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*
Department of Trauma Surgery, University of Freiburg, Freiburg, Germany;
Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109;
Department of Pediatrics, University of Cincinnati School of Medicine, Cincinnati, OH 45229;
§
Department of Physiology, University of Nebraska School of Dentistry, Lincoln, NE 68198; and
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Department of Surgery, University of Louisville School of Medicine, Louisville, KY 40292
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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in bronchoalveolar lavage fluids by 61%
(p = 0.008). Treatment of rats with anti-rat
MCP-1 or anti-rat RANTES had no effect on the development of lung
injury. In animals pretreated intratracheally with blocking Abs to
MCP-1, RANTES, or MIP-1ß, significant reductions in the
bronchoalveolar lavage content of these chemokines occurred, suggesting
that these Abs had reached their targets. Conversely, exogenously
MIP-1ß, but not RANTES or MCP-1, caused enhancement of the lung
vascular leak. These data indicate that MIP-1ß, but not MCP-1 or
RANTES, plays an important role in intrapulmonary recruitment of
neutrophils and development of lung injury in the model employed. The
findings suggest that in chemokine-dependent inflammatory responses in
lung CC chemokines do not necessarily demonstrate redundant
function. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and MIP-1ß (1). These subunits are
highly homologous. Because both MIP-1 and MIP-1ß show in vitro
chemotactic activity for monocytes and demonstrate nearly identical
binding characteristics for macrophages together with an ability to
cross-compete for binding to monocytes, a common receptor on monocytes
for MIP-1 and MIP-1ß has been suggested (2).
MIP-1, but not MIP-1ß, stimulates the secretion of TNF-,
IL-1, and IL-6 from peritoneal macrophages in vitro
(3). MIP-1 has been shown to contribute to leukocyte
recruitment and lung injury in models of lung inflammation
induced by deposition of IgG immune complexes (4) and by
airway instillation of bacterial LPS (4, 5). Under these
conditions MIP-1 seems to function as an autocrine stimulator of
macrophages, causing enhanced secretion of TNF-. Little is known
about the functions of MIP-1ß during in vivo inflammatory
reactions. Monocyte chemotactic protein-1 (MCP-1) is derived from mononuclear cells and other cell sources, including alveolar macrophages, and has chemotactic activity for monocytes (6, 7, 8, 9). Lung inflammatory injury in rats induced by IgA immune complexes is macrophage dependent but neutrophil independent. In this model, blockade of MCP-1 attenuates the development of lung injury (7). It has been suggested that MCP-1 may also enhance the inflammatory response to other stimuli (10). However, whether MCP-1 affects neutrophil-dependent acute lung inflammation is unknown.
RANTES has been characterized as a chemoattractant for monocytes and
lymphocytes (11). In a model of endotoxemia in mice,
RANTES was shown to be up-regulated in lung after i.p. injection of LPS
(12). Furthermore, the expression of RANTES in vivo was
dependent on the production of TNF-.
In the current study we sought to determine whether the rat CC
chemokines, MIP-1ß, MCP-1, and RANTES, were involved in the acute
inflammatory response following intrapulmonary deposition of IgG immune
complexes. All three chemokines were present in BAL fluids during lung
inflammation. Studies employing blocking Abs indicated that MIP-1ß
contributed significantly to lung production of TNF- and to
recruitment of neutrophils and full development of lung injury. In
contrast to the effects of in vivo blockade of MIP-1ß, Ab-induced
blockade of MCP-1 or RANTES had no effect on lung neutrophil
recruitment or the severity of lung injury, even though it could be
shown in vivo that the BAL levels of MCP-1 and RANTES were
substantially reduced in the presence of these Abs. In companion
experiments exogenous administration of MIP-1ß, but not of MCP-1 or
RANTES, caused enhanced lung injury. Thus, these data demonstrate a
role for MIP-1ß, but not for MCP-1 or RANTES, in IgG immune
complex-induced lung injury, suggesting that the biological functions
of CC chemokines in lung inflammatory reactions are not necessarily
interchangeable.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rabbit polyclonal IgG anti-BSA was purchased from ICN
Biomedicals (Costa Mesa, CA). Rabbit polyclonal Ab to rat MCP-1 was
produced as described previously (7). Rabbit polyclonal
IgG anti-rat RANTES was produced in rabbits and purified as IgG
using protein G affinity chromatography. By Western blot analysis,
anti-MCP-1 did not cross-react with MIP-1 or RANTES, and
anti-RANTES did not cross-react with MIP-1 or MCP-1. Goat
polyclonal IgG anti-murine MIP-1ß was purchased from R&D Systems
(Minneapolis, MN). The ability of anti-MCP-1 IgG to block the in
vitro chemotactic activity for mononuclear cells has been reported
(7), while the ability of anti-RANTES to cause delayed
rejection of cardiac allografts and xenografts in rats has also been
noted (13, 14).
IgG immune complex-induced alveolitis
Specific pathogen-free male Long-Evans rats (275–300 g; Charles River Breeding Laboratories, Portage, MI) were anesthetized with ketamine HCl (150 mg/kg i.p.). In positive control animals 2.5 mg of rabbit polyclonal IgG anti-BSA in a volume of 300 µl of PBS, pH 7.4, was instilled via an intratracheal catheter during inspiration. Immediately thereafter, 10 mg of BSA in 0.5 ml of PBS was injected i.v. Negative control rats received 2.5 mg of anti-BSA IgG intratracheally in the absence of an i.v. infusion of 10 mg of BSA. For analysis of pulmonary vascular permeability, trace amounts of 125I-labeled BSA were injected i.v. Four hours after initiation of the inflammatory reactions, rats were exsanguinated, the pulmonary circulation was flushed via the pulmonary artery with 10 ml of phosphate (PBS), and the lungs were surgically removed. The extent of lung injury was quantified by calculating the lung permeability index (dividing the amount of radioactivity ([125I]BSA) in the PBS-perfused lungs by the amount of radioactivity in 1.0 ml of blood obtained from the inferior vena cava at the time of sacrifice). Effects of in vivo blockade of MIP-1ß or RANTES in the lung injury model were assessed by the intratracheal instillation of 400 µg of anti-MIP-1ß, anti-RANTES, or nonspecific goat or rabbit IgG together with anti-BSA. In vivo blockade of MCP-1 was achieved by i.v. injection of 0.5 ml of anti-MCP-1 serum or 0.5 ml of normal rabbit serum just before infusion of BSA, because this is the protocol that caused suppression of other types of inflammatory injury in lung models of granulomatous vasculitis or in IgA immune complex-induced alveolitis (7, 15). In other experiments, where indicated, 400 µg of rabbit anti-rat MCP-1 IgG or preimmune IgG were given intratracheally together with anti-BSA. For the animal studies, the n values noted in the figure legends represent the number of rats used in each group, indicated by vertical bars. In other studies experimental results were indicative of similar findings from at least two separate and independent experiments.
RNA extraction for cloning
Whole lungs were dissected and immediately frozen in liquid nitrogen 2–4 h after initiation of immune complex deposition. Total RNA was extracted from lung homogenates using a guanidinium isothiocyanate/chloroform-based technique (RNA STAT-60, Tel-Test, Friendswood, TX) followed by isopropanol precipitation. Poly(A) mRNA was purified on oligo(dT)-cellulose. First-strand cDNAs were constructed from 1.0 µg of poly(A) mRNA in a RT reaction primed with a poly(dT) primer (cDNA Cycle Kit, Invitrogen, San Diego, CA). Cloning of rat MIP-1ß cDNA was obtained using these first strands as templates by PCR with the following oligonucleotide primers: 5' primer (5'-ATG AAG CTC TGC GTG TCT-3') and 3' primer (5'-TCA GTT CAA CTC CAA GTC A-3'). The PCR product of the predicted size (279 bp) was ligated into a pCRII vector (TA Cloning Kit, Invitrogen) and sequenced followed by submission of the results to GenBank (accession no. UO6434).
MIP-1ß protein expression
NdeI (5') and BamHI (3') restriction enzyme sites were added to the 5' and 3' ends of the cDNA encoding the mature protein for MIP-1ß by PCR for ligation into the pET15b expression vector (Novagen, Madison, WI). After ligation, the pET-rMIP-1ß plasmids were transformed into competent Nova Blue Escherichia coli. After plasmid purification proper orientation of the insert was confirmed by restriction digest analysis and sequencing. Purified pET-rMIP-1ß plasmid was then used for transformation of the E. coli strain BL21(DE3)pLysS. Following induction of the protein with 0.4 mM isopropyl ß-D-thiogalactopyranoside (Life Technologies, Grand Island, NY), the cells were lysed into 50 mM NaH2PO4 (pH 8.0), 300 mM NaCl, and 10 mM imidazole. The supernatant fluid was applied to a Ni-NTA column (Qiagen, Santa Clarita, CA), and the protein was eluted according to the manufacturer’s instructions, dialyzed in PBS, and concentrated using Centricon concentrators (Amicon, Beverly, MA). Rat PBMC and alveolar macrophages were evaluated for chemotactic responses to CC chemokines as previously described (4). FMLP was used as a reference as a positive control.
RT-PCR cloning of rat RANTES
First-strand cDNAs were constructed using 1 mg of mRNA with a RT reaction primed with poly(dT) primer (cDNA Cycle Kit, Invitrogen). Amplification was performed using these first strands as templates in a PCR employing the following primers: 5' primer (5'-ACC AATG AAG ATC TCT GCA GCT-3') and 3' primer (5'-ATC CTA GCT CAT CTC CAA ATA-3'). PCR products were then ligated into a pCRII vector according to the manufacturer’s instructions (TA Cloning Kit, Invitrogen). TA One Shot competent E. coli cells (TA Cloning Kit, Invitrogen) were transformed with ligation reaction mixtures and grown up for plasmid purification. Plasmids that provided a template for a successful PCR were sequenced by the University of Michigan Core Facility using the T7 and SP6 promoter regions of the pCRII vector and compared with the known human and murine sequences. The procedure was duplicated on a product cloned from mRNA obtained from lungs of a second rat undergoing intrapulmonary deposition of IgG immune complexes.
Recombinant expression of rat RANTES
HindIII (5') and BamHI (3') restriction enzyme sites were added to the ends of the cDNA encoding the mature protein for rat RANTES by PCR. The amplified product was then used for ligation into the pET23a vector (Novagen). Competent Nova Blue E. coli was transformed with the ligated pET-rRANTES plasmids. After confirmation of proper orientation by restriction digest analysis, purified plasmids were used for transformation of BL21(DE3)pLysS E. coli strains. These bacterial cultures were used to optimize isopropyl ß-D-thiogalactopyranoside-stimulated expression of recombinant rat RANTES.
Rabbit polyclonal anti-rat RANTES
Polyclonal rabbit anti-rat RANTES was raised against the expression product in 3-kg New Zealand White rabbits repeatedly immunized with 50–500 µg of rat RANTES emulsified in Freund’s adjuvant. For determination of serum titers of anti-RANTES Ab, an indirect ELISA was used. Briefly, 96-well Immulon 4 ELISA plates (Dynex Technologies, Chantilly, VA) were coated with soluble recombinant rat RANTES (5 µg/ml) overnight at 4°C. The plates were blocked with 2% BSA in PBS for 1 h before addition of serial dilutions of rabbit anti-RANTES serum. The plates were washed, and 100 µl/well HRP-conjugated goat anti-rabbit Ab (1/3000 dilution; Bio-Rad, Richmond, CA) was added to the wells. Reactions were developed with o-phenylenediamine dihydrochloride substrate and stopped by addition of 3 M H2SO4. Titers were determined by measuring absorbance at 490 nm. The ability of this Ab to neutralize recombinant rat and human RANTES was demonstrated by the chemotaxis assay (as described below).
Pulmonary expression of MIP-1ß, MCP-1, and RANTES mRNA
Pulmonary cytoplasmic RNA was isolated as previously described
(4), fractionated electrophoretically in a 1%
formaldehyde gel, and transferred to a nylon membrane (Micron
Separations, Westboro, MA). For analysis of MIP-1ß mRNA, a
MIP-1ß-specific oligomer (5'-GGC CAC AAG CAG GAG GAG AGA GAA GGC AGA
CAC-3') was end labeled with [
-32P]ATP. For
analysis of MCP-1 or RANTES mRNA, plasmids containing the confirmed rat
cDNAs for MCP-1 or RANTES were used to generate radiolabeled
[32P]dCTP probes by PCR using specific
oligoprimer pairs. The primer sequences for MCP-1 were 5'-ATC AGC TAG
CCT CCA CCA CTA TGC-3' and 5'-CTA AAC CTT ACA CTA CGA TCG GGT GG-3'.
The primer sequences for RANTES were 5'-ACC ATG AAG ATC TCT GCA GCT-3'
and 5'-ATA AAC CTC TAC TCG ATC CTA-3'. Northern blots were hybridized
at 65°C for 16 h. Autoradiography of the blots was performed at
-70°C on Kodak BioMax MR film (Eastman Kodak, Rochester,
NY).
Pulmonary expression of MIP-1ß, MCP-1, and RANTES proteins
Lung expression of MIP-1ß, MCP-1, and RANTES was assessed by Western blot analysis. Lungs were obtained from rats 4 h after intrapulmonary deposition of IgG immune complexes (positive controls) or from rats 4 h after receiving anti-BSA intratracheally but with the omission of i.v. infused BSA (negative controls). The lungs were homogenized, and the material was centrifuged for 30 min at 14,000 x g. Supernatant fluids were subjected to electrophoretic separation in a nonreducing 10% polyacrylamide gel and transferred to a nitrocellulose membrane. Nonspecific binding sites were blocked with TBS (40 mM Tris, pH 7.6, and 300 mM NaCl) containing 5% nonfat dry milk for 12 h at 4°C. Membranes were then incubated with 2 µg/ml IgG Ab to the relevant CC chemokine in TBS with 0.1% Tween 20 (TBST). After three washes in TBS with 0.1% Tween 20, membranes were incubated in a 1/25,000 dilution of HRP-conjugated donkey anti-goat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) for detection of MIP-1ß. For RANTES and MCP-1, HRP-conjugated goat anti-rabbit IgG was employed. Immunoreactive proteins were detected by enhanced chemiluminescence according to manufacturer’s instructions.
Chemotaxis assay
This assay was described previously (4, 7). Rat PBMC and rat alveolar macrophages were labeled with 2',7'-bis-[2-carboxyethyl]-5-[and 6]-carboxy-fluorescein acetoxymethyl ester (Molecular Probes, Eugene, OR). Labeled cells (44 µl; 2.25 x 105 cells/well) were then loaded into upper compartments of 96-well minichambers (NeuroProbe, Cabin John, MD). Test samples (in 33 µl) were added to the bottom compartments. The top and bottom compartments were separated by a polycarbonate membrane with a pore size of 3 µm, and the chambers were incubated at 37°C for 2 h. The numbers of cells that migrated through the polycarbonate filter (on the bottom surface of the membrane) were measured with a cytofluorometer (Cytofluor II, PerSeptive Biosystems, Framingham, MA), using an excitation wavelength of 485 nm and an emission wavelength of 530 nm and expressed as fluorescence units.
Lung myeloperoxidase (MPO) content
Whole lungs obtained at the time of sacrifice were immediately frozen in liquid nitrogen. After mechanical homogenization in a Polytron homogenizer (Tebmar, Cincinnati, OH) at an instrument setting of 4 for 40 s at 5°C, MPO content was quantitated by measuring the rate of decomposition of H2O2 (per minute) in the presence of O-dianisidine at an absorbance of 460 nm (16).
TNF- levels in BAL fluids
The BAL fluid content for TNF- was measured using the highly
sensitive WEHI cell cytotoxicity assay as previously reported
(17).
Analysis of CC chemokines in BAL fluids in the presence of blocking Abs
To determine whether the blocking Abs to CC chemokines reached their targets, 400 µg of Ab IgG to MCP-1, RANTES, or MCP-1 or 400 µg of preimmune IgG were instilled intratracheally with the anti-BSA IgG followed by i.v. infusion of 10 mg of BSA, according to procedures described above. Negative controls received 400 µg of preimmune IgG intratracheally together with the anti-BSA, but the i.v. infusion of BSA (10 mg) was omitted. At the time of sacrifice, BAL fluids (3.0 ml) were collected (see above). To remove IgG from the BAL fluids, because IgG would interfere with the ELISA measurement, BAL fluids were subjected to centrifugation in Ultrafree centrifugal filter and tube devices (Millipore, Bedford, MA) with a cut-off of 50 kDa. The pass-through fluid was then analyzed for the appropriate CC chemokine by indirect ELISA as described above. All reagents were purchased from R&D Systems. For MIP-1ß detection, coating Ab (1 µg/ml) followed by biotinylated Ab (200 ng/ml) was used. For MCP-1 detection, coating Ab and biotinylated Ab (2 µg/ml and 200 ng/ml, respectively) were used. An ELISA detection kit was used for RANTES detection, using instructions of the manufacturer.
Exogenous administration of CC chemokines
To extend studies featuring the in vivo use of Abs to MIP-1ß. MCP-1 and RANTES, recombinant CC chemokines (5.0 µg each), were added to intratracheally administered anti-BSA, and the permeability index was measured at 4 h according to technical details provided above and in Results. Recombinant mouse MIP-1ß was obtained from R&D, while recombinant rat MCP-1 and RANTES were obtained from PeproTech (Rocky Hill, NJ).
Densitometric analysis
Northern blots were exposed for 3 days on a phosphorimaging screen and quantitated by laser densitometry using ImageQuant software and PhosphorImager 445 SI (both obtained from Molecular Dynamics, Sunnyvale, CA).
Statistical analysis
All values are expressed as the mean ± SEM. Data were analyzed with a one-way ANOVA, and individual group means were compared using Student-Newman-Keuls test. Differences were considered significant when p < 0.05. For calculation of the percent change, negative control values were subtracted from positive control group.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cloned cDNA for rat MIP-1ß (GenBank accession no. UO6434) had a 279-nucleotide open reading frame that shared 89% homology with the published murine oligonucleotide sequence (1). The open reading frame encoded a 93-aa protein with a putative 26-aa signal peptide; the mature protein consisted of 67 aa. The deduced amino acid sequence shared 84% homology with the published sequence of the murine protein.
Expression of rat recombinant (rr) MIP-1ß
rrMIP-1ß was expressed in BL21(DE3)pLysS E. coli,
analyzed by 15% SDS-PAGE, and stained with Coomassie blue. The
expressed product showed a band that aligned closely with the 14.3-kDa
marker together with slower migrating species, perhaps representing
multimers (Fig. 1
A), formation
of which would be promoted by the four cysteine residues present in
MIP-1ß. Western blot analysis showed a band for rrMIP-1ß (Fig. 1B, lane 2) that closely aligned with the
14.3-kDa marker compared with the position of rmMIP-1ß, which
appeared between the 6.2- and 14.3-kDa markers (Fig. 1B,
lane 1).
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Rat rMIP-1ß was purified from BL21(DE3)pLysS E. coli
supernatants at a final stock concentration of 300 µg/ml. The
chemotactic activity of rrMIP-1ß for rat PBMC was assessed and
compared with the PBML responses to FMLP. Rat rMIP-1ß had significant
chemotactic activity for rat PBMB over a range from 3.8 x
10-13 to 3.8 x 10-9
M, with peak activity at 3.8 x 10-10 M
(Fig. 2). The characteristic fall in
chemotactic activity at high concentrations of chemoattractant was
found. PBMC demonstrated peak chemotactic responses to 10 nM
FMLP.
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Pulmonary expression of MIP-1ß mRNA during IgG immune
complex-induced injury was determined as a function of time (0–48 h)
by Northern blot analysis. Equal loading of RNA was suggested by the
28S marker (Fig. 3A). MIP-1ß
mRNA was undetectable in lungs of animals sacrificed at time zero (Fig. 3, B and C). However, up-regulation of MIP-1ß
mRNA was observed as early as 30 min after the intrapulmonary
deposition of immune complexes. Peak expression of MIP-1ß mRNA
occurred at 6 h, similar to the time courses for expression of
mRNA for MIP-2, MIP-1, and cytokine-induced neutrophil
chemoattractant (CINC) in inflamed rat lung (18, 19, 20).
Enhanced expression of mRNA for MIP-1ß was observed for as long as
24 h. By 48 h, MIP-1ß mRNA returned to nearly undetectable
levels. A similar pattern of expression was noted in two additional,
independent experiments.
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Expression of MIP-1ß in lung was assessed by Western blot
analysis of whole lung homogenates obtained 4 h after initiation
of lung injury. Homogenates (50, 100, 300, and 500 µg total protein)
were separated in SDS-PAGE under nonreducing conditions. In these
experiments, 25 ng of rmMIP-1ß was used as a positive control.
Anti-MIP-1ß recognized a protein in rat lung homogenates (300 and 500
µg) in a position that aligned with the slowest migrating of three
bands for rmMIP-1ß (Fig. 4). It is
assumed that the rmMIP-1ß preparation contained isoforms of MIP-1ß.
Western blot analysis of MIP-1ß in lung homogenates (Fig. 4) also
revealed the presence of slower migrating species of material reactive
with anti-MIP-1ß. Homogenates (500 µg of protein) obtained from
lungs of rats undergoing IgG immune complex deposition but sacrificed
at time zero failed to demonstrate reactivity for MIP-1ß in Western
blots.
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To determine whether MIP-1ß contributed to IgG immune
complex-induced lung injury, rats were treated with MIP-1ß blocking
Ab. Administration of 400 µg of anti-MIP-1ß (given
intratracheally with the anti-BSA) significantly reduced the
permeability index by 37% (p = 0.012) compared
with otherwise unmanipulated positive controls or compared with
positive controls also receiving 400 µg of preimmune IgG
intratracheally with the anti-BSA (Fig. 5A). Anti-MIP-1ß reduced IgG
immune complex-induced increases in lung MPO by 65%
(p = 0.047) compared with otherwise
unmanipulated positive controls (Fig. 5B). Compared with the
positive control group receiving preimmune IgG, which caused elevated
lung levels of MPO, treatment with anti-MIP-1ß depressed the MPO
content by nearly 80% (Fig. 5B). BAL fluids from negative
control lungs (in which the i.v. infusion of BSA was omitted) contained
virtually no detectable TNF- (Fig. 5C). In contrast,
levels of TNF- in BAL fluids from IgG immune complex-injured lungs
(at 4 h) were decreased by 61% (p =
0.008) in the presence of anti-MIP-1ß (Fig. 5C),
whereas BAL content of TNF- was not significantly reduced by
treatment with 400 µg of preimmune goat IgG compared with that in the
otherwise nonmanipulated positive controls. The data suggest that
MIP-1ß, like MIP-1 (4), is required for the full
generation of TNF- and the subsequent recruitment of neutrophils and
full development of lung injury.
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Pulmonary expression of MCP-1 mRNA during IgG immune
complex-induced lung injury was determined as a function of time (0–8
h) by Northern blot analysis. Equal loading of RNA was suggested by the
28S marker (Fig. 6A). MCP-1
mRNA was undetectable in lungs at time zero (Fig. 6, B and
C). Up-regulation of MCP-1 mRNA was detected by 1 h and
progressively increased, reaching maximal levels 4 h after
induction of lung injury. Peak expression of MCP-1 mRNA was maintained
for as long as 8 h. Pulmonary expression of MCP-1 protein was
assessed by ELISA analysis of BAL fluids. Levels of MCP-1 protein were
virtually undetectable in negative controls (<2 ng/ml; Fig. 6D) in which i.v. infusion of BSA was omitted. Four hours
after initiation of IgG immune complex-induced lung injury, MCP-1
levels in BAL fluids increased to 12.3 ± 0.3 ng/ml
(p < 0.05).
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To determine whether MCP-1 contributed to lung injury induced by
IgG immune complexes, rats were injected i.v. with 0.5 ml of normal
rabbit serum or 0.5 ml of rabbit anti-rat MCP-1 serum just before
infusion of BSA. This protocol has been shown to protect against
MCP-1-dependent models of acute lung injury in rats (7, 15). Treatment with anti-MCP-1 did not reduce IgG immune
complex-induced increases in the lung permeability index (Fig. 7A). Similarly, anti-MCP-1
had no effect on lung recruitment of neutrophils as determined by lung
MPO content (Fig. 7B). Additional studies were performed
with intratracheal administration of 400 µg of anti-MCP-1 IgG
(rabbit) or preimmune rabbit IgG. Similar to the protocols for in vivo
use of anti-MCP-1 and anti-RANTES, 400 µg of anti-MCP-1
or 400 mg of preimmune IgG was added to the anti-BSA preparation,
and the mixture was instilled intratracheally. Using the procedures
described above, the permeability index was measured at 4 h in two
positive control groups receiving either preimmune IgG or
anti-MCP-1 IgG. Six animals were in each group. The permeability
index values for the positive control groups receiving either preimmune
IgG or anti-MCP-1 IgG were 0.65 ± 0.05 and 0.69 ± 0.05,
respectively (nonsignificant). Thus, although MCP-1 mRNA is
up-regulated during IgG immune complex-induced lung inflammation (Fig. 7), it apparently does not contribute to the development of lung injury
in the lung injury model.
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Pulmonary expression of RANTES mRNA during IgG immune
complex-induced lung injury was determined by Northern blot analysis as
a function of time (0–8 h). Equal loading of RNA was suggested by the
28S marker (Fig. 8A). RANTES
mRNA was constitutively expressed in lungs of animals undergoing immune
complex deposition but sacrificed at time zero (Fig. 8, B
and C). Changes in RANTES mRNA were not consistently
detected in lung extract over an 8-h period following induction of the
lung inflammatory reactions.
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Intratracheal administration of 400 µg of anti-rat RANTES
(coinstilled with anti-BSA) did not reduce the increase in the lung
permeability index in the lung inflammatory model compared with the
effect of intratracheal coinstillation (with anti-BSA) of 400 µg
of preimmune IgG (Fig. 8
D). No protection from injury was
found despite the fact that anti-rat RANTES blocked the chemotactic
properties of rrRANTES (Table I), and, as
shown (below) in Table II, the presence
of anti-RANTES IgG prevented detection of RANTES in BAL fluids from
inflamed lungs. As shown in Table I, both rat PBMC and rat alveolar
macrophages responded chemotactically to FMLP and rrRANTES, although
the chemotactic responses of alveolar macrophages to rrRANTES were more
impressive. In part this was due to the relatively high background
values (in the presence of the buffer, HBSS) found with rat PBMC. In
the presence of anti-RANTES, the chemotactic responses to RANTES
were profoundly suppressed. This same Ab significantly prolonged the
rejection time of cardiac allografts and xenografts in rats (13, 14). The data suggest that although RANTES is constitutively
expressed in lung at the mRNA level, its levels are not altered during
IgG immune complex-induced lung injury. The failure of anti-RANTES
to reduce neutrophil influx and to protect against lung injury suggests
that RANTES does not contribute directly or indirectly to the
development of lung injury in the model employed.
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To extend the results with blocking Abs to MIP-1ß, MCP-1, or
RANTES (see below), another series of experiments was completed in
which 5.0 µg of MIP-1ß, MCP-1, or RANTES was added to the
anti-BSA IgG, which was administered intratracheally. The
permeability index was determined 4 h later. As shown by the data
in Table III (Expt A), the copresence of
RANTES did not alter the permeability index in a statistically
significant manner, while MIP-1ß significantly increased (by 44%;
p = 0.02) the lung permeability index. The copresence
of MCP-1 (Table III, Expt B), like that of RANTES, failed to cause any
increase in the vascular permeability index in this lung injury model.
These observations, which are the reverse of the effects of blocking
Abs employed in vivo, strengthen the conclusion in this model of lung
injury that MIP-1ß, but not MCP-1 or RANTES, participates in events
leading to lung injury.
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Animals (n = 4/group) undergoing IgG immune
complex deposition received 400 µg of preimmune IgG or 400 µg of
anti-MCP-1, anti-RANTES, or anti-MIP-1ß IgG
intratracheally together with the anti-BSA, as described above. At
the time of sacrifice, BAL fluids were obtained, and ELISA was
performed for quantitation of the three CC chemokines (MCP-1, MIP-1ß,
and RANTES). Experimental protocols are described above. The results
are shown in Table II. As shown by the data, the BAL levels of MCP-1
were reduced from 46.1 ± 1.91 to 17.4 ± 1.77 when the
latter group received intratracheal instillation of anti-MCP-1;
this difference was statistically significant and represented nearly an
80% reduction when the negative control values are subtracted from the
positive control groups. In the case of RANTES the increase from
74.0 ± 4.02 to 312 ± 10.4 occurring in negative and
positive controls, respectively, was totally eliminated in the presence
of anti-RANTES. In fact, BAL levels of RANTES fell to less than
detectable levels (<3 pg/ml). In the case of MIP-1ß, BAL levels in
the negative and positive controls were 2.75 ± 0.35 and 7.50
± 1.41 pg/ml, respectively. In the presence of anti-MIP-1ß,
levels fell to 0.73 ± 0.04 pg/ml. Thus, it appears that
intratracheal delivery of anti-CC chemokines found their targets in
lung, causing substantial reductions in BAL levels of these CC
chemokines.
To determine whether the administration of anti-MIP-1ß affected
BAL levels of MIP-1 (which is known to be required for the full
expression of injury in this model (18)), BAL fluids (as
described in Table II, in which IgG anti-MIP-1ß IgG was employed
in vivo) were evaluated by ELISA for MIP-1 content. The negative
control values for MIP-1 were 4.00 ± 0.35 pg/ml, while the
positive control values in animals treated with preimmune IgG or
anti-MCP-1 IgG (400 µg intratracheally) were 110 ± 10.5 and
103 ± 6.15 pg/ml, respectively. In positive controls receiving
400 µg of anti-MIP-1ß intratracheally, the MIP-1 values fell
to 34.7 ± 5.52 pg/ml, suggesting that full in vivo expression of
MIP-1 depends on MIP-1ß.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, is
up-regulated during IgG immune complex-induced lung injury
(4). In this model the development of lung injury is
dependent upon the intrapulmonary recruitment of neutrophils
(22). Treatment with blocking Ab to MIP-1 significantly
reduced both lung recruitment of neutrophils and the increases in
vascular permeability induced by formation of IgG immune complexes
(4). These effects were associated with marked reductions
in BAL levels of TNF-, suggesting that MIP-1 functions as an
autocrine activator of alveolar macrophages, facilitating their
production of TNF-. The reduction of lung neutrophil recruitment
observed under conditions of MIP-1 blockade could in part be
attributed to neutralization of MIP-1, which also possesses
chemotactic activity for neutrophils (23). As well,
reduced lung neutrophil accumulation mediated by MIP-1 blockade may
be a result of reduced intrapulmonary production of TNF- and
subsequent up-regulation of vascular adhesion molecules. Generation of
TNF- (and IL-1) during IgG immune complex-induced lung injury is the
primary stimulus for the up-regulation of the adhesion molecules,
ICAM-1 and E-selectin, on pulmonary vascular endothelial cells
(24, 25). In this model both ICAM-1 and E-selectin are
required for lung neutrophil recruitment (26). The current
study demonstrates a similar role for MIP-1ß in IgG immune
complex-induced lung injury. Unlike MIP-1, MIP-1ß demonstrates no
chemotactic activity for neutrophils (27). Thus, it is
likely that, under conditions of MIP-1ß blockade, reduced lung
neutrophil recruitment is due to reduced pulmonary production of
TNF- and subsequent reduction in the up-regulation of adhesion
molecules in the pulmonary vasculature. Our in vivo data contrast with
previous in vitro studies that failed to demonstrate an effect of
MIP-1ß on macrophage production of TNF- (3). Together
with the current findings the fact that the earlier studies used
peritoneal macrophages could suggest organ-specific macrophage
responses to MIP-1ß.
The role of MCP-1 during acute inflammation is unclear. In IgA immune
complex-induced lung injury, blockade of MCP-1 by i.v. administration
of anti-MCP-1 greatly reduced the extent of tissue injury
(7). In this model lung injury is monocyte/macrophage
dependent, suggesting that MCP-1 may function as an activator of
pulmonary macrophages. Also, in pulmonary granulomatous vasculitis
occurring after the i.v. infusion of glucan, this inflammatory reaction
was greatly reduced by Ab to rat MCP-1 (15). During acute
endotoxemia in mice, i.v. administration of MCP-1 reduced plasma levels
of TNF- and improved survival rates that were associated with
increased IL-10 production (28). In the same studies
neutralization of MCP-1 augmented TNF- production, reduced IL-10
production, and decreased survival, suggesting that MCP-1 may under
special circumstances have anti-inflammatory effects. In the
current study we show that MCP-1 is up-regulated in lung shortly after
the deposition of IgG immune complexes. However, blockade of MCP-1 had
no effect on pulmonary neutrophil recruitment or the extent of lung
injury. Whether the biological effects of MCP-1 are species specific or
are specific for the type of inflammatory condition under scrutiny
remains to be determined.
Expression of RANTES mRNA is constitutive in lung (Fig. 8). Induction
of lung inflammation by IgG immune complexes did not lead to measurable
up-regulation of RANTES mRNA beyond the constitutive level. RANTES
demonstrates chemotactic activity for monocytes, T lymphocytes of the
memory/helper phenotype, and eosinophils (11, 29). No
significant chemotactic activity for neutrophils has been detected, but
RANTES has the ability to activate basophils and cause histamine
release (30). Ab to rat RANTES has caused significant
prolongation in the survival of hearts allografted from Brown Norway
rats into Lewis rats (13) and of hamster hearts
xenografted into rats (14). However, in the IgG immune
complex model of lung injury described in the current studies,
treatment with anti-RANTES failed to reduce lung neutrophil
accumulation and did not affect the increases in lung vascular
permeability. The lack of an effect of anti-RANTES on acute lung
inflammation occurred despite the fact that this preparation was also
shown to potently block the chemotactic activity of rrRANTES for PBMC
and alveolar macrophages and to reduce BAL levels of RANTES below
measurable levels. Intravenous protocols for anti-MCP-1 and
anti-RANTES blocked the production of IgA immune complex-induced
lung injury (7) and rejection of allografted hearts in
rats, respectively (see above). Although it is possible that in the IgG
immune complex model of lung injury insufficient Ab gained access to
lung RANTES and MCP-1, this seems unlikely in view of the effects of
these Abs on BAL levels of these two chemokines. Therefore, it appears
that RANTES as well as MCP-1 have no demonstrable role in the
development of acute lung inflammation induced by IgG immune
complexes.
The experiments featuring the exogenous administration of MIP-1ß,
RANTES, or MCP-1 (together with the anti-BSA) indicate that only
MIP-1ß enhances lung injury as measured by the change in lung
vascular extravasation of [125I]albumin (Table III). These observations correlate inversely with the blocking effects
of anti-MIP-1ß on reducing parameters of inflammatory lung injury
(Fig. 6), whereas anti-MCP-1 (Fig. 7) and anti-RANTES (Fig. 8)
failed to influence the intensity of inflammatory lung injury triggered
by intrapulmonary deposition of IgG immune complexes. Collectively,
these data suggest that in this model of lung injury only one of the
three CC chemokines participates in the pathogenesis of lung
injury.
In acute lung inflammatory injury in rats, there appears to be a
somewhat late peak in the expression of mRNA for MIP-1
(18), MIP-1ß and MCP-1 (this report), MIP-2 (19, 20), and CINC (20). Despite this, in the case of
MIP-2 and CINC proteins, which were precisely quantitated by ELISA in
BAL fluids, the expression of CINC protein peaked at 2 h, while
the peak for MIP-2 was between 2 and 4 h. The apparent discrepancy
between lung mRNA levels and BAL chemokine content could well be due to
the fact that the BAL content of chemokines probably derives from
alveolar macrophages, while much of the mRNA measured in whole lung
extracts derives from the interstitial compartment, which includes
macrophages and other cell types capable of expressing chemokines. It
should also be noted that beyond 4 h after initiation of IgG
immune complex-induced lung injury in rats, no further increases occur
in injury (measured as [125I]albumin
extravasation), in lung buildup of neutrophils (myeloperoxidase
content), and in BAL levels of TNF- (20). Accordingly,
the focus on changes occurring between 0 and 4 h seems most
relevant to the pathogenesis of events linked to inflammatory injury in
the lung.
This report as well as previously published data indicate that some CC
chemokines, namely MIP-1 and MIP-1ß, play significant roles in the
induction of acute inflammatory reactions in lung. The data suggest
that the functions of MIP-1 and MIP-1ß during acute lung
inflammation induced by IgG immune complexes may be separate from their
leukocyte chemoattractant properties. It appears that both MIP-1 and
MIP-1ß operate as autocrine activators of alveolar macrophages,
facilitating the acute inflammatory process. In contrast, other CC
chemokines, including MCP-1 and RANTES, do not appear to have any
relevant role in the development of acute lung injury, despite being
constitutively expressed (RANTES) or up-regulated (MCP-1) during lung
inflammation.
The pathogenesis of the lung inflammatory reactions triggered by
intrapulmonary deposition of IgG immune complexes in rats is probably
dependent on engagement of FcRs in lung macrophages. Knockout mice
lacking FcR showed profoundly depressed acute inflammatory responses
in reversed passive Arthus reactions compared with those of wild-type
mice (31). Engagement of FcRs in these inflammatory models
would be expected to be required for activation of tissue macrophages
and their generation of a wave of cytokines and chemokines. In the lung
inflammatory reactions described in the current report, BAL macrophages
showed immunostaining for MIP-1ß (data not shown), similar to our
earlier studies of MIP-2 and CINC in the same inflammatory model
(20). Regarding the role of complement in these
inflammatory models, complement depletion involving reduction of serum
C3 to <3% of normal levels or blockade of C5a with Ab profoundly
suppressed the lung-damaging inflammatory response, reduced BAL levels
of TNF-, and greatly diminished up-regulation of vascular ICAM-1
(32, 33). Why C3-deficient (knockout) mice appear to
respond to immune complex-induced alveolitis (34) is
unclear, but it is possible that in the absence of C3,
noncomplement-derived C5-cleaving enzymes that are known to be abundant
in neutrophils (35, 36) may replace the
complement-generated C5 convertases.
|
Acknowledgments |
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|
Footnotes |
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2 Address correspondence and reprint requests to Peter A. Ward, Department of Pathology, University of Michigan Medical School, M5240 Medical Science I, Box 0602, 1301 Catherine Road, Ann Arbor, MI 48109-0602. E-mail address:
3 Abbreviations used in this paper: MIP-1, macrophage inflammatory protein-1; MCP-1, monocyte chemotactic protein-1; MPO, myeloperoxidase; rr, rat recombinant; CINC, cytokine-induced neutrophil chemoattractant.
Received for publication July 30, 1999. Accepted for publication December 13, 1999.
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References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and ß and monocyte chemotactic and activating factor utilize common and unique receptors on human monocytes. J. Immunol. 150:3022.[Abstract]
(MIP-1) in acute lung injury in rats. J. Immunol. 54:4793.
mediates lung leukocyte recruitment, lung capillary leak, and early mortality in murine endotoxemia. J. Immunol. 155:1515.[Abstract]
(MIP-1) in acute lung injury in rats. J. Immunol. 154:4793.[Abstract]
regulates in vivo intrapulmonary expression of ICAM-1. Am. J. Pathol. 142:1739.[Abstract]
and -1ß. J. Immunol. 150:4550.[Abstract]
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