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*
Department of Traumatology, University of Freiburg, Freiburg, Germany;
Department of Anesthesiology, University Hospital, Zurich, Switzerland; and
Department of Pathology, University of Michigan, Ann Arbor, MI 48109
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and the CXC
chemokine, macrophage inflammatory protein-2 (MIP-2). Alveolar
macrophages exhibited cytokine responses to both sICAM-1 and
immobilized sICAM-1, while rat PBMCs failed to demonstrate similar
responses. Exposure of alveolar macrophages to sICAM-1 resulted in
NF
B activation (which was blocked by the presence of the aldehyde
peptide inhibitor of 28S proteosome and by genistein, a tyrosine kinase
inhibitor). As expected, cross-linking of CD18 on macrophages with Ab
resulted in generation of TNF- and MIP-2. This response was also
inhibited in the presence of the proteosome inhibitor and by genistein.
Alveolar macrophages showed adherence to immobilized sICAM-1 in a
CD18-dependent manner. Finally, airway instillation of sICAM-1
intensified lung injury produced by intrapulmonary deposition of IgG
immune complexes in a manner associated with enhanced lung production
of TNF- and MIP-2 and increased neutrophil recruitment. Therefore,
through engagement of ß2 integrins, sICAM-1 enhances
alveolar macrophage production of MIP-2 and TNF-, the result of
which is intensified lung injury after intrapulmonary disposition of
immune complexes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In IgG immune complex-induced lung injury in rats, inflammatory injury is neutrophil dependent and requires ICAM-1, CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1), and E- and L-selectin. Interestingly, in the lung a compartmentalized role for the adhesion has been found, with LFA-1 being important on the vascular side and Mac-1 being important on the airway side (10, 11). Although blocking of ICAM-1 in the vasculature or in the distal airway compartment inhibited full development of lung injury, anti-CD11a was only protective when given i.v., and anti-CD11b was only protective when given intratracheally. These data suggest a role for ICAM-1 in the distal airway compartment (perhaps involving ICAM-1 on alveolar type II epithelial cells or on alveolar macrophages). ICAM-1 could function as a stimulus for alveolar macrophages via their ß2 integrin content and in the vascular compartment as a central adhesion molecule required for neutrophil recruitment from the blood.
In the current study we investigated the ability of rat sICAM-1
to activate alveolar macrophages and to enhance lung injury. Production
of TNF- and MIP-2 by alveolar macrophages and PBMCs was monitored
following exposure to sICAM-1. Both of these cytokines are known to
play a role in IgG immune complex-mediated lung injury (12, 13). The
intracellular signal transduction pathways of cells exposed to sICAM-1
were partially analyzed using inhibitors of NF-B activation and of
tyrosine kinase activity. Binding of sICAM-1 to surfaces of alveolar
macrophages and adhesion of alveolar macrophages to immobilized sICAM-1
were also demonstrated. Our results indicate that sICAM-1 binds to and
activates macrophages in a CD18-dependent manner. The signaling induced
by this interaction appears to be NF-B and tyrosine kinase
dependent. In rat lungs undergoing injury during deposition of IgG
immune complexes, the instillation of sICAM-1 significantly enhanced
lung injury in a manner associated with enhanced recruitment of
neutrophils and increased lung levels of TNF- and MIP-2. Since
sICAM-1 has been detected in body fluids of humans, it may have
important biologic roles in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Except where noted, all reagents were purchased from Sigma (St. Louis, MO).
Protein expression of ICAM-1
A soluble fragment of ICAM-1 was expressed by means of pet15b (Novagen, Madison, WI)-transformed Escherichia coli that were stimulated with isopropyl ß-D-thiogalactoside (IPTG) (14). The ICAM-1 derivative consisted of the three N-terminal domains responsible for its interactions with ß2 integrins (15, 16). Nickel affinity chromatography facilitated rapid purification of the recombinant ICAM-1 using imidazole. A 200 µg/ml stock solution of sICAM-1 containing 0.01% thimerosal was maintained at 4°C. Abs to sICAM-1 were obtained in rabbits as described previously (14).
Determination of endotoxin content
A Limulus amebocyte lysate assay QCL-1000 was used according to manufacturer’s instructions (BioWhittaker, Walkersville, MD). The LPS concentration of the 200 µg/ml stock solution of sICAM-1 was 120 ng/ml. Since most sICAM-1 applications entailed sICAM-1 concentrations from 0.125 to 2 µg/ml, the working concentrations of LPS did not exceed 1 ng/ml. Using this concentration of LPS, only modest amounts of MIP-2 (61.1 ± 1.71 ng/ml) were produced by rat alveolar macrophages compared with background expression by macrophages (40.7 ± 1.34 ng/ml).
Northern blots
RNA (12 µg) extracted from whole lungs of IgG immune complex-injured rats (at 4 h) were electrophoretically fractionated in a 1% agarose formaldehyde gel and then transferred to a nylon membrane (Zetabind, Cuno, Meriden, CT). 32P-labeled dCTP probes were generated using the PCR primers specific for the partial length cDNA templates of ICAM-1. Radioactivity of the probes was determined by scintillation counting. Probes containing 1.5 x 107 cpm were applied to the blot, with hybridization occurring at 65°C for 16 h. Autoradiography of the blots was performed at -70°C for 24 to 48 h on Kodak X-OMAT-AR film (Eastman Kodak, Rochester, NY). Quantitation of autoradiographs was performed using an AMBIS Image Analysis System (San Diego, CA).
Isolation of PBMC
PBMC were isolated from the venous blood of rats by separation over Ficoll-Paque (Pharmacia, Uppsala, Sweden) followed by a lysis step to remove contaminating erythrocytes. The cells were then washed twice with 0.9% sodium chloride containing 0.1% BSA, resuspended in DMEM (Life Technologies, Gaithersburg, MD) containing 10% FBS, 1% L-glutamine, 1% antibiotic/antimycotic, and 1% nonessential amino acids (DMEM) and then plated at a concentration of 1 x 106 cells/ml. After 1-h incubation, media were removed, and cells were exposed to the indicated stimuli.
In vitro stimulation of alveolar macrophages
Alveolar macrophages were obtained from normal rats by bronchoalveolar lavage (BAL). The cells were then added to 48-well plates (Corning, Corning, NY) and cultured for 1 h in DMEM. Wells were loaded with 1 x 106 cells in a total volume of 1 ml. After the incubation period, media were removed, and stimuli were added. The supernatant fluids were collected after 4 h. The concentrations of Abs and activating reagents used in the assays are reported for each experiment. When immobilized sICAM-1 was used as a stimulus of cells, sICAM-1 was applied directly to the microtiter wells. Immobilization of sICAM-1 was achieved by incubation of 100 µl of sICAM-1/well in carbonate buffer (pH 9.6) overnight at 4°C. Before use, the wells were rinsed with PBS, pH 7.4.
Protein degradation
The sICAM-1 was denatured by heating the protein solution for 5 min at 95°C. This was followed by a 5-min centrifugation at 14,000 rpm. For digestion of sICAM-1, the protein stock solution was incubated with 10 µg/ml proteinase K at 37°C overnight. To demonstrate the extent of digestion, the treated and untreated proteins were evaluated by SDS-PAGE, followed by staining with Coomassie blue.
Adhesion assays
This assay was adapted from a method previously described (17). The sICAM-1 was immobilized on 96-well Immulon 4 ELISA plates (Fisher, Pittsburgh, PA) by incubation in carbonate buffer at 4°C overnight. The plates were blocked before use for 1 h with 2% BSA in PBS. Alveolar macrophages were obtained by BAL and were incubated for 30 min at 37°C in DMEM with 1 µM 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (Molecular Probes, Eugene, OR). Residual 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester was removed by centrifugation of the cells, which were resuspended in HBSS (with Ca2+ and Mg2+). The cells were incubated on the ICAM-1-coated plates for 10 min at 37°C. The plates were washed four to six times with 0.9% NaCl, and the fluorescence was measured with a Cytofluor II (PerSeptive Biosystems, Framingham, MA).
Cell-based ELISA
Alveolar macrophages were isolated by BAL and cultured in 96-well microtiter plates (Costar, Cambridge, MA) in DMEM. Cells (2 x 105/well) were loaded, resulting in >95% of cells adhering to form a monolayer after 1 h. These monolayers were incubated with different concentrations of sICAM-1 in DMEM for 45 min. Each incubation step was conducted at 37°C and was followed by careful washing with PBS. Both the rabbit anti-rat-ICAM-1 detector Ab (14) and a preimmune IgG fraction were used at a concentration of 50 µg/ml. The incubation time with this Ab was 1 h. This was followed by an incubation period of 45 min with an anti-rabbit horseradish peroxidase-conjugated Ab (Amersham, Arlington Heights, IL) in a dilution of 1/5000. The assay was developed by addition of o-phenylenediamine dihydrochloride substrate. The developing reaction was stopped by adding 50 ml of 3 M H2SO4, and the OD at 490 nm was analyzed by a MicroELISA Autoreader (Bio-Tek Instruments, Winooski, VT).
Animal model of IgG immune complex-mediated alveolitis
Male Long-Evans rats (275–300 g; specific pathogen free; Harlan Industries, Rochester, MI) were anesthetized with i.p. ketamine (175 mg/kg). Injury was induced by the intratracheal instillation of rabbit polyclonal IgG (1.25 mg) rich in Ab to BSA (anti-BSA) in the presence or the absence of added sICAM-1 in a volume of 300 µl of PBS via an intratracheal catheter during inspiration. Immediately thereafter, 10 mg of BSA with trace amounts of [125I]BSA (as a quantitative marker of permeability) was injected i.v. Rats were sacrificed 4 h later, the pulmonary circulation was flushed, and lung injury was quantitated by increases in vascular permeability. For calculations of the permeability index, the amount of radioactivity ([125I]BSA) remaining in the saline perfused lungs was compared with the amount of radioactivity present in 1.0 ml of blood obtained from the inferior vena cava at the time of sacrifice. Negative control animals received the same volume of PBS intratracheally instead of anti-BSA.
Bronchoalveolar lavage
For cell isolation, 10 ml of PBS was gently instilled into the lung via a tracheal catheter and then withdrawn. This process was repeated five times. The cells were analyzed by manual microcytometry. Neutrophils were easily distinguished from mononuclear cells (lymphocytes and macrophages). In each preparation the cells were 100% viable and contained only minor contaminating cells (>95% purity). For neutrophil cell counts, 5 ml of PBS were instilled into the lung, withdrawn, and reinstilled repeatedly (three times). After centrifugation at 400 x g, the cell pellet underwent a lysis step to remove contaminating RBCs.
ELISA for MIP-2
A sandwich ELISA was used as previously described (18). Briefly, 50 µl of a 10 µg/ml MIP-2 Ab solution in carbonate coating buffer (pH 9.6) was applied to coat a 96-well Immulon 4 ELISA plate (Fisher, Pittsburgh, PA). The coating procedure was conducted overnight. A 2% BSA solution in PBS was used to block specific binding (30 min at 37°C). Following a washing step, 100 µl/sample was added per well, and incubation was performed for 1 h at 37°C. Biotinylated Ab (100 µl, diluted 1/750) was added and incubated for 1 h at 37°C. Following a 30-min incubation with a streptavidin-horseradish peroxidase conjugate (Pierce, Rockford, IL), the assay was developed by addition of the substrate o-phenylenediamine dihydrochloride. The developing reaction was stopped by adding 50 µl of 3 M H2SO4.
TNF- assay
Samples were diluted 1/10 to 1/1000. One hundred microliters of
samples (BAL fluids or cell culture supernatant fluids) were added to
96-well microtiter plates (Costar). WeHi cells (WeHi 164, subclone 13)
in a volume of 0.1 ml/well were added in a final concentration of
5 x 105 cells/ml in the presence of actinomycin D
(Life Technologies; 0.5 µg/ml). In a separate set of wells, a
standard consisting of serially diluted recombinant rat TNF-
(Biosource, Camarillo, CA; starting at a concentration of 100 pg/ml)
was added. The cells were incubated at 37°C for 20 h, then 20
µl of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
from a 5 mg/ml stock solution were added per well. This was followed by
another 4-h incubation period. Then 150 µl of supernatant fluids per
well were carefully removed, and 100 µl of acidified isopropanol
(isopropanol in 0.04 N HCl) was added. The plate was then wrapped in
foil and left on the bench top overnight. The OD at 550 nm was analyzed
by a MicroELISA Autoreader (Bio-Tek).
Nuclear extraction and electrophoretic mobility shift assay (EMSA)
Alveolar macrophages were harvested by BAL and were plated on
100-mm tissue culture plates (1 x 107 cells/plate).
Cells were incubated with vehicle (DMSO) or with the aldehyde inhibitor
of NF-B (30 µg/ml) for 15 min before the addition of sICAM-1 (500
ng/ml). After 1 h, cells were harvested by scraping, and nuclear
extracts were prepared as previously described (19). Protein
concentrations were determined by bicinchoninic acid assay with
trichloroacetic acid precipitation using BSA as a reference standard
(Pierce). The double-stranded NF-B consensus oligonucleotide (5'-AGT
GAG GGG ACT TTC CCA GGC-3'; Promega) was end labeled with
[32P]ATP (3000 Ci/mmol at 10 mCi/ml; Amersham, Arlington
Heights, IL). Binding reactions containing equal amounts of protein (5
µg) and 35 fmol (
50,000 cpm, Cherenkov counting) of
oligonucleotide were performed for 30 min in binding buffer (4%
glycerol, 1 mM MgCl2, 0.5 mM EDTA (pH 8.0), 0.5 mM DTT, 50
mM NaCl, 10 mM Tris (pH 7.6), and 50 µg/ml poly(dI-dC); Pharmacia,
Piscataway, NJ). Reaction volumes were held constant at 15 µl.
Reaction products were separated in a 4% polyacrylamide gel and
analyzed by autoradiography.
Statistical analyses
As appropriate, the percent protection or reduction in injury was calculated by first subtracting the background negative control values from both the positive control values and the values obtained from positive control animals given various treatments. The data were analyzed with one- or two-way analysis of variance. Additional Student’s t tests were performed to determine the significance of differences between means of individual groups. All values were expressed as the mean ± SEM unless otherwise indicated. Statistical significance was defined when p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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After IPTG-induced expression of sICAM-1 in BL21(DE)pLysS E.
coli, the carboxyl-terminal six-histidine tag facilitated rapid
purification using a nickel column for metal ion affinity
chromatography (20). Elutions were obtained with increasing
concentrations of imidazole (100–500 mM), providing an enriched
fraction of sICAM-1, as demonstrated by SDS-PAGE and staining with
Coomassie blue (Fig. 1
, elution 2).
Material from this elution procedure was used for the experiments
described below.
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To obtain information related to up-regulation of lung ICAM-1, RNA
from lungs of animals sacrificed at different time points after
intrapulmonary deposition of IgG immune complexes was extracted and
analyzed by Northern blot analysis (Fig. 2, A and B). Since
different isoforms of sICAM-1 have been described, we sought to
determine whether differently spliced ICAM-1 mRNAs were present. Only
one distinct mRNA band for ICAM-1 could be detected, suggesting that a
single mRNA species is expressed. ICAM-1 mRNA demonstrated constitutive
expression followed by up-regulation at 2 and 4 h. At 6 h,
mRNA expression for ICAM-1 declined to background levels (Fig. 2).
Approximately equal loading was confirmed by the 18S (lower) and 28S
(upper) RNA bands (Fig. 2C).
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Binding of sICAM-1 to monolayers of rat alveolar macrophages was
demonstrated by means of a cell-based ELISA. The cells were incubated
with different concentrations of sICAM-1 (7.8–2000 ng/ml), and then
detector anti-ICAM-1 IgG or preimmune IgG was added to the
monolayers. The OD of the monolayers was directly proportional to the
sICAM-1 concentrations added to the wells, indicating binding and
immobilization of the Ab to ICAM-1 present on surfaces of macrophages
(Fig. 3). Differences between background
values (0.202 ± 0.008) and those values of sICAM-1-treated cells
were observed at a concentration of 125 ng/ml (0.246 ± 0.027) or
greater. In macrophages exposed to 2000 ng of sICAM-1, the OD (at 490
nm) reached a value of 0.929 ± 0.075. Preimmune IgG was used as
control Ab, in which case no increase in binding to cell monolayers was
observed at any of the concentrations, indicating that the differences
were not a result of binding of Ab via Fc receptors of macrophages. The
data shown are representative of two separate and independent
experiments that produced similar results.
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by sICAM-1
When alveolar macrophages were incubated with increasing
concentrations of sICAM-1, production of TNF- and the CXC chemokine,
MIP-2, was observed (Fig. 4). For these
studies both unheated and heated (5 min at 95°C) sICAM-1 were used.
Over a dose range of 125 to 2000 ng sICAM-1/ml, there were progressive
increases in the amounts of MIP-2 and TNF- produced, with peak
levels of 373 ± 15.2 ng/ml and 6170 ± 1367 pg/ml,
respectively (Fig. 4). At the highest dose (2.0 µg) of sICAM-1
employed, cytokine responses were equivalent to those obtained with 10
ng LPS/ml. Heated sICAM-1 was considerably less effective than unheated
sICAM-1. To assess whether the production of cytokines could be due to
contamination of sICAM-1 with LPS, sICAM-1 was degraded by either heat
denaturation or digestion with proteinase K. The results are shown in
Table I. Both forms of treatment with
sICAM-1 resulted in a significant decrease in the cytokine-inducing
effects of sICAM-1. In the case of MIP-2, inhibition was 60% when
sICAM-1 was treated with the proteinase and 64% when sICAM-1 was heat
denatured. In the case of TNF-, reductions were 90 and 100%,
respectively. This experiment was repeated four times with two
different sICAM-1 preparations (n = 3–6/group) and
produced similar results (data not shown).
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and employing
rat PBMC, incubation with sICAM-1 caused generation of small amounts of
MIP-2 and no generation of TNF- (Table II). In the case of MIP-2, there was no
statistically significant difference between treatment with unheated or
heat-inactivated sICAM-1. In the case of TNF-, no increases occurred
with any of the sICAM-1 preparations. In fact, in all such cases
TNF- levels were less than those found with untreated PBMC. As
expected, LPS was an effective stimulus for PBMC generation of MIP-2 or
TNF-. Thus, PBMC, in contrast to alveolar macrophages, show poor
cytokine responses to sICAM-1 under the conditions employed.
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To investigate the ability of sICAM-1 to activate NF-B in
macrophages, cells were incubated with sICAM-1 (3.0 µg/ml) in the
absence or the presence of the aldehyde proteasome inhibitor (PSI),
Z-Ile-Glu(O-tBu)-Ala-Leu-H, and activation of NF-B was
assessed by EMSA. PSI has been shown to block the chymotrypsin-like
activity of the multicatalytic proteinase complex (20S proteasome),
preventing NF-B translocation by stabilizing the complex of
phosphorylated IB- with NF-B (21, 22). Alveolar macrophages
underwent a 15-min preincubation period with PSI before exposure to
sICAM-1. Using EMSA, exposure of macrophages to sICAM-1 (500 ng/ml)
caused NF-B activation; the shift was diminished in the presence of
PSI (Fig. 5). The amount of total protein
added to each lane was 5 µg. The effects of PSI on cytokine
production in sICAM-1-stimulated macrophages was studied concurrently.
The control groups (with or without sICAM-1) were incubated with DMSO,
since PSI required similar amounts of DMSO for PSI solubility. Under
conditions identical with those described in Figure 5, pretreatment of
macrophages with PSI caused an 80% reduction
(p < 0.001) in levels of MIP-2 (to 52.3
± 1.9 ng/ml) and an 81% decrease (p < 0.01)
in TNF- levels (to 737 ± 340 pg/ml; Fig. 6).
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(Fig. 7). Positive controls were incubated with
sICAM-1 at a concentration of 500 ng/ml in the presence of DMSO. In the
presence of sICAM-1, MIP-2 levels rose nearly threefold above negative
control values, to 134 ± 7.50 ng/ml. In the presence of
genistein, MIP-2 levels dropped 64% (p <
0.02), to 76.5 ± 5.52 ng/ml. The TNF- level in the positive
controls was 1276 ± 124 pg/ml. Pretreatment with genistein
reduced TNF- levels by 89% (p < 0.004), to
512 ± 77 pg/ml (Fig. 7). This experiment demonstrates that MIP-2
and TNF- production stimulated by sICAM-1 is genistein
sensitive.
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It has been demonstrated that monocyte integrin cross-linking with
Abs can mimic ligand binding, resulting in cell activation (24).
Similar results were observed in alveolar macrophages incubated with
anti-CD18 (WT-3; Fig. 8). At
anti-CD18 doses of 10 and 20 µg/ml, MIP-2 levels in the
supernatant rose to 189 ± 15 and 469 ± 19 ng/ml,
respectively. These values were significantly different
(p < 0.05) compared with the effect of the
subclass-matched, control MOPC-21 Ab. MIP-2 concentrations in the
supernatant fluids of macrophages treated with 10 and 20
µg/MOPC-21/ml were 115 ± 4.07 and 139 ± 16.2 ng/ml,
respectively, values close to background MIP-2 expression (108 ±
4.78 ng/ml).
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. As expected,
addition of the proteasome inhibitor in a concentration of 30 µg/ml
resulted in a 39% reduction in MIP-2 levels in supernatant fluids of
macrophages that had been activated with 20 µg/ml anti-CD18 (with
DMSO, the vehicle for PSI). Positive controls generated 187 ±
8.64 ng MIP-2/ml, while PSI-treated cells produced 132 ± 9.77
ng/ml; background MIP-2 expression was 31.4 ± 2.74 ng/ml. The
addition of genistein (at a concentration of 30 µg/ml) decreased
anti-CD18-induced MIP-2 production by 43% (to 126 ± 9.77
ng/ml). With respect to TNF- levels, addition of anti-CD18
caused the production of 1423 ± 46.0 pg TNF-/ml, significantly
more than that by cells coincubated with the same amount of MOPC 21
(216 ± 7.00 pg/ml) or with no treatment (249 ± 2.00 pg/ml).
Pretreatment of macrophages with PSI followed by addition of
anti-CD18 resulted in a 72% drop (to 575 ± 194 pg/ml), while
addition of genistein caused a 47% reduction (to 870 ± 98 pg/ml;
Fig. 9). These data indicate that stimulation of alveolar macrophages
with anti-CD18 causes cytokine production in a manner that is
susceptible to inhibitors that also suppress sICAM-1-induced production
of MIP-2 and TNF-.
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Since sICAM-1 can bring about activation of alveolar
macrophages in vitro, we sought to determine whether sICAM-1 would
enhance the inflammatory response in IgG immune complex-induced lung
injury. The lung permeability index of the negative control animals was
0.23 ± 0.03 (n = 5; Fig. 10A). When 50 µg of sICAM
was instilled into rat lungs, the lung permeability index (0.25 ±
0.04; n = 5) was not statistically different from that
for the negative control group. Positive control animals injured with
IgG immune complexes together with heat-inactivated sICAM-1 had a
vascular permeability index of 0.37 ± 0.03 (n =
10). Instillation of IgG immune complexes together with 50 µg of
unheated sICAM-1 resulted in a 62% increase in the lung permeability
index, to 0.46 ± 0.03 (p < 0.04;
n = 10), compared with that in positive controls. The
number of BAL neutrophils in these experimental groups was also
assessed. In negative control animals the total number of BAL
neutrophils was 2.2 ± 0.25 x 105 (Fig. 10B). Administration of sICAM-1 alone (50 µg) caused
increased (p < 0.03) numbers of neutrophils,
to 4.2 ± 0.45 x 105 cells. Deposition of IgG
immune complexes in the presence of heat-inactivated sICAM-1 in lung
resulted in 5.6 ± 1.5 x 105 neutrophils. The
coinstillation of sICAM-1 with anti-BSA further increased the
neutrophil count in BAL fluids by 131%, to 13 ± 2.2 x
105 cells (p < 0.02).
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and
MIP-2 levels in BAL fluids. The TNF- level in negative controls was
519 ± 29.1 pg/ml. Instillation of sICAM-1 alone into otherwise
normal lungs did not affect TNF- production (698 ± 227 pg/ml),
but significantly increased TNF- production occurred when sICAM-1
was coinstilled with the anti-BSA, rising by 97% to 25,765 ±
4,741 pg/ml (Fig. 11A).
Immune complexes alone caused a TNF- level of 14,857 ± 2,423
pg/ml. There was virtually no MIP-2 present in lavage fluid of negative
control animals (3.35 ± 2.7 ng/ml). The sICAM-1 alone increased
MIP-2 levels to 173 ± 11.6 ng/ml (Fig. 11B). In the
positive controls also given heat-inactivated sICAM-1 (Fig. 11B), the MIP-2 level rose to 304.9 ± 25.2 ng/ml. When
immune complexes and unheated sICAM-1 were coinstilled, MIP-2 levels
increased by an additional 29%, to 393.5 ± 31.78 ng/ml
(p < 0.04). These data demonstrate that
sICAM-1 enhances IgG immune complex-induced lung injury, associated
with increased lung production of MIP-2 and TNF- and greater
accumulation of neutrophils, and intensifies the level of lung injury.
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by fluid phase and solid phase
sICAM-1
Alveolar macrophages were incubated with sICAM-1 in fluid phase or
in solid phase, and the expressions of MIP-2 and TNF- were compared.
The results are summarized in Table III.
Both fluid phase and solid phase sICAM-1 induced MIP-2 responses in a
dose-dependent manner. The responses to solid phase sICAM-1 appeared to
be consistently higher than those responses to fluid phase sICAM-1,
although statistical significance between the two responses was only
reached at the highest dose (2000 ng/ml). There was a dose-response
relationship for MIP-2 production for both fluid phase and solid phase
presentation of sICAM-1. In the case of TNF- responses, the response
to 1000 ng of sICAM-1 (fluid phase) was twofold above background; at
the same dose of solid phase sICAM-1, the response was nearly eightfold
greater than the TNF- response to fluid phase sICAM-1 (Table III).
Thus, alveolar macrophages can respond to either fluid phase or solid
phase sICAM-1; the latter appears to induce a more robust
response.
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These experiments were designed to determine whether alveolar
macrophages would adhere to solid phase sICAM-1 and to assess whether
such adherence was CD18 dependent. The sICAM-1 was immobilized on
plastic surfaces as described above, and alveolar macrophages were
exposed for 10 min to coated plates. As shown by the data in Figure 12A, macrophages adhered to
plates treated with 0.5 to 2.0 µg of sICAM-1 when compared with
adhesion in the absence of immobilized sICAM-1. Approximately 56% of
macrophages bound to surfaces exposed to 2 µg of immobilized sICAM-1
compared with binding of 18% of macrophages to uncoated plates. Under
the same conditions, the effect of anti-CD18 on adherence of
macrophages to immobilized sICAM-1 adhesion was evaluated. Macrophages
underwent 15 min of preincubation with anti-CD18 10 µg/ml (clone
WT-3, Endogen, Woburn, MA) or with an isotype-matched mouse IgG1
(MOPC-21) at the same concentration. Using 1.0 or 2.0 µg of sICAM-1
for immobilization, the presence of anti-CD18 totally abolished the
response to 1.0 µg of sICAM and reduced by 94% the adhesion response
to wells exposed to 2.0 µg of sICAM-1 (Fig. 12B). Thus,
adhesion of alveolar macrophages to immobilized sICAM-1 requires CD18.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Both the binding
interactions and cytokine production appear to be CD18 dependent. In
contrast to the responses of macrophages, PBMCs were not stimulated by
sICAM-1, although production of MIP-1 after cell exposure to
endothelial cell ICAM-1 has been described (3). Under the experimental
conditions employed in the current studies, alveolar macrophages were
considerably more responsive to sICAM-1 than were PBMCs.
There were suggestions that the cytokine responses of macrophages to
sICAM-1 may be optimized with the use of immobilized sICAM-1,
especially at the higher concentrations. Steric changes in immobilized
sICAM-1 may provide better access to ß2 ligands on
surfaces of alveolar macrophages. We have also obtained insights into
the relevant intracellular pathways triggered by sICAM-1 binding. It
has been suggested that NF-B translocation might be an essential
step in cell activation following adherence (30, 31). It is known that
the promoter regions for both MIP-2 and TNF- contain a consensus
motif for NF-B. In alignment with these reports, MIP-2 and TNF-
production by alveolar macrophages stimulated with sICAM-1 was
associated with NF-B activation. Interference with NF-B
activation using the aldehyde peptide inhibitor suppressed activation
of NF-B as well as cytokine formation. Our data also suggest that
tyrosine kinases may play a role in intracellular signaling following
binding of soluble ICAM-1 to ß2 integrins of macrophages.
This would be consistent with the conclusions of others (32, 33).
Cross-linking of ß2 integrins with Abs has been shown to
stimulate cells and mimic receptor/ligand interactions (24). In our
studies, exposure of macrophages to sICAM-1 or to monoclonal
anti-CD18 Ab (WT-3) also induced generation of MIP-2 and TNF-.
As expected, inhibition of NF-B translocation by the aldehyde
peptide inhibitor of proteasome and blocking of tyrosine kinase
activity by genistein inhibited the cytokine responses induced by
anti-CD18.
What was of special interest in these studies was whether
sICAM-1 would demonstrate proinflammatory effects in lung. To
evaluate this question, we used the rat model of IgG immune
complex-induced lung injury. MIP-2 and TNF- have been shown to play
a vital role in the development of inflammatory lung injury, being
necessary for neutrophil recruitment (13, 34, 35, 36). In the current
studies, airway instillation of sICAM-1 led to a small accumulation of
BAL neutrophils, but did not cause evidence of lung injury (as
determined by extravascular leak of albumin). Under the same conditions
no production of TNF- was measurable in BAL fluids, while small
amounts of MIP-2 could be measured. In contrast, intratracheal
coadministration of sICAM-1 with the anti-BSA intra-alveolarly
significantly increased neutrophil accumulation, which was associated
with enhanced lung injury, as defined by intensified vascular
permeability. These effects could be directly linked to augmented
levels of MIP-2 (by 29%) and TNF- (by 97%) levels in BAL fluids.
Our experiments also clearly showed that sICAM-1 can directly induce
macrophage activation in vitro, causing NF-B activation and
generation of cytokines. Since sICAM-1 can be shed from
surfaces of endothelial cells and released into the vascular
compartment and may be shed from surfaces of alveolar epithelial cells
and macrophages (both of which are known to express ICAM-1 on their
surfaces); this raises the question of the biologic relevance of this
phenomenon. Recent reports have suggested that sICAM-1 and E-selectin
can interfere with leukocyte binding to counter-receptors on
endothelial cells or synovial cells (37, 38). This suggests that
sICAM-1, if present in adequate amounts, may function as an
anti-inflammatory component, especially by limiting leukocyte
adhesion to endothelial cells. Thus, sICAM-1 shed into the vascular
compartment may lead to a self-limiting mechanism of the inflammatory
response. Alternatively, shedding of sICAM-1 into the distal airway
compartment may, in the presence of an already activated inflammatory
response, cause intensified injury due to enhanced production of
cytokines by lung macrophages. In general, these data suggest that in
lung, sICAM-1 may function as a proinflammatory or
anti-inflammatory mediator depending on the anatomic location of
the shed sICAM-1.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Peter A. Ward, Department of Pathology, University of Michigan Medical School, 1301 Catherine St., Box 0602, Ann Arbor, MI 48109-0602. E-mail address:
3 Abbreviations used in this paper: sICAM-1, soluble ICAM-1; MIP-2, macrophage inflammatory protein-1; BAL, bronchoalveolar lavage; EMSA, electrophoretic mobility shift assay; PSI, proteosome inhibitor; IPTG, isopropyl ß-D-thiogalactoside.
Received for publication April 2, 1998. Accepted for publication May 27, 1998.
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