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Departments of
* Pathology and
Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 48109;
Veterans Administration Medical Center, Ann Arbor, MI 48105; and
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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15,000–20,000 receptors/cell. Recombinant mC5a competed effectively
with binding of [125I]rmC5a to MDMEC. Enhanced binding of
C5a occurred, as well as increased mRNA for C5aR, after in vitro
exposure of MDMEC to LPS, IFN-
, or IL-6 in a time- and
dose-dependent manner. By confocal microscopy, C5aR could be detected
on surfaces of MDMEC using anti-C5aR Ab. In vitro expression of
macrophage inflammatory protein-2 (MIP-2) and monocyte chemoattractant
protein-1 (MCP-1) by MDMEC was also measured. Exposure of MDMEC to C5a
or IL-6 did not result in changes in MIP-2 or MCP-1 production, but
initial exposure of MDMEC to IL-6, followed by exposure to C5a,
resulted in significantly enhanced production of MIP-2 and MCP-1 (but
not TNF-
and MIP-1). Although LPS or IFN- alone induced some
release of MCP-1 and MIP-2, pre-exposure of these monolayers to LPS or
IFN-, followed by addition of C5a, resulted in synergistic
production of MIP-2 and MCP-1. Following i.v. infusion of LPS into
mice, up-regulation of C5aR occurred in the capillary endothelium of
mouse lung, as determined by immunostaining. These results support the
hypothesis that C5aR expression on MDMEC and on the microvascular
endothelium of lung can be up-regulated, suggesting that C5a in the
co-presence of additional agonists may mediate pro-inflammatory effects
of endothelial cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1) have been shown to induce endothelial injury in the
presence of neutrophils, in part due to endothelial cell-enhanced
expression of adhesion molecules (4), appearance in plasma
of chemokines (5, 6), and increased permeability of
endothelial monolayers (7). During sepsis and multiple organ dysfunction syndrome, increased levels of the complement activation product, C5a, occur in plasma (8, 9, 10, 11, 12). C5a induces its inflammatory functions by interacting with specific receptors (C5aR) that belong to the rhodopsin family of seven-transmembrane G protein-coupled receptors (13, 14, 15). Traditionally, C5aR expression was thought to be present only on hemopoietic cells bone marrow cells (16), neutrophils (17), monocytes (18), and eosinophils (19). However, recent studies have demonstrated the presence of C5aR on non-myeloid cells, including human lung and liver (20, 21, 22), rodent type II alveolar epithelial cells (23), astrocytes (24), kidney tubular epithelial cells (25), mesangial cells (26), and hepatocyte-derived cell lines (27, 28). Various studies have further documented that C5aR expression is up-regulated in some organs under pathologic conditions. Enhanced C5aR expression has been reported in pyogenic granulomas of human skin (29), Huntington disease (30), allergic encephalomyelitis (31), and the inflamed central nervous system (32). The presence of C5aR on cultured endothelial cells has been controversial. Although C5a has been shown to induce P-selectin expression and secretion of von Willebrand factor (vWF)3 (33) and to increase tissue factor activity in HUVECs (34), the presence of specific C5a binding sites on HUVEC has been debated (35).
The current studies were designed to characterize the binding of
recombinant mC5a (rmC5a) to mouse dermal microvascular endothelial
cells (MDMEC). The ability of C5a to bind to MDMEC was consistent with
a ligand-receptor interaction. In addition, since there is abundant
evidence that apart from complement activation, LPS and early response
cytokines, such as IL-6 and IFN-, have been implicated in the
inflammatory process (36, 37, 38), we evaluated the effects of
these mediators on C5aR expression of MDMEC. Enhanced binding of rmC5a
and increased expression of mRNA for C5aR were found in MDMEC following
exposure to LPS, IL-6, or IFN-. Increased C5aR protein was also
detected on these cell surfaces, using C5aR Ab. These changes were
associated with enhanced production of macrophage inflammatory
protein-2 (MIP-2) and monocyte chemoattractant protein-1 (MCP-1) after
addition of C5a. We also show evidence for increased expression of C5aR
protein in vivo in small pulmonary vessels and capillaries of mice
following LPS infusion, extending the in vitro data.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For all experiments, 6- to 8-wk-old mice (B10D2/nSn-J; The Jackson Laboratory, Bar Harbor, ME) were used.
Reagents
RPMI 1640, heat-inactivated FBS, penicillin-streptomycin,
fungizone, and L-glutamine were purchased from Life
Technologies (Grand Island, NY). Endothelial cell growth supplement was
obtained from Collaborative Biomedical (Bedford, MA), recombinant mouse
IFN- and recombinant mouse IL-6 were purchased from R&D Systems
(Minneapolis, MN), and dispase II neutral protease was obtained from
Roche (Indianapolis, IN). Recombinant human C5a, LPS (Escherichia
coli, serotype 0111:B4) and all other reagents were obtained from
Sigma (St. Louis, MO).
Anti-mouse C5aR Abs (anti-mC5aR)
A 37-aa peptide from the N terminus of the mouse C5aR (MDPIDNSSFEINYDHYGTMDPNIPADGIHLPKRQPGDC) was synthesized using an PE Applied Biosystem 430A peptide synthesizer (Foster City, CA) as previously described (39). The peptide was then coupled to keyhole limpet hemocyanin by the glutaraldehyde method and used for immunization of rabbits and production of immunoreactive antisera. The anti-peptide-specific Ab was purified by affinity chromatography using the synthetic peptide coupled to cyanogen bromide-activated Sepharose 4B (Pharmacia, Piscataway, NJ).
Characterization of anti-mC5aR by flow cytometric analysis
The expression of mC5aR was evaluated by direct
immunofluorescence staining of whole blood using an established
lysis/wash procedure (BD PharMingen, San Diego, CA). Flow cytometric
analysis was conducted immediately after blood collection. Anti-mC5aR
IgG (3 µg) in 100 µl staining buffer (PBS with 0.1% sodium azide
and 1% FBS) was incubated with 100 µl mouse whole blood in the
presence of different concentrations of the above-described 37-aa
peptide from the N terminus of C5aR or pepstatin for 1 h on ice.
After washing, resuspended cells were incubated with 1 µg of
FITC-labeled rat anti-rabbit Ab (BioSource, Camarillo, CA) in 100
µl staining buffer for 30 min on ice. Erythrocytes were lysed for 10
min by addition of 1x FACS lysing solution (BD PharMingen). After
washing, the leukocytes were resuspended in a fixation solution (0.5%
paraformaldehyde prepared in PBS with 0.1% sodium azide). The cells
were analyzed using a flow cytometer (Coulter, Miami, FL). Granulocytes
were gated by the typical forward and side light scatter profiles. We
identified the gated population as granulocytes by staining of whole
blood with an FITC-labeled rat granulocyte marker, HIS48 (BD
PharMingen), revealing that 
90% of gated cells were
granulocytes.
Preparation and characterization of rmC5a
E. coli BL21(DE3) pLysS-competent cells (Novagen, Madison, WI) transformed with the mouse C5a construct in pET 15b (Novagen) were grown to an OD of 0.6 (at a wavelength of 600 nm), then induced with IPTG for 3 h (optimal induction time) at 30°C and either stored at -80°C or processed immediately. The recombinant protein was purified over an Ni2+ column (Qiagen, Valencia, CA). The presence of rmC5a was confirmed by Western blotting. For subsequent experiments, endotoxin was removed from the rmC5a (endotoxin, <0.5 pg/ml) essentially as described by Aida and Pabst (40).
MDMEC culture
Mouse dermal microvascular endothelial cells were isolated from the ear dermis of 5- to 8-wk-old mice. Ears were removed, split, and incubated in dispase II (5 mg/ml in PBS) for 45 min, after which the epidermis was removed and discarded. Endothelial cells were expressed from the dermal sheets and placed into growth medium (RPMI medium supplemented with endothelial cell growth factor, 20% heat-inactivated FCS, L-glutamine, streptomycin-penicillin, and fungizone) in gelatin-coated tissue culture dishes by applying lateral pressure with the blunt end of a scalpel according to a previously described method (41). Cells grown to confluence in 4–6 days were trypsinized and used at 90–95% confluence for all experimental studies (passage 1). Cultured cells were characterized by a cobblestone appearance and specific staining for vWF as well as flow cytometric determination of uptake of 1,1'-dioctadexyl-3,3,3',3'-tetramethyl-indocrbocynanine perchlorate according to previously described methods (42, 43, 44). The results reported were obtained using several separate isolates of endothelial cells, as indicated for each experiment.
Binding of [125I]C5a to stimulated MDMEC
To assess C5a binding by C5aR, binding assays were performed on
MDMEC monolayers grown to 90–95% confluence in six-well plates
(Corning, Elmina, NY) at passage 1. Cells (1.4 x
106 cells/well) were left unstimulated or were
stimulated for various lengths of times at 37°C with different
concentrations of IL-6, IFN-, or LPS in RPMI 1640 medium
supplemented with 0.1% BSA. Recombinant mouse C5a was labeled with
125I using the chloramine T-based method as
previously described (45). This method involves gentle
oxidation and preserves the functional (chemotactic) activity of C5a
(45). Cells were placed on ice and incubated for 30 min in
2 ml/well of HBSS containing 0.5% BSA. Cell monolayers were then
washed with cold Dulbecco’s PBS, followed by incubation in assay
buffer (HBSS and 0.1% BSA) containing different concentrations of
[125I]C5a (sp. act., 28 µCi/µg) in a final
volume of 1.0 ml/well for 20 min on ice. After this incubation period
with the radiolabeled ligand, cell monolayers were washed three times
with cold Dulbecco’s PBS. Subsequently, cell monolayers were lysed
with 1% SDS and 0.1% Nonidet P-40, using 1.0 ml/well each. The
cell-bound [125I]rmC5a in the lysates was
determined in a gamma-counter (1261 Multi; Wallac, Gaithersburg,
MD). Data for the stimulation assays are presented as absolute values
(counts per minute). For all binding assays each condition was run in
triplicate and repeated at least three or more times.
C5aR expression in MDMEC by laser scanning confocal microscopy
Mouse dermal microvascular endothelial cells were grown at
passage 1 on four-well Lab-Tek chamber slides (Nalge Nunc, Naperville,
IL). Cells were stimulated with LPS, IL-6, or IFN- in the amounts
indicated for 6 h at 37°C. After fixation with 2%
paraformaldehyde and incubation for 10 min at 37°C, the cells were
washed with 50 mM glycine in PBS for 5 min at room temperature to
remove any remaining paraformaldehyde, followed by washing in binding
buffer (PBS with 0.5% human IgG; Calbiochem, San Diego, CA). The cells
were incubated with either affinity-purified rabbit anti-C5aR (5
µg/ml) or rabbit preimmune IgG (5 µg/ml; Jackson
ImmunoResearch, West Grove, PA), goat anti-mouse vWF (8
µg/ml; Enzyme Research, South Bend, IN), or goat IgG (8 µg/ml;
Calbiochem, San Diego, CA) for 1 h at 37°C. The cells were then
washed again and incubated simultaneously with FITC-labeled rat
anti-rabbit IgG (1/100; BioSource, Santa Clarita, CA) and ALEXA
Fluor 594-labeled donkey anti-goat IgG (H+L; 10 µg/ml; Molecular
Probes, Eugene, OR) for double labeling for 1 h in the dark. The
slides were then covered with Vector shield anti-fading mounting
medium (Vector, Burlingame, CA) and visualized using a confocal
fluorescence microscope (LSM 510; Zeiss, Jena, Germany). Both
projection view and optical sections were developed electronically and
processed digitally as previously reported (46).
Identification of C5aR mRNA in MDMEC
Total RNA was extracted from nonstimulated and stimulated cultures of confluent MDMEC monolayers (at passage 1) in 100-mm culture dishes using TRIzol reagent (Life Technologies) according to the manufacturer’s instructions. Before RT-PCR, RNA samples were treated with RQ1 RNase-free DNase (Promega, Madison, WI) to remove any traces of contaminating genomic DNA. Five micrograms of total RNA was used for reverse transcription using Superscript II RNase H- reverse transcriptase (Life Technologies, Grand Island, NY). PCR was performed with the following primers: 5' prime primer, 5'-TAT AGT CCT GCC CTC GCT CAT-3'; and 3' prime primer, 5'-TCA CCA CTT TGA GCG TCT TGG-3'. cDNA amplification was achieved using the following protocol: hot start for 5 min at 94°C, 40 cycles with melting temperature of 94°C, annealing temperature of 60°C, and extension temperature of 72°C, each for 1 min, followed by a final extension at 72°C for 8 min (Amp PCR system 9700; PerkinElmer, Norwalk, CT). The RT-PCR product was separated on a 1.2% agarose gel and visualized by ethidium bromide staining. The predicted size of the cDNA product designed from the middle region of the C5aR (nt 373–781) was 409 bp as previously described (23, 29). GAPDH housekeeping gene primers (5'prime primer, 5'-GCC TCG TCT CAT AGA CAA GAT G-3'; and 3'prime primer, 5'-CAG TAG ACT CCA CGA CAT AC-3') were also used in RT-PCR to confirm equal loading. Experiments were conducted in which total RNA from MDMEC was amplified with different cycle numbers to GAPDH and C5aR primers to assure that DNA bands after 40 cycles of amplification were detected within the linear part of the amplifying curves. To further rule out DNA contamination, we performed internal controls where all steps except the reverse transcriptase step were repeated. The results indicated no contamination of the samples with DNA (data not shown).
C5aR expression by immunostaining of mouse lung after i.v. endotoxin infusion
Mice (n = 3/group) were anesthetized by i.p. injection of a mixture of Ketaset (Fort Dodge Animal Health, Fort Dodge, IA) and Rompun (Bayer, Shawnee Mission, KS) at doses of 1.66 and 0.033 mg/g body weight, respectively. LPS dissolved in sterile Dulbecco’s PBS was injected i.v. into the dorsal penile vein at a dose of 10 mg/kg body weight and administered in a volume of 0.1 ml/10 g body weight. Control animals were injected with DPBS. Animals were sacrificed at 6 h by lethal injection of Ketaset, and after exsanguination the thoracic cavities were opened to isolate the lungs. Lungs from control and LPS-injected mice were then frozen in OCT Tissue-Tek compound (Miles, Elkhart, IN). Glass slides with tissue sections of 4–5 µm thickness were prepared and stored at -80°C. Samples were fixed in acetone at 4°C for 10 min and stained using EnVision, peroxidase, rabbit kit (DAKO, Carpinteria, CA) according to the manufacturer’s instructions. All incubations took place in a humid chamber at room temperature and were preceded and followed by three washes with TBST. After a 5-min blocking step with peroxidase blocking reagent (DAKO), the tissue samples were incubated for 30 min at room temperature with rabbit anti-C5aR Ab or rabbit IgG (Jackson ImmunoResearch) at a concentration of 1.6 µg/ml, followed by a 30-min incubation with the secondary peroxidase-conjugated anti-rabbit Ab according to the supplier’s instructions. The last incubation step was performed using the diaminobenzidene substrate chromogen system from DAKO. The reaction was stopped after exactly 5 min. The nuclei were counterstained with hematoxylin according to standard protocols.
Effects of C5a, IL-6, IFN-, and LPS on MIP-2 and MCP-1
production by MDMEC
Detection of MIP-2 and MCP-1 in supernatant fluids of
nonstimulated and stimulated MDMECs was performed using ELISA kits
(BioSource) according to the manufacturer’s instructions (detection
limit, 5–10 pg/ml). For all ELISA studies, cells were plated in
24-well plates (Corning, Elmina, NY) and used at passage 1 (90–95%
confluence). To investigate the synergistic effects of Il-6, IFN-,
or LPS with or without additional stimulation of C5a on MDMEC
monolayers, we designed the studies as follows. Cells were first
incubated for 3 h with various mediators (IL-6, LPS, IFN-, or
C5a alone). The cell monolayers were then washed with PBS and again
incubated with one of the above-mentioned agonists for 4 h. At the
end of the second incubation, cell supernatants were collected and
assayed by ELISA.
Statistical analyses
All values are expressed as the mean ± SEM. Datasets in groups with equal variances were analyzed using one-way ANOVA. Individual group means were then compared with the Student-Newman-Keuls multiple comparison test. In groups containing unequal variances, the Kruskal-Wallis ANOVA was performed, followed by Dunnett’s method for multiple comparisons.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine binding of [125I]rmC5a to
MDMEC (1.4 x 106 cells/well), competition
and saturation assays were performed. As shown in Fig. 1
A, binding with a constant
concentration of radioligand (200 pM
[125I]rmC5a) could be competed against by
progressively increasing concentrations of unlabeled rmC5a. Additional
binding experiments using 1.0 nM radiolabeled mC5a in the presence of
cold mC5a as well as other peptides, IL-6 and fMLP (each at a
concentration of 10-8 M)) showed that while C5a
reduced the binding of [125I]C5a by 31%, IL-6
reduced the binding by 4%, and fMLP did not reduce the binding,
suggesting specific binding of radiolabeled mC5a to MDMEC.
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B). The plateau of binding was reached between 7
and 9 nM [125I]rm C5a. The half-maximal
inhibition of [125I]rm C5a binding was
3.63 nM, defining the apparent Kd50
for binding C5a to MDMEC and predicting a number of 15,000–20,000
binding sites/cell. Up-regulation of [125I]rm C5a binding to activated MDMEC
For all subsequent stimulation binding assays,
[125I]rmC5a (4000–5000 cpm/well) was used
at a concentration of 3.6 nM according to the calculated
Kd50 (Fig. 1). The dose-response curves were
established by stimulating MDMEC monolayers for 6 h (37°C) with
different concentrations of IL-6, IFN-, or LPS, (Figs. 2A, 3A, and
4A). MDMEC exposure to any of these three mediators resulted
in significantly increased binding of
[125I]rmC5a to the cell monolayers as a
function of time of incubation and concentration of the mediator,
suggesting up-regulation of C5aR expression. Treatment of MDMEC with
IL-6 (Fig. 2A) demonstrated the highest increase (>4-fold)
in [125I]rmC5a binding at an IL-6 concentration
of 0.5 ng/ml, while IFN- induced maximal effects on enhanced binding
at a concentration of 25 IU/ml (Fig. 3A). C5a binding to MDMEC
stimulated with LPS peaked at 1 µg/ml (Fig. 4A). Following exposure at
higher concentrations of any of these three mediators,
[125I]rmC5a binding actually declined (Figs. 2A, 3A, and 4A). In contrast to the
effects of LPS, IL-6, and IFN-, cells treated with IL-4 and IL-10 at
concentrations of 0.5 and 50 ng/ml failed to show up-regulation of
binding of [125I]mC5a to MEC (data not
shown).
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, or LPS. All three mediators were
used at their optimal concentrations (based on the data in Figs. 2A, 3A, and4A). Binding was
measured at various time points (Figs. 2B, 3A,
and 4B). Exposure of cells to IL-6 significantly increased
[125I]rmC5a binding as early as 2 h, with
maximal binding (>3-fold) after 6-h exposure of cells to IL-6 (Fig. 2B). Similarly, MDMEC exposure to IFN- appeared to reach
a plateau phase for maximal increase in binding by 4–24 h (Fig. 3B). Exposure of MDMEC to LPS led to a rapid and sustained
increase in [125I]rmC5a binding, with a plateau
phase reached between 2–18 h of stimulation, declining thereafter
(Fig. 4B). These data indicate that increased binding of C5a
to MDMEC treated with IL-6, LPS. and IFN- occurs in a dose- and
time-dependent manner. Enhanced mRNA for C5aR in stimulated MDMEC
To assess C5aR mRNA expression, primers for mC5aR were designed
(as described above), and RT-PCR was performed using total RNA isolated
from unstimulated and stimulated MDMEC after exposure of cells to
agonists for various lengths of times, as indicated (Fig. 5). For all conditions,
equivalent loading for the different templates was verified
using primers to GAPDH (Fig. 5, lower bands). All panels in
Fig. 5 indicate that nonstimulated MDMEC express no detectable mRNA
for mouse C5aR (control). Cell monolayers were stimulated with the
optimal concentrations of IL-6, IFN-, and LPS, as determined in
Figs. 2–4. Exposure of MDMEC to IL-6 (0.5 ng/ml) alone caused
increased mRNA levels for C5aR at 3, 6, and 14 h (Fig. 5), while
exposure to LPS (1 µg/ml) also caused enhanced mRNA expression at all
three time points (Fig. 5), especially at 3 and 14 h (Fig. 5, A and B). Exposure to IFN- (25 IU/ml) caused
increased mRNA levels for C5aR at 3 and 6 h, while mRNA declined
by 14 h. Stimulation of MDMEC with LPS together with either
IFN- or IL-6 caused decreased mRNA expression for C5aR at 14 h
compared with LPS treatment alone. We also investigated the effects of
C5a on up-regulation of C5a mRNA expression in MDMEC. In a dose range
from 0.5 ng to 10 µg/ml, C5a did not up-regulate C5aR mRNA in MDMEC
monolayers after incubation for 6 h at 37°C (data not
shown).
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Mouse C5aR expression was evaluated by direct immunofluorescence
staining of whole blood (Fig. 6). The
anti-C5aR IgG (3 µg) was incubated with increasing concentrations
of mC5aR peptide or pepstatin ranging from 0.2–2 µM. Under these
conditions mC5aR peptide dramatically decreased the ability of
anti-C5aR to detect C5aR on neutrophils; the control value of
18.5 ± 1.3 mean fluorescence intensity (MFI) fell by 93% at a
dose of 0.2 µM C5aR peptide (to 1.82 ± 0.2 MFI), by 97% at a
dose of 1 µM (to 1.22 ± 0.5 MFI), and by 99% at a dose of 2
µM (to 0.82 ± 0.09 MFI). In contrast, 0.2–2 µM pepstatin did
not significantly reduce the ability to detect C5aR (<5%), suggesting
specific reactivity of anti-mC5aR IgG to the N terminus of
mC5aR.
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Using immunostaining, studies were performed to determine C5aR
protein expression on the surfaces of unstimulated and stimulated MDMEC
monolayers (Fig. 7). Incubating
unstimulated and stimulated cells with preimmune rabbit IgG or goat IgG
alone or in combination caused little or no immunostaining (Fig. 7, A and C). Using Ab to C5aR with unstimulated
MDEMEC, C5aR was detectable, but variable, over surfaces of cells (Fig. 7B, top). Similarly, Ab to vWF demonstrated cell
surface distribution on unstimulated MDMEC as detected by the ALEXA
Fluor 594 Red label (Fig. 7B, middle). When the
dual use of both anti-C5aR and anti-vWF Abs featured
simultaneous examination on unstimulated MDMEC, there was a blending of
the fluorescent labels, resulting in a yellow color (Fig. 7B, bottom).
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, LPS, or IL-6
for 6 h at 37°C, increased expression of C5aR was shown by
enhanced FITC staining (Fig. 7, D–F, top) as
also in the case of vWF staining (Fig. 7, D–F,
middle). Due to the more intense FITC staining, more green
than yellow color was found (Fig. 7, D–F,
bottom) compared with the yellow color in unstimulated cells
when Abs (to C5aR and vWF) were simultaneously used, indicating
up-regulation of C5aR surface expression. C5aR up-regulation in mouse lung after i.v. infusion of LPS
To examine in vivo expression of C5aR protein in normal mice and
in mice receiving LPS i.v., frozen lung sections were obtained from the
two groups and analyzed by immunostaining. Lungs were removed at 6
h, sectioned, and stained. Consistent with a previous report
(47), weak staining for C5aR was found in bronchial and
alveolar epithelial cells as well as on the endothelium of larger
pulmonary blood vessels in tissues from control (normal mice; data not
shown). The use of normal rabbit IgG failed to result in immunostaining
of LPS-injured as well as normal lung (Fig. 8A; magnification, x20;
isotype IgG control). No positive staining for C5aR was found on small
vessels and capillaries in lung sections from normal mice (Fig. 8B; magnification, x100). However, in lungs from
LPS-infused mice, staining for C5aR protein was present in lung
capillary endothelium (Fig. 8, C (magnification, x20) and
D (magnification, x100)). Similar to the previously
reported finding that bronchial epithelial cells from lungs injected
intratracheally with LPS showed increased staining for C5aR protein
(47), we observed increased C5aR presence in alveolar
epithelial cells (Fig. 8D) compared with normal lung (Fig. 8B).
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, or IL-6
It has been shown that MCP-1 and MIP-2 production can be induced
in microvascular endothelial cells by various proinflammatory mediators
(48, 49, 50, 51). To determine whether exposure of MDMEC to C5a
affects chemokine production, MCP-1 and MIP-2 levels were evaluated in
supernatant fluids from MDMEC that were first exposed for 3 h at
37°C to either buffered salt solution or to C5a, IL-6, IFN-, or
LPS alone. The monolayers were then washed and subsequently exposed to
one of the above-mentioned agonists, as indicated, for 4 h at
37°C. Table I shows MIP-2 and MCP-1
production by MDMEC stimulated with an agonist alone or in combination.
Optimal doses of C5a, IL-6, IFN-, or LPS were selected according to
the data in
Figs. 2–4. Unstimulated MDMEC produced low levels of MIP-2
(130 pg/ml; Table I) and MCP-1 (200 pg/ml; Table I). Exposure to
C5a alone (100 ng/ml) did not increase the low levels of MIP-2 or
MCP-1. Similarly, incubating MDMEC with IL-6 alone (1 ng/ml) had no
effect on chemokine levels (Table I). However, prestimulation with C5a,
followed by addition of IL-6, failed to enhance MIP-2 or MCP-1
production. In striking contrast, when MDMEC were first incubated with
IL-6, followed by stimulation with C5a, the chemokine response
increased 2- to 6-fold (Table I, Expt. A).
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, IL-1, LPS, or IFN- alone could
induce MCP-1 in HLMEC as well as HUVEC, although HLMEC were more
sensitive to IL-1 and LPS, producing significantly more MCP-1. To
evaluate the synergistic effects of LPS treatment combined with
C5a stimulation, MDMEC monolayers were incubated with LPS (10
ng/ml) with or without subsequent exposure to C5a (Table I). Although
stimulation of MDMEC with LPS alone enhanced MCP-1 and MIP-2 production
(3- to 10-fold), an additional increment was observed when LPS exposure
was followed by addition of C5a (Table I, Expt. B). In this case,
chemokine levels rose 6- to 17-fold. Similar to the LPS effects,
IFN- alone (at a concentration of 25 IU/ml) increased MCP-1 and
MIP-2 levels in MDMEC 2- to 3-fold (Table I, Expt. C). Pre-exposure of
the endothelial monolayers with IFN-, followed by addition of C5a,
resulted in 4- to 5-fold increases in chemokine release. We also
studied TNF- production and MIP-1 production in MDMEC under
similar conditions, but found no increase in levels of TNF- or
MIP-1 (data not shown). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, which is probably related to up-regulation of C5aR. We
calculated a Kd50 of 3.6 nM and
15,000–20,000 binding sites/resting cell. The C5aR on MEC binds C5a
with an affinity similar to that of the C5aR expressed on human
neutrophils (Kd50 = 2 nM)
(17). Fewer C5aR appear to be expressed on MEC than on
granulocytes (100,000–200,000 receptors/cell) (17). C5aR
expression on MEC could be up-regulated 3- to 4-fold by IL-6,
IFN-, or LPS, resulting in an increase in binding sites to
60,000–80,000/stimulated cell. The increased binding of C5a correlates
with increased levels of mRNA for C5aR in MDMEC stimulated with the
above-mentioned agonists. At the protein level, increased C5aR
expression could be demonstrated by in vitro immunostaining of treated
MDMEC as well as the capillary network in lung tissue sections from
mice infused with LPS. It has been reported that IFN- induces C5aR
expression in the monocytic U937 cell line and in related myeloblastic
cell lines (52). In vivo treatment of rats and mice with
IL-6 has led to increased C5aR mRNA expression in lung and hepatocytes
(53, 54).
In a mouse model of i.p. injection of LPS, up-regulation of mRNA for
C5aR occurred predominantly in bronchial epithelial cells
(20). Staining for C5aR protein was also found in alveolar
epithelial cells and in lung vascular walls (47). Similar
C5aR expression has been described in human lung from patients with
cystic fibrosis (20). Our data reinforce the recent
findings that C5aR expression can be detected in non-myeloid cells.
Besides evidence that larger vessels express C5aR, we now show that the
microvascular endothelium in the mouse lung stains positively for C5aR
after infusion of LPS. We have also demonstrated C5aR expression after
stimulation of isolated primary cultures of MDMEC with proinflammatory
mediators, such as IL-6, IFN-, or LPS. Activation of vascular
endothelial cells is an early critical event in the development of an
inflammatory response. Endothelial cell activation results in enhanced
expression of surface molecules required for adhesion of circulating
inflammatory cells and is thought to result in the release of
pro-inflammatory mediators from both endothelial cells as well as
adherent leukocytes (55). In studies with HUVEC,
complement-derived membrane attack complex was found to have the
capability to up-regulate adhesion molecules (ICAM-1, E-selectin)
(56). Additionally, we have recently demonstrated in an in
vivo study that blockade of C5a ameliorates coagulation/fibrinolytic
protein changes in a rat model of sepsis (57), leading to
improved survival and suggesting that complement may play an important
role in activation of endothelium during inflammation.
Assuming that C5aR up-regulation allows C5a directly to trigger
proinflammatory events on the microvascular endothelium, we
investigated the effects of C5a alone or in combination with IL-6, LPS,
or IFN- on chemokine release from MDMEC. It has been shown that
various proinflammatory mediators can up-regulate endothelial chemokine
products. TNF- induces MIP-2 production in brain microvascular
endothelial cells (48). Responses of brain, lung, or
dermal microvascular endothelial cells as well as HUVEC with MCP-1
production after stimulation with IL-1
, TNF-, IFN-, or
endothelial growth factor have also been reported
(49, 50, 51). While an earlier study demonstrated
down-regulation of IL-8 production from HUVEC after stimulation with
C5a for 48 h (58), we found a synergistic effect of
C5a on MDEMC pre-exposed to LPS, IFN-, or, especially, IL-6, with
resultant enhanced induction of chemokines. Our data suggest that even
though LPS or IFN- alone has the ability to induce production of
MIP-2 and MCP-1 by MDMEC, the subsequent exposure to C5a evokes
additional mediator production. Interestingly, and similar to C5a, IL-6
by itself did not alter chemokine production during stimulation, but
the sequential addition of IL-6 followed by C5a revealed enhanced
release of MCP-1 and MIP-2 (but not MIP-1 or TNF-). These
findings support the hypothesis that IL-6, which is released very early
into the plasma during an acute inflammation (59, 60),
induces changes that cause these cells to become hyper-responsive to
C5a. Our studies suggest a close relationship between C5a binding and
cytokine release, namely MIP-2 and MCP-1, from MDMEC, resulting in
recruitment of cells to the inflamed endothelium. These findings are
similar to previous reports suggesting that the membrane attack
complex, C5b-9, has the ability to enhance the production of IL-8 and
MCP-1 by HUVEC (61). MIP-2 is a member of the CXC
subfamily of chemokines and a powerful neutrophil chemotactic factor,
whereas MCP-1, a member of the CC chemokine subfamily, appears to be
mainly involved in the recruitment of lymphocytes and monocytes. While
C5a alone could not directly induce MIP-2 and MCP-1 from MDMEC, C5a
synergistically enhanced chemokine release from MDMEC following
exposure of endothelial cells to IL-6, IFN-, or LPS, resulting in
enhanced secretion of MIP-2 as well as MCP-1. Synergistic effects of
LPS and C5a were also recently described for rat liver macrophages
(Kupffer cells) (62). Just as we found that C5a alone
failed to increase chemokine production, Schieferdecker et al.
(62) found that C5a (in contrast to LPS) could not induce
IL-6 synthesis from Kupffer cells, but synergistically enhanced
LPS-dependent IL-6 production.
In conclusion, our studies provide evidence that C5aR can be induced in
microvascular endothelial cells, rendering these cells responsive to
C5a (Fig. 9). C5aR expression on MDMEC
can be up-regulated by LPS, IL-6, or IFN-. This results in the
ability of MDMEC to produce chemokines and increased C5aR mRNA levels.
Under such conditions, these cells become more sensitive to the agonist
effects of C5a, resulting in an increased production of chemokines such
as MIP-2 and MCP-1. The release of these chemokines in the presence of
C5a leads to enhanced transmigration of neutrophils through the
endothelial barrier. The specific effects of C5a and, especially, the
inducibility of C5aR on the capillary endothelium, support the idea
that anaphylatoxins play an important role in the activation of the
microvascular endothelium and the process of transmigration. Complement
or its activation products may therefore become a primary target for
anti-inflammatory drugs that block C5aR function.
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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, 1301 Catherine Road, 5240 Medical Science I Building, Ann Arbor, MI 48109-0602. E-mail address: pward{at}umich.edu
3 Abbreviations used in this paper: vWF, von Willebrand factor; C5aR, C5a receptor; HLMEC, human lung microvascular endothelial cells; mC5aR, mouse C5aR; MCP-1, monocyte chemoattractant protein-1; MDMEC, mouse dermal microvascular endothelial cells; MFI, mean fluorescence intensity; MIP-2, macrophage inflammatory protein-2; rmC5aR, recombinant mouse C5aR; [125I]rmC5a, 125I-labeled rmC5a; [125I]C5a, 125I-labeled C5a.
Received for publication June 6, 2002. Accepted for publication September 18, 2002.
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