent manner
and was directly related to the number of PBMCs present. Although neither anti-human IL-8 nor IL-1 mAb inhibited HMVEC-L
cytotoxicity, anti-human TNF-
a
mAb inhibited both the cytotoxicity and IL-8 accumulation completely. When HMVEC-L were
incubated with supernatants from SEA-treated PBMCs, HMVEC-L cytotoxicity was comparable with HMVEC-L incubated with
SEA and PBMCs at the same time. Although high concentrations of purified PMNs induced HMVEC-L lysis in a dose-dependent
manner, the effect of PMNs was not changed in the presence of SEA. These findings suggest that TNF-
a
secreted by SEA-
stimulated PBMCs plays a leading role in HMVEC-L injury. The Journal of Immunology, 1998, 161: 5627–5632.
A
cute respiratory distress syndrome (ARDS)
3
is an acute
deterioration of lung function that occurs in association
with severe clinical disorders such as sepsis (1). Al-
though the pathogenesis of ARDS remains unclear, the interaction
between polymorphonuclear cells (PMNs), their toxic products
such as myeloperoxidase, and cytokines derived from PBMCs and
pulmonary endothelium are thought to increase microvascular per-
meability to plasma proteins. The resultant lung edema is consid-
ered a major component of ARDS (2–4). Sepsis is one of the
clinical disorders that is most often associated with ARDS (5, 6),
and many studies have examined the possible role of endotoxin
from Gram-negative bacteria, and in particular the cell wall com-
ponent LPS, in the development of ARDS (7–9). However, there
is a significant group of patients who develop ARDS associated
with Gram-positive bacteria such as Staphylococcus aureus which
do not produce LPS (10). Staphylococcal enterotoxin A (SEA) is
a 28-kDa exoprotein produced by several strains of S. aureus (11).
SEA is known to induce TNF-
a
and IL-1
b
in human monocytic
cells (12–15). Proinflammatory cytokines such as TNF-
a
and
IL-1
b
can stimulate the production of IL-8 (15), and there are
reports that describe TNF-
a
increasing the permeability of the en-
dothelium (16–18) and being associated with the acute lung injury
(19). IL-8, a potent PMN chemotactic and activating factor, has
also been implicated in the pathogenesis of ARDS (20). Further-
more, we have found that IL-8 concentrations are higher in the
lungs of patients with ARDS associated with sepsis than in non-
septic ARDS patients (21), and that i.v. SEA increases the IL-8
concentration of plasma and epithelial lining fluid in rabbits (22).
In these studies, we examined the mechanism of lung endothelial
cell injury associated with SEA.
Materials and Methods
Human subjects
All work involving human subjects was approved by the Institutional Hu-
man Subjects Committee at the University of Texas Health Center.
Cell culture
Human lung microvascular endothelial cells (HMVEC-L) (Clonetics, San
Diego, CA) were maintained in EGM medium containing human epidermal
growth factor (10 ng/ml), bovine brain extract (12 mg/ml), gentamicin
sulfate (50 mg/ml), amphotericin-B (50 ng/ml) (Clonetics), and 10% FCS
(Sigma, St. Louis, MO) at 37°C in a humidified atmosphere containing 5%
CO
2
. HMVEC-L were grown as monolayers in tissue culture flasks. Cells
were passaged when they reached 70–80% confluence using trypsin
(0.025%)/EDTA (0.01%) in HBSS (Clonetics), centrifuged at low speed
(220 3 g for 5 min), and resuspended in fresh medium. HMVEC-L were
maintained for no longer than 3 wk.
Preparation of human PMNs and PBMCs
Human blood from healthy donors was anticoagulated with heparin (El-
kins-Sinn, Cherry Hill, NJ). PMNs were isolated by dextran (Pharmacia,
Piscataway, NJ) sedimentation and E lysis using the method of Boyum (23)
as modified in our earlier studies (24, 25) and were further purified in
gradients of Ficoll-Hypaque (density 1.114; ICN Biomedicals, Costa Mesa,
CA) (26) for cytotoxic assays. The isolated PMNs were $99% pure and
viable. PBMCs were also isolated in gradients of Ficoll-Hypaque. The
isolated PBMCs were $98% pure and 99% viable.
*Department of Biochemistry, University of Texas Health Center, Tyler, TX 75710;
and
†
Department of Medicine, Saga Medical School, Saga, Japan
Received for publication December 1, 1997. Accepted for publication July 7, 1998.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This study was supported by National Institutes of Health Grants R29 HL55622 (to
E.J.M.) and R29 HL56768 (American Heart Association, Texas Affiliate) (to A.K.).
2
Address correspondence and reprint requests to Dr. Edmund J. Miller, Department
of Biochemistry, University of Texas Health Center, Highway 271 at Highway 155,
Tyler, TX 75710.
3
Abbreviations used in this paper: ARDS, acute respiratory distress syndrome; PMN,
polymorphonuclear cell; SEA, staphylococcal enterotoxin A; HMVEC-L, human lung
microvascular endothelial cell(s); MFI, mean fluorescence intensity.
Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00
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51
Cr release cytotoxicity assay
The assay was performed as described previously (27). In brief, HMVEC-L
monolayers in 96-well plates were incubated with 2 Ci/well of Na
2
51
CrO
4
(DuPont-New England Nuclear, Wilmington, DE) alone or with indicated
concentrations of SEA (Toxin Technology, Sarasota, FL) overnight at
37°C. Following the incubation, the wells were washed three times and
incubated in culture medium for an additional 30 min at 37°C to allow
spontaneous lysis of marginally viable cells. After washing twice, a 100-
m
l
aliquot of SEA, purified mouse IgG1 anti-IL-8 mAb (R&D Systems, Min-
neapolis, MN), anti-TNF-
a
mAb, anti-IL-1
b
mAb (Biosource Interna-
tional, Camarillo, CA), freshly isolated PBMCs, and/or PMNs were added
to each well. The cells were cultured for 21 h, and the radioactivity in the
supernatants was counted using a gamma radiation spectrometer. Each well
received culture medium alone or 2% SDS (EM Industries, Cherry Hill,
NJ) to determine spontaneous and maximum release, respectively. Percent
lysis was calculated using the following formula: % Lysis 5 ([experimen-
tal cpm 2 spontaneous cpm]/[maximum cpm 2 spontaneous cpm]) 3 100.
Quantitation of IL-8
IL-8 accumulation in the supernatants was quantitated using an ELISA as
described previously (28, 29). The assay employed an anti-IL-8 mAb
(IgG1) (purified from ascites that had been developed using HB9467 hy-
bridoma cells (American Type Culture Collection, Manassas, VA, with
permission from Dr. E. J. Leonard, National Cancer Institute, Frederick,
MD)) (28) and rabbit polyclonal anti-human IL-8 polyclonal antiserum
(Upstate Biotechnology, Lake Plasid, NY) followed by swine anti-rabbit
Igs conjugated with horseradish peroxidase (Dako, Carpinteria, CA). The
immunoassay was specific for IL-8 and did not cross-react with other mem-
bers of the
a
-chemokine family (29).
Flow cytometric analysis
Flow cytometric analysis was performed as described previously with
some modifications (30). Briefly, PMNs that had been freshly isolated us-
ing dextran and Ficoll-Hypaque were incubated with FITC-labeled anti-
CD16 mAbs (Exalpha, Boston, MA) for 30 min at 4°C to identify the PMN
population. Cells were then washed three times with cold PBS and incu-
bated with phycoerythrin-labeled mAbs for Mac-1 (CD11b; Monosan,
Uden, Netherlands) or L-selectin (CD62L; Exalpha) for an additional 30
min at 4°C. Cells were washed again three times and analyzed by FACScan
(Becton Dickinson, Mountain View, CA). Control leukocytes were pre-
pared by hypotonic lysis of freshly isolated blood and incubated with mAbs
as described above.
Mean fluorescence intensity (MFI) was calculated, and the percent stimu-
lation of expression was calculated using the following formula: % Stim-
ulation 5 ([MFI of purified PMNs 2 MFI of control leukocytes]/MFI of
control leukocytes) 3 100.
Statistics
Data are expressed as mean values 6 SD. Significant differences between
the means of two groups were assessed using the Student t test. Data were
considered statistically significant if p values were #0.05. The experiments
were performed at least twice with at least four replicate cultures per
experiment.
Results
Cytotoxic effect toward HMVEC-L coincubated with SEA,
PBMCs, and/or PMNs
Endothelial cell injury was estimated as the release of chromium
from prelabeled HMVEC-L. When HMVEC-L were incubated
with PBMCs and SEA, HMVEC-L cytotoxicity increased signif-
icantly compared with the incubation with PBMCs alone (p ,
0.0001) (Fig. 1). Alternatively, when HMVEC-L were incubated
with PMNs in the presence of SEA, there was no increase in en-
dothelial cell lysis as compared with the incubation with PMNs
alone. There was also no additional increase in cytotoxicity when
PBMCs and SEA were incubated with PMNs. However, when
HMVEC-L and SEA were incubated with PBMCs, the percentage
of cell lysis was increased in a dose-dependent manner. There were
significant differences from control cultures (incubated without
PBMCs) at concentrations $7.5 3 10
4
cells/ml of PBMCs (p ,
0.007) (Fig. 2A). When HMVEC-L and PBMCs were incubated
with SEA, the percentage of cell lysis also increased in a dose-
dependent manner. There were significant differences from control
FIGURE 1. Cytotoxic effect of SEA, PBMCs, and/or PMNs toward
HMVEC-L. HMVEC-L were incubated with PBMCs (1.5 3 10
5
cells/ml)
and/or PMNs (8.5 3 10
5
cells/ml) in the presence or absence of 10 ng/ml
of SEA for 21 h. Next, percent lysis was calculated as described in Ma-
terials and Methods.
FIGURE 2. Cytotoxic effect of different concentrations of PBMCs and
SEA toward HMVEC-L. A, HMVEC-L were incubated with 10 ng/ml of
SEA and indicated concentrations of PBMCs for 21 h. B, HMVEC-L were
incubated with 1.5 3 10
5
cells/ml of PBMCs and indicated concentrations
of SEA for 21 h.
5628 SEA INDUCES TNF-
a
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cultures (incubated without SEA) at concentrations $0.1 ng/ml of
SEA (p , 0.01) (Fig. 2B).
IL-8 accumulation in supernatants of HMVEC-L incubated with
SEA, PBMCs, and/or PMNs
SEA contributed to IL-8 production and secretion when it was
incubated with PBMCs (Fig. 3). When HMVEC-L and PBMCs
were incubated with SEA, IL-8 accumulation was increased sig-
nificantly compared with incubation in the absence of SEA (p 5
0.0003). The addition of PMNs did not contribute to the accumu-
lation of IL-8 regardless of the presence of SEA or PBMCs. In-
terestingly, HMVEC-L produced and secreted IL-8 in the absence
of any additional stimuli. When HMVEC-L and SEA were incu-
bated with PBMCs, IL-8 accumulation was increased and was re-
lated to the number of PBMCs added; there were significant dif-
ferences from control cultures (incubated without PBMCs) at
concentrations $1.5 3 10
4
cells/ml of PBMCs (p , 0.0001)
(Fig. 4A). When HMVEC-L and PBMCs were coincubated in the
presence of SEA, IL-8 accumulation also increased in a dose-de-
pendent manner, and there were significant differences from con-
trol cultures (incubated without SEA) at concentrations $0.001
ng/ml of SEA (p , 0.0001) (Fig. 4B).
Effect of supernatants of PBMCs incubated with or without SEA
To determine whether the cytotoxic factor and/or the stimulus for
IL-8 production was present in the medium, or whether cell contact
was required, PBMCs were incubated for 21 h in the presence or
absence of SEA. Next, supernatants were collected and incubated
with HMVEC-L. The cytotoxic effects induced by the coincuba-
tion of HMVEC-L, PBMCs, and SEA were due to soluble factors
released into the medium. When HMVEC-L were incubated with
SEA alone (no PBMCs), there was no significant increase in cy-
totoxicity compared with the incubation with medium alone (Fig.
5A). When HMVEC-L were incubated with supernatants that had
been incubated with PBMCs in the presence or absence of SEA,
the percentage of cell lysis was increased significantly compared
with the incubation with medium alone ( p , 0.0001 and p 5
0.0261, respectively). Furthermore, supernatants from SEA-stim-
ulated PBMCs were equally cytotoxic to HMVEC-L compared
with when SEA, PBMCs and HMVEC-L were present at the same
time (p 5 0.61). IL-8 accumulation in the supernatants before and
after incubation with HMVEC-L is shown in Fig. 5B. Both
PBMCs and HMVEC-L produced and secreted IL-8, and SEA
increased the accumulation only when PBMCs had been incubated
previously in the medium.
Effect of cytokine neutralizing Abs on SEA-induced cytotoxicity
and IL-8 accumulation
51
Cr-labeled HMVEC-L were incubated with SEA and PBMCs in
the presence of neutralizing Abs. When anti-IL-8 mAb was used,
the percentage of cell lysis did not change significantly (Fig. 6A).
However, when anti-TNF-
a
mAb was incubated with HMVEC-L,
SEA, and PBMCs, the percentage of cell lysis was decreased in a
dose-dependent manner; in addition, there were significant differ-
ences from control cultures (incubated without anti-TNF-
a
mAb)
at concentrations $5
m
g/ml of anti-TNF-
a
mAb (p , 0.02) (Fig.
6B). The SEA-induced cytotoxicity was completely inhibited when
anti-TNF-
a
mAb was coincubated at concentrations of $20
m
g/ml
(p 5 0.267). Anti-IL-
b
mAb, another proinflammatory cytokine
neutralizing Ab, was also tested (Fig. 6C). In this case, there was
no significant change in cytotoxicity from control cultures grown
in the absence of anti-IL-1
b
mAb.
IL-8 accumulation in the culture supernatants from the coincu-
bation of HMVEC-L, SEA, and PBMCs in the presence of anti-
TNF-
a
mAb was also quantitated (Fig. 7). The accumulation of
IL-8 was inhibited by anti-TNF-
a
mAb in a dose-dependent man-
ner and was completely inhibited at concentrations $1
m
g/ml.
FIGURE 3. IL-8 accumulation in supernatants of HMVEC-L incubated
with PBMCs and/or PMNs in the presence or absence of SEA. HMVEC-L
were incubated with PBMCs (1.5 3 10
5
cells/ml) and/or PMNs (8.5 3 10
5
cells/ml) in the presence or absence of SEA (10 ng/ml). Supernatants were
collected and assayed by ELISA.
FIGURE 4. IL-8 accumulation in supernatants of HMVEC-L incubated
with PBMCs and SEA. A, HMVEC-L were incubated with 10 ng/ml of
SEA and indicated concentrations of PBMCs. B, HMVEC-L were incu-
bated with 1.5 3 10
5
cells/ml of PBMCs and indicated concentrations of
SEA. Supernatants were collected and assayed by ELISA.
5629The Journal of Immunology
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Effect of PMNs on HMVEC-L
HMVEC-L were incubated with various concentrations of PMNs
(Fig. 8). In the absence of any added stimulants, the percentage of
cell lysis increased with the number of PMNs added; there were
significant differences from control cultures (incubated without
PMNs) at concentrations $1 3 10
6
cells/ml of PMNs (p , 0.002).
Expression of Mac-1 and L-selectin on the surface of PMNs
Flow cytometry was performed to determine whether PMNs were
activated by the purification procedure (Table I). The expression of
Mac-1 (CD11b) was increased and the expression of L-selectin
(CD62L) was decreased in CD16
1
cells.
Discussion
The loss of endothelial integrity by cytolysis is a common mech-
anism causing increased vascular permeability, and vascular dam-
age plays an important role in the pathogenesis of vasculitis asso-
ciated with ARDS (31). Infectious diseases are most commonly
associated with ARDS, and Seidenfeld et al. showed that 36% of
infection-induced ARDS were due to Gram-positive cocci (1).
In this study, we describe a mechanism for SEA-induced endo-
thelial damage. TNF-
a
, which was produced and secreted by SEA-
stimulated PBMCs, is an essential component of endothelial in-
jury. To our knowledge, this is the first report that demonstrates a
mechanism of SEA-induced endothelial damage.
Many previous reports have described PMN-dependent endo-
thelial damage and increase in endothelial permeability (9, 16, 32).
However, ARDS has been reported in patients who are neutropenic
(33–36), suggesting that PMNs are not essential for its develop-
ment. We found that the SEA-induced cytotoxic effect occurred
when the toxin was incubated with PBMCs alone and was inde-
pendent of the presence of PMNs. However, the cytotoxicity was
dependent upon the concentrations of both SEA and PBMCs. Fur-
thermore, supernatants from PBMCs incubated with SEA also in-
duced the same level of cytotoxicity in HMVEC-L. These data
support the hypothesis that the ability of SEA to cause endothelial
cell lysis is not related to the presence of PMNs, suggesting a
possible mechanism of endothelial injury in neutropenic patients.
FIGURE 5. Effect of supernatants of PBMCs incubated in the presence
or absence of SEA on HMVEC-L cytotoxicity and IL-8 release. PBMCs
(1.5 3 10
5
cells/ml) were cultured with or without SEA (10 ng/ml) for
21 h; next, supernatants were collected. A, Supernatants were incubated
with radiolabeled HMVEC-L for an additional 21 h, and cytotoxicity was
calculated. B, IL-8 accumulation of supernatants before (open bars) and
after (hatched bars) incubation with HMVEC-L were quantitated.
FIGURE 6. Effect of cytokine neutralizing Abs on the HMVEC-L cy-
totoxicity induced by PBMCs and SEA. Radiolabeled HMVEC-L were
incubated with PBMCs (1.5 3 10
5
cells/ml), SEA (10 ng/ml), and indi-
cated concentrations of neutralizing Abs. Next, the percentage of cell lysis
was calculated. A, Coincubation with anti-IL-8 mAb. B, Coincubation with
anti-TNF-
a
mAb. C, Coincubation with anti-IL-1
b
mAb.
5630 SEA INDUCES TNF-
a
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IL-8 has been identified as a potent PMN chemotactic and ac-
tivating factor (37, 38). Our previous study showed that the con-
centration of IL-8 in the airspaces is elevated in patients with
ARDS. Additionally, the IL-8 in the lungs of patients with ARDS
associated with sepsis reached greater concentrations than in non-
septic ARDS patients (20, 21). In this study, we found that IL-8
was produced and secreted by both PBMCs and HMVEC-L with-
out any added stimulants. Although the accumulation of IL-8 was
increased in parallel with the cytotoxicity when HMVEC-L were
incubated with PBMCs and SEA, neutralizing Ab for IL-8 did not
reduce the cytotoxicity. These data suggest that IL-8 is not directly
responsible for HMVEC-L injury.
TNF-
a
and IL-1
b
, which are known IL-8 inducers, are produced
and released by monocytic cells in response to SEA (12–15). Also,
both TNF-
a
and IL-1
b
have been reported to be associated with
ARDS (19). In particular, TNF-
a
increases pulmonary vascular
permeability independent of neutrophils (39). Although the addi-
tion of anti-IL-1
b
mAb did not affect HMVEC-L cytotoxicity,
when anti-TNF-
a
mAb was coincubated with HMVEC-L, SEA,
and PBMCs, the cytotoxicity decreased in a dose-dependent man-
ner and was inhibited completely by adding 20
m
g/ml of the Ab.
The accumulation of IL-8 was also inhibited completely by adding
1
m
g/ml of anti-TNF-
a
mAb. These data suggest that TNF-
a
is
essential for the HMVEC-L cytotoxic reaction and the increase in
the accumulation of IL-8. Since TNF-
a
also induces gene expres-
sion and the secretion of monocyte chemoattractant protein-1 by
human endothelial cells (40), it is also possible that the migration
of monocytes to a focus of inflammation accelerates the production
and secretion of TNF-
a
, which could worsen the endothelial
injury.
The interaction between toxic products from PMNs such as my-
eloperoxidase and pulmonary endothelium is thought to increase
microvascular permeability to plasma proteins, and the resultant
lung edema is considered to be a major component of ARDS (41,
42). Furthermore, it has been shown previously that i.v. adminis-
tration of IL-8 to rabbits induced changes in the lung histology that
were consistent with ARDS (43), and that IL-8 also plays a sig-
nificant role in PMNs adherence to and transmigration through
vascular endothelium (44). As shown in Fig. 8, high concentrations
of purified PMNs induced HMVEC-L lysis in a dose-dependent
manner. Our PMN-purification protocol, which is a standard
method for in vitro study, caused an increase of Mac-1 as de-
scribed previously (45), as well as a decrease of L-selectin expres-
sion on the cell surface (Table I). These changes in adhesion mol-
ecules were also noted on IL-8-activated PMNs (46). Therefore, it
is possible that the activation of PMNs by the purification proce-
dure participates in the PMN-induced cytotoxic effect. However,
as shown in Fig. 1, despite any purification-induced activation,
PMNs did not enhance the cytotoxic effect of PBMCs for endo-
thelial cells. These data indicate that PBMCs play an important
role in the pulmonary endothelial cytotoxicity induced by SEA.
In conclusion, we have demonstrated a mechanism of SEA-in-
duced human lung endothelium injury. TNF-
a
, which is secreted
by SEA-induced PBMCs, injures HMVEC-L and stimulates the
production and secretion of IL-8 from PBMCs and HMVEC-L.
Because PMNs can be cytotoxic to the endothelium, it is suggested
that the accumulation of PMNs due to IL-8 may accelerate the
cytotoxicity of HMVEC-L. From the findings we have reported
here, it is expected that antagonists of TNF-
a
may have an im-
portant role in the treatment of SEA-induced pulmonary injury.
References
1. Fein, A., M. Lippman, and H. Holtzman. 1983. The risk factors, incidence, and
prognosis of ARDS following septicaemia. Chest 40:84.
2. Windsor, A. C. J., P. G. Mullen, A. A. Powler, and H. J. Sugerman. 1993. Role
of the neutrophil in adult respiratory distress syndrome. Br. J. Surg. 138:10.
3. Murray, J. F., M. A. Matthay, J. M. Luce, and M. R. Flick. 1988. An expanded
definition of the adult respiratory distress syndrome. Am. Rev. Respir. Dis. 138:
720.
4. Said, S. I., and H. D. Foda. 1989. Pharmacologic modulation of lung injury. Am.
Rev. Respir. Dis. 139:1553.
5. Pepe, P. E., R. T. Potkin, D. H. Reus, C. D. Hudson, and C. J. Carrico. 1982.
Clinical predictors of the adult respiratory distress syndrome. Am. J. Surg. 144:
124.
6. Fowler, A. A., R. F. Hamman, J. T. Good, B. Benson, B. David, D. Eberke,
T. C. Petty, and T. M. Hyers. 1983. Adult respiratory distress syndrome: risk with
common predispositions. Ann. Intern. Med. 98:593.
7. Yokoi, K., N. Mukaida, A. Harada, Y. Watanabe, and K. Matsushima. 1997.
Prevention of endotoxemia-induced acute respiratory distress syndrome-like lung
injury in rabbits by a monoclonal antibody to IL-8. Lab. Invest. 76:1203.
8. Strieter, R. M., S. L. Kunkel, H. J. Showell, D. G. Remick, S. H. Phan,
P. A. Ward, and R. M. Marks. 1989. Endothelial cell gene expression of a neu-
trophil chemotactic factor by TNF
a
, LPS, and IL-1
b
. Science 243:1467.
FIGURE 7. Effect of anti-TNF-
a
neutralizing Ab on IL-8 accumulation
when HMVEC-L were incubated with PBMCs and SEA. HMVEC-L were
incubated with 10 ng/ml of SEA, 1.5 3 10
5
cells/ml of PBMCs, and in-
dicated concentrations of anti-TNF-
a
mAb for 21 h.
FIGURE 8. Effect of PMNs on HMVEC-L cytotoxicity. HMVEC-L
were incubated with indicated concentrations of PMNs without any stim-
ulants for 21 h; the percentage of cell lysis was calculated as described in
Materials and Methods.
Table I. Effect of density gradient centrifugation on PMNs
a
Expression % Stimulation
Mac-1 (CD11b) 64.8 6 8.1
L-selectin (CD62L) 223.8 6 8.8
a
Cell staining using mAbs and analysis by FACScan were performed as described
in Materials and Methods.
5631The Journal of Immunology
at NSTL China Trial on June 18, 2013http://www.jimmunol.org/Downloaded from
9. Schneeberger, P. M., P. van Langevelde, K. P. M. van Kessel,
C. M. J. E. Vandenbroucke-Grauls, and J. Verhoef. 1994. Lipopolysaccharide
induces hyperadhesion of endothelial cells for neutrophils leading to damage.
Shock 2:296.
10. Seidenfeld, J. J., D. F. Pohl, R. C. Bell, G. D. Harris, and W. G. Johanson, Jr.
1986. Incidence, site, and outcome of infections in patients with the adult respi-
ratory distress syndrome. Am. Rev. Respir. Dis. 134:12.
11. Iandlo, J. J. 1989. Genetic analysis of extracellular toxins of Staphylococcus
aureus. Annu. Rev. Microbiol. 43:309.
12. Bjo¨rk, L., J. Andersson, M. Ceska, and U. Anderson. 1992. Endotoxin and Staph-
ylococcus aureus enterotoxin A induce different patterns of cytokines. Cytokine
4:513.
13. Grossman D., R. G. Cook, J. T. Sparrow, J. A. Mollick, and R. R. Rich. 1990.
Dissociation of the stimulatory activities of staphylococcal enterotoxins for T
cells and monocytes. J. Exp. Med. 172:1831.
14. Trede, N. S., A. V. Tsytsykova, T. Chatila, A. E. Goldfeld, and R. S. Geha. 1995.
Transcriptional activation of the human TNF-
a
promoter by superantigen in hu-
man monocytic cells. J. Immunol. 155:902.
15. Strieter R. M., S. W. Chensue, M. A. Basha, T. J. Standiford, J. P. Lynch,
M. Baggioni, and S. L. Kunkel. 1990. Human alveolar macrophage gene expres-
sion of interleukin-8 by tumor necrosis factor-
a
, lipopolysaccharide, and inter-
leukin-1
b
. Am. J. Respir. Cell Mol. Biol. 2:321.
16. Gibbs, L. S., L. Lai, and A. B. Malik. 1990. Tumor necrosis factor enhances the
neutrophil-dependent increase in endothelial permeability. J. Cell. Physiol. 145:
496.
17. Goldman, S. E., X. Ding, and J. Campbell-Washington. 1993. TNF-
a
induces
endothelial cell F-actin depolymerization, new actin synthesis, and barrier dys-
function. Am. J. Physiol. 264:C894.
18. Stephens, K. E., A. Ishizaka, J. W. Larrick, and T. A. Raffin. 1988. Tumor ne-
crosis factor causes increased pulmonary permeability and edema: comparison to
septic acute lung injury. Am. Rev. Respir. Dis. 137:1364.
19. Suter, P. M., S. Suter, E. Girardin, P. Roux-Lombard, G. E. Grau, and
J. M. Dayer. 1992. High bronchoalveolar levels of tumor necrosis factor and its
inhibitors, interleukin-1, interferon, and elastase, in patients with adult respiratory
distress syndrome after trauma, shock, or sepsis. Am. Rev. Respir. Dis. 145:1016.
20. Miller, E. J., A. B. Cohen, S. Nagao, D. Griffith, R. J. Maunder, T. R. Martin,
J. P. Wiener-Kronish, M. Sticherling, E. Christophers, and M. A. Matthay. 1992.
Elevated levels of NAP-1/interleukin-8 are present in the airspaces of patients
with the adult respiratory distress syndrome and are associated with increased
mortality. Am. Rev. Respir. Dis. 146:427.
21. Miller, E. J., A. B. Cohen, and M. A. Matthay. 1996. Elevated interleukin-8 levels
in pulmonary edema fluid of patients with ARDS from sepsis. Crit. Care Med.
24:1448.
22. Miller, E. J., A. B. Cohen, and B. T. Peterson. 1996. Peptide inhibitor of inter-
leukin-8 reduces staphylococcal enterotoxin A-induced neutrophil trafficking to
the lung. Inflamm. Res. 45:393.
23. Boyum, A. 1968. Isolation of mononuclear cells and granulocytes from human
blood: isolation of mononuclear cells by one centrifugation and of granulocytes
by combining centrifugation and sedimentation at 1 3 g. Scand. J. Clin. Lab.
Invest. 21:97.
24. MacArthur, C. K., E. J. Miller, and A. B. Cohen. 1987. A peptide secreted by
human alveolar macrophages which releases neutrophil granule contents. J. Im-
munol. 139:3456.
25. Hayashi, S., A. Kurdowska, E. J. Miller, M. E. Albright, B. E. Girten, and
A. B. Cohen. 1995. Synthetic hexa- and heptapeptides that inhibit IL-8 from
binding to and activating human blood neutrophils. J. Immunol. 154:814.
26. Ferrante, A., and Y. H. Thong. 1978. A rapid one-step procedure for purification
of mononuclear and polymorphonuclear leukocytes from human blood using
modification of the Hypaque-Ficoll technique. J. Immunol. Methods 24:389.
27. Hayashi, S., A. Kurdowska, A. B. Cohen, M. D. Stevens, N. Fujisawa, and
E. J. Miller. 1997. A synthetic peptide inhibitor for
a
-chemokines inhibits the
growth of melanoma cell lines. J. Clin. Invest. 99:2581.
28. Sylvester, I., J. A. Rankin, T. Yoshimura, S. Tanaka, and E. J. Leonard. 1990.
Selection of neutrophil attractant/activation protein by lipopolysaccharide-stim-
ulated lung macrophages determined by both enzyme-linked immunosorbent as-
say and N-terminal sequence analysis. Am. Rev. Respir. Dis. 141:683.
29. Miller, E. J., S. Nagao, F. K. Carr, J. M. Noble, and A. B. Cohen. 1996. Inter-
leukin-8 (IL-8) is a major neutrophil chemotaxin from human alveolar macro-
phages stimulated with staphylococcal enterotoxin A (SEA). Inflamm. Res. 45:
386.
30. Naitoh, K., Y. Ichigi, K. Miyake, A. Muraguchi, and M. Kimoto. 1994. Signal
transmission through MHC class II molecules in a human B lymphoid progenitor
cell line: different signaling pathways depending on the maturational stages of B
cells. Microbiol. Immunol. 38:967.
31. Bechard, D. E., R. P. Fairman, D. B. Hinshaw, A. A. Fowler, and F. L. Glauser.
1990. In vivo interleukin-2-activated sheep lung lymph lymphocytes increase
ovine vascular endothelial permeability by non-lytic mechanisms. Eur. J. Cancer.
26:1074.
32. Bratt, J., and J. Palmblad. 1997. Cytokine-induced neutrophil-mediated injury of
human endothelial cells. J. Immunol. 159:912.
33. Laufe, M. D., R. H. Simon, A. Flint, and J. B. Keller. 1986. Adult respiratory
distress syndrome in neutropenic patients. Am. J. Med. 80:1022.
34. Maunder, R. J., R. C. Hackman, E. Riff, R. K. Albert, and S. C. Springmeyer.
1986. Occurrence of the adult respiratory distress syndrome in neutropenic pa-
tients. Am. Rev. Respir. Dis. 133:313.
35. Braude S., J. Apperly, T. Krausz, J. M. Goldman, and D. Royston. 1985. Adult
respiratory distress syndrome after allogenic bone mallow transplantation. Lancet
1:1239.
36. Ognibene, F. P., S. E. Martin, M. M. Parker, T. Schlesinger, P. Roach, C. Burch,
J. H. Shelhamer, and J. E. Parrillo. 1986. Adult respiratory distress syndrome in
patients with severe neutropenia. N. Engl. J. Med. 315:547.
37. Baggioni, M., A. Waltz, and S. K. Kunkel. 1989. Neutrophil-activating peptide-
1/interleukin-8, a novel cytokine that activates neutrophils. J. Clin. Invest. 84:
1045.
38. Matsushima, K., and J. J. Oppenheim. 1989. Interleukin-8 and MCAF: novel
inflammatory cytokines inducible by IL-1 and TNF. Cytokine 1:2.
39. Horvath, C. J., T. J. Ferro, G. Jesmok, and A. B. Malik. 1988. Recombinant tumor
necrosis factor increases pulmonary vascular permeability independent of neu-
trophils. Proc. Natl. Acad. Sci. USA 85:9219.
40. Brown, Z., M. E. Gerritsen, W. W. Carley, R. M. Strieter, S. L. Kunkel, and
J. Westwick. 1994. Chemokine gene expression and secretion by cytokine-acti-
vated human microvascular endothelial cells: differential regulation of monocyte
chemoattractant protein-1 and interleukin-8 in response to interferon-
g
.
Am. J. Pathol. 145:913.
41. Windsor, A. C. J., P. G. Mullen, A. A. Powler, and H. J. Sugerman. 1993. Role
of the neutrophil in adult respiratory distress syndrome. Br. J. Surg. 138:10.
42. Murray, J. F., M. A. Matthay, J. M. Luce, and M. R. Flick. 1988. An expanded
definition of the adult respiratory distress syndrome. Am. Rev. Respir. Dis. 138:
720.
43. Standiford, T. J., S. L. Kunkel, and R. M. Strieter. 1993. Interleukin-8: a major
mediator of acute pulmonary inflammation. Reg. Immunol. 5:134.
44. Larsen, C. G., A. O. Anderson, E. Appella, J. J. Oppenheim, and K. Matsushima.
1989. The neutrophil-activating protein (NAP-1) is also chemotactic for lympho-
cytes. Science 243:1045.
45. Kujipers, T. W., A. T. J. Tool, C. E. van der Schoot, L. A. Ginsel,
J. J. M. Onderwater, D. Roos, and A. J. Verhoeven. 1991. Membrane surface
antigen expression on neutrophils: a reappraisal of the use of surface markers for
neutrophil activation. Blood 78:1105.
46. Detmers, P. A., D. E. Powell, A. Walz, I. Clark-Lewis, M. Baggiolini, and
Z. A. Cohn. 1991. Differential effects of neutrophil-activating peptide 1/IL-8 and
its homologues on leukocyte adhesion and phagocytosis. J. Immunol. 147:4211.
5632 SEA INDUCES TNF-
a
-MEDIATED INJURY OF LUNG ENDOTHELIAL CELLS
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