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Department of Surgery, Duke University, Durham, NC 27710
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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as an intermediate
step. The results indicate that the expression of chemokine genes in
endothelial cells occurs as a function of differential responses to
complement and may in part be conditioned by the availability of
IL-1. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Endothelial cells produce chemokines in response to a variety of
endogenous and exogenous stimuli, including IL-1 (7, 8, 9, 10), TNF- (7, 9, 11), low density lipoprotein (12), endotoxin (7), and complement
(13). The expression of chemokines by endothelial cells differs
depending on the stimulators applied, and this differential expression
may contribute to the selectivity of cell recruitment in various types
of inflammation, since only a limited set of immune cells is sensitive
to each chemokine (14).
The present study examined the mechanisms and kinetics by which
complement might induce the expression of chemokine genes in
endothelial cells, thus influencing inflammatory reactions. To this
end, we used cultured porcine aortic endothelial cells as a model for
the donor, and human serum as a source of anti-endothelial cell Abs
and complement that are known to be involved in xenograft rejection
(15). Using such a model, it was earlier observed that complement
stimulates endothelial cells with loss of barrier and anticoagulant
functions (16, 17, 18). We studied the complement-mediated regulation of
expression of chemokines
MCP-1,4 IL-8, and RANTES in
cultured endothelial cells because of discrete as well as overlapping
functional characteristics of these molecules. For example, IL-8
attracts and activates neutrophils (19, 20), while MCP-1 and RANTES
attract and activate monocytes and subsets of T cells (21, 22, 23, 24, 25). We show
here that complement differentially induces the expression of the
MCP-1, IL-8, and RANTES genes as a direct consequence of its action on
endothelial cells and through an intermediate step, the synthesis and
elaboration of IL-1.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Samples of human blood from healthy individuals were used as a
source of anti-endothelial cell Abs and complement (26). Blood
collected in sterile, pyrogen-free containers was allowed to clot at
4°C, and the serum fraction was separated and stored as aliquots at
-80°C until used. The titer of Abs in serum was determined by ELISA,
as described previously using cultured porcine endothelial cells as
target (27). Human serum immunodepleted of C8 and purified human C8
were purchased from Quidel (San Diego, CA). Recombinant human IL-1
was obtained from Genzyme (Boston, MA). IL-1RA and anti-IL-1
neutralizing Abs were obtained from R & D Systems (Rockford, IL).
Neutralizing anti-human IL-8 Ab was a gift from Dr. Nikolai N.
Voitenok (Foundation for Fundamental Research, Minsk, Republic of
Belarus). Anti-human MCP-1 and anti-human RANTES Abs
(R & D Systems, Minneapolis, MN) were gifts from Dr. Terry O.
Harville, Duke University (Durham, NC). Soluble complement receptor-1
(sCR1) was obtained from T-Cell Sciences (Cambridge, MA). Actinomycin D
was purchased from Boehringer Mannheim (Indianapolis, IN).
Cycloheximide and LPS (LPS, Esherichia coli serotype
0111:B4) were obtained from Sigma (St. Louis, MO). Porcine IL-8,
IL-1, IL-1ß, and TNF- primers were provided by Dr. Robin S.
Monroe (Duke University). Porcine IL-1 cDNA was obtained from Dr.
Soheyla Saadi and Robert A. Holzknecht (Duke University). Human IL-8
cDNA was obtained from Drs. Joost J. Oppenheim and Kouji Matsushima
(National Cancer Institute, National Institutes of Health, Frederick,
MD). Human MCP-1 cDNA was obtained from Dr. Barrett Rollins
(Dana-Farber Cancer Institute, Boston, MA). Human RANTES cDNA was
obtained from Dr. Thomas J. Schall (DNAX Research Institute, Palo Alto,
CA).
Endothelial cells
Endothelial cells were explanted from porcine aortae and
cultured in DMEM supplemented with 10% heat-inactivated FBS, 4 mM
L-glutamine, 50 U/ml penicillin, and 50 µg/ml
streptomycin (Life Technologies, Grand Island, NY) as previously
described (28, 29). The endothelial cells were characterized by their
morphology, including a cobblestone appearance when confluent, and by
the ability to take up acetylated low density lipoprotein (28). During
our initial screening of a number of primary endothelial cell lines for
their capacity to induce various cytokine genes in response to human
serum, we identified an endothelial cell line that failed to express
IL-1, IL-1ß, and TNF- in response to complement. These cells
had normal morphologic characteristics and sensitivity to complement as
measured by deposition of normal amounts of iC3b after incubation with
human serum and by a normal level of complement-mediated cytotoxicity
(data not shown) (30). Experiments were performed with cells at
passages 4 to 10 that were grown to confluence in 60-mm tissue culture
dishes (Corning, Corning, NY).
Stimulation of endothelial cells
Confluent monolayers of endothelial cells were washed twice with
DMEM and then incubated with human serum diluted in DMEM. Human serum
that had been heated to 56°C for 30 min to inactivate complement was
used as a control. Endothelial cells were also stimulated with LPS,
IL-1, or TNF- in DMEM containing heat-inactivated FBS, as
discussed in the text.
Northern blot analysis
Total RNA was obtained by the single step method, using acidic
guanidinium thiocyanate (31). Samples of total RNA (10 µg) were
electrophoresed, blotted by capillary transfer onto nylon membranes
(Amersham, Arlington Heights, IL), and cross-linked to membranes by UV
irradiation (CL 1000 Ultraviolet Crosslinker, UVP, San Gabriel, CA) as
described previously (32). Blots were prehybridized at 68°C in
QuikHyb (Stratagene, CA) containing 200 µg of herring sperm DNA and
were hybridized for 2 h with 32P-labeled probes at
68°C in the same buffer. Blots were washed in 1x SSC/0.5% SDS at
23°C followed by high stringency washes in 0.1% SSC/0.1% SDS at
50°C and were exposed to Kodak BIOMAX film (Eastman Kodak, Rochester,
NY). Blots were stripped by exposure to boiling 0.1% SDS before
reprobing. The probes used were the RT-PCR fragment of porcine IL-8
(GAA TTC CTT CCA AAC TGG CTG TTG CC and AAG CTT CTG TAC AAC CTT CTG CAC
CC) (33), actin (ATG TTT GAG ACC TTC AAC AC and CAC GTC ACA CTT CAT GAT
GGA) (34), the PCR fragment of porcine IL-1 (GAA TTC CAA CAT ACA GCT
TCC AGA GC and AAG CTT TGG GTG TCT CAG GCA GCT CC) (35), the PCR
fragment of porcine IL-1ß (GAA TTC CCA TAG TAC CTG AAC CCG CC and AAG
CTT TCA TCG GCT TCT CCA CTG CC) (36), and the PCR fragment of TNF-
(GAA TTC CGT TGT AGC CAA TGT CAA AGC C and AAG CTT CCA GGT AGA TGG GTT
CGT ACC) (37). RNA loading was assessed by staining the blots for 5 min
with 0.01% methylene blue in 0.3 M sodium acetate. Quantitative
analysis of blots was accomplished using a Betascope (Betagen, Waltham,
MA) or an Ultroscan XL laser densitometer (LKB, Gaithersburg, MD).
Metabolic labeling, immunoprecipitation, and ELISA
Endothelial cells were labeled with [35S]methionine and [35S]cysteine (DuPont-New England Nuclear, Boston, MA) as described previously (32). Aliquots of labeled culture supernatants were immunoprecipitated using anti-human MCP-1, anti-human IL-8, and anti-human RANTES Abs. Samples containing immunoprecipitated proteins were electrophoresed in 15% discontinuous SDS-polyacrylamide gels according to the method of Laemmli (38). Proteins were visualized by fluorography (39) using Kodak BIOMAX film. The levels of MCP-1, IL-8, and RANTES proteins in the culture supernatant of stimulated endothelial cells were quantitated in duplicate using human chemokine ELISA kits obtained from Endogen (Woburn, MA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cultured porcine aortic endothelial cells expressed low levels of
MCP-1, IL-8, and RANTES mRNAs under resting conditions (Fig. 1
). To begin to determine how activation
of complement might induce the expression of various chemokine genes in
endothelial cells, the pattern of expression of MCP-1, IL-8, and RANTES
genes in response to human serum containing xenoreactive natural Ab and
complement was investigated. As shown in Figure 1 and Table I (columns
on left), exposure of endothelial cells to human serum induced a
notable increase in the levels of MCP-1, IL-8, and RANTES mRNAs.
However, the rate of increase in IL-8 and MCP-1 mRNAs was different
from the rate of induction of the RANTES gene in response to
complement. Thus, IL-8 and MCP-1 mRNAs were increased as early as
6 h, while RANTES mRNA was not expressed until 12 h had
elapsed. The rate of induction of MCP-1, IL-8, and RANTES mRNAs was a
function of the amount of complement applied to the endothelial cells,
as MCP-1 and IL-8 mRNAs were not up-regulated until 12 h when
cells were exposed to a 10% concentration of human serum, and lower
concentrations of human serum induced no up-regulation of these
transcripts.
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We examined whether human serum-induced expression of MCP-1, IL-8,
and RANTES genes was due to complement activation. Endothelial cells
treated with varying concentrations of heat-inactivated human serum did
not express increased levels of MCP-1, IL-8, and RANTES mRNAs (Fig. 1
, A and B). To test whether the heat-labile
component was complement, endothelial cells were treated with human
serum in the presence or the absence of sCR1, which inhibits C3
convertase. As shown in Figure 2A, sCR1 abolished human
serum-induced up-regulation of MCP-1, IL-8, and RANTES genes. It was
previously demonstrated that complement activation and assembly of
membrane attack complex were required for complement-induced
up-regulation of the IL-1 gene (17). To investigate whether the
assembly of membrane attack complex (MAC) induces the initial
expression of MCP-1 and IL-8 mRNAs, the induction of these transcripts
was examined in endothelial cells stimulated with a high concentration
(20%) of C8-depleted human serum. As shown in Figure 2, B
and C, human serum depleted of C8 did not induce MCP-1 and
IL-8 mRNAs. However, the C8-depleted serum reconstituted with purified
C8 induced IL-8 and MCP-1 mRNAs. Taken together, these results (Figs. 1 and 2) suggest that complement activation leading to assembly of MAC on
endothelial cells causes expression of MCP-1, IL-8, and RANTES mRNAs.
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We next examined whether porcine endothelial cells that expressed
high levels of mRNA for MCP-1, IL-8, and RANTES genes also secreted the
corresponding proteins. Endothelial cells stimulated with human serum
were labeled with [35S]methionine and
[35S]cysteine for 2 h, and the chemokines secreted
from the cells were immunoprecipitated. SDS-PAGE analysis of the
immunoprecipitates revealed high levels of IL-8, MCP-1, and RANTES in
the supernatants of the stimulated cells but very low levels in
controls (Fig. 3). Consistent with the
later induction of RANTES mRNA, culture medium was found to contain
RANTES protein at 16 h, but not at 3 h, following stimulation
(Fig. 3C). The sizes of these porcine chemokines, estimated
by migration in polyacrylamide gels, were similar to those of human
chemokines (40). The levels of MCP-1, IL-8, and RANTES proteins were
quantitated by ELISA in the culture supernatant of human
serum-stimulatedendothelial cells. Table II shows one
experiment that is representative of four conducted. As was seen with
immunoprecipitation analysis, high levels of IL-8, MCP-1, and RANTES
were detected in the culture supernatants of the stimulated endothelial
cells, while low levels of MCP-1 and undetectable levels of IL-8 and
RANTES were seen in controls. A lower concentration (10%) of human
serum induced MCP-1, IL-8, and RANTES at 16 h following
stimulation; higher concentrations (20%) of human serum induced an
early increase in MCP-1 and IL-8 but not RANTES. Although
immunoprecipitation and ELISA have different levels of sensitivity, the
results by ELISA were similar to those obtained with
immunoprecipitation.
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The early induction of MCP-1 and IL-8 mRNAs by an increased level
of human serum may be a direct consequence of the action of complement.
Conversely, the apparent delay in induction (>12 h) of MCP-1, IL-8,
and RANTES mRNAs by human serum under some conditions suggested that
these genes might be regulated by intermediary factors produced by the
endothelial cells in response to primary stimuli. Previous work
indicated that the physiology of endothelial cells was potentially
regulated by endogenous IL-1 (41). Specifically, it was shown that
the induction of cyclo-oxygenase in endothelial cells was mediated by
synthesis of IL-1 and its action on endothelial cells. In addition,
previous work from our laboratory indicated that following stimulation
of porcine endothelial cells with complement, the induction of tissue
factor was mediated by IL-1 produced by the endothelial cells in
response to MAC (17). Our analysis using PCR and Northern blots
revealed that endothelial cells express very little IL-1ß mRNA and
undetectable levels of TNF- mRNA in response to human serum (data
not shown). Thus, we considered the possibility that IL-1 might be a
potential factor regulating the late phase induction of chemokine
genes. To directly test whether the expression of MCP-1, IL-8, and
RANTES mRNAs is determined in part by expression of IL-1, porcine
aortic endothelial cells were stimulated with human serum in the
absence or the presence of neutralizing Ab against human IL-1 or in
the presence of IL-1RA. As Figure 4
shows, the delayed induction of MCP-1 and IL-8 mRNAs by 10 and 20%
human serum was strongly inhibited by anti-IL-1 Ab (91 and 80%
inhibition, respectively). On the other hand, the early (at 3 h)
up-regulation of MCP-1 and IL-8 mRNAs by 20% human serum was minimally
inhibited by the anti-IL-1 Ab (25% inhibition). Similar levels
of inhibitory response were observed with the inclusion of IL-1RA (Fig. 4). These results suggest that MCP-1 and IL-8 genes are induced by two
mechanisms, one involving an initial phase that is IL-1 independent
and another late phase that is dependent upon synthesis of IL-1 and
its action on endothelial cells. In contrast, up-regulation of RANTES
mRNA was always inhibited by anti-IL-1 Ab or IL-1RA, suggesting
that RANTES gene induction by complement requires the synthesis and
availability of IL-1.
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, IL-1ß, or TNF- (Fig. 5A and data not shown) in
response to complement. During routine culture, these cells
constitutively expressed low levels of MCP-1 and IL-8 mRNAs but not
IL-1 or RANTES mRNA (Fig. 5A). Stimulation of these cells
with a high concentration (20%) of human serum induced heightened
levels of MCP-1 and IL-8 mRNAs but negligible levels of IL-1 and
RANTES mRNAs. This is in contrast to the expression of all three
chemokine mRNAs in normal IL-1-expressing cells (compare Fig. 5, A with B; Table I).
Following exposure to lower concentrations (10%) of human serum,
IL-1-nonexpressing cells did not express MCP-1, IL-8, or RANTES mRNA
(data not shown), while IL-1-expressing cells did so (Fig. 4 and Table I). Furthermore, the anti-IL-1 Ab that significantly inhibited
late induction of MCP-1, IL-8, and RANTES mRNAs in IL-1-expressing
cells (Fig. 5B) did not alter the levels of MCP-1 and IL-8
mRNAs in IL-1-nonexpressing cells (Fig. 5A).
IL-1-nonexpressing cells were able to respond normally to other types
of stimulation, as treatment of the cells with rIL-1 (Fig. 6), LPS (Fig. 6), or rTNF- (data not
shown) induced RANTES as well as MCP-1 and IL-8 mRNAs. Taken together,
these results demonstrate that RANTES gene induction by complement for
the most part depends on the production of endogenous IL-1, while
MCP-1 and IL-8 genes are induced directly by complement.
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We investigated whether initial induction of MCP-1 and IL-8 mRNAs
in endothelial cells by complement was the result of transcriptional or
post-transcriptional mechanisms. Endothelial cells that did not express
IL-1 in response to complement were stimulated with 15% human serum
for 2 h and were further cultured for additional time with or
without actinomycin D (Fig. 7). MCP-1 and
IL-8 mRNAs decayed similarly in actinomycin D-treated and untreated
cells. These experiments suggest that the human serum-induced increase
in MCP-1 and IL-8 mRNA levels resulted from induction of MCP-1 and IL-8
gene transcription. We then investigated whether the initial expression
of MCP-1 and IL-8 genes required de novo protein synthesis, since these
genes are induced as a direct consequence of the action of complement.
As shown in Figure 8, incubation with
cycloheximide alone for 3 h led to an increased basal level of
expression of MCP-1 and IL-8 mRNAs. Incubation with cycloheximide
followed by stimulation with 15% human serum resulted in a further
increase in the levels of MCP-1 and IL-8 mRNAs compared with those in
endothelial cells treated with either cycloheximide or 15% human serum
alone. These results demonstrate that de novo protein synthesis is not
required for the initial induction of MCP-1 and IL-8 gene transcription
in endothelial cells stimulated with a high concentration of human
serum. Further, we extended our studies to check the effects of
cycloheximide to ascertain the role of protein synthesis in the late
phase (beyond 12 h) expression of MCP-1, IL-8, and RANTES mRNAs in
10% human serum-stimulated endothelial cells. Cycloheximide alone
induced the expression of MCP-1, IL-8, and RANTES mRNAs similar to the
levels up-regulated by human serum in the presence or the absence of
cycloheximide (data not shown). These results suggest that protein
synthesis, presumably the synthesis of endonucleases, is an important
factor in degrading the mRNA synthesized during stimulation and
determining the kinetics of these responses.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(17, 45). Due to alterations of the physiologic status induced
by complement, the endothelial cells loose barrier function and thereby
may amplify inflammatory responses in part by directing the trafficking
of inflammatory cells. The association of cellular infiltration with
humoral immune responses involving activation of complement may well
depend upon the production of a cascade of chemokines by endothelial
cells. Thus, the pattern of expression of chemokine genes that occurs
in response to complement or other inflammatory mediators may
contribute to the kinetics and selectivity of cell recruitment, since a
limited set of immune cells is sensitive to each chemokine (5, 14).
Although MAC is thought to induce MCP-1 and IL-8 genes, the mechanisms
leading to the induction of these genes and the pattern of chemokine
gene expression that ensues are uncertain (13). The present report
addresses the relationship between activation of complement and
differential expression of chemokines MCP-1, IL-8, and RANTES in
endothelial cells. Both MCP-1 and IL-8 responded early with a prolonged
induction as a result of high level complement activation. RANTES
expression, on the other hand, occurred late regardless of the quantity
of complement components generated. The late phase induction of the
MCP-1, IL-8, and RANTES genes indicates that the level of induction of
intermediary factors by complement could regulate the expression of
these chemokine genes. Two phases of chemokine gene induction are
clearly separated by inhibition of IL-1. In the absence of any role
for IL-1ß or TNF-, inclusion of anti-IL-1 Ab or IL-1RA only
minimally inhibited the expression of MCP-1 and IL-8 genes during the
early phase, but prevented later expression of MCP-1, IL-8, and RANTES
genes. Based on this finding, we reasoned that the incomplete
inhibition of expression of chemokines genes by anti-IL-1 Ab or
IL-1RA during the early phase of stimulation reflects a primary
response to complement. In other words, both complement and IL-1
rapidly synthesized by complement might have simultaneously influenced
the expression of IL-8 and MCP-1 genes during the early phase in cells
exposed to higher concentrations of human serum. IL-1-dependent late
phase induction of these chemokine genes suggests that the IL-1 gene
is more sensitive to complement-induced signaling pathways.
Additionally, it may be due to IL-1 inducing its own biosynthesis (46).
The dependence of MCP-1, IL-8, and RANTES expression on endogenous
IL-1 activity suggests that factors governing local availability of
IL-1, such as regional blood flow, could play an important role in
inflammation. Consequently, the present study indicates that the
expression of chemokines in endothelial cells occurs as a function of
differential responses to complement. Consistent with these
observations, it has been demonstrated that early induction of the IL-8
and MCP-1 genes and delayed induction of RANTES occur in a number of
cell types, including endothelial cells, in response to stimulation by
a variety of other agents (7, 8, 9, 10, 47, 48). The pattern of induction of chemokine genes would appear to correlate with in vivo cellular infiltration associated with acute vascular and cellular rejection. It has been well documented that IL-8 predominantly attracts and activates neutrophils, whereas MCP-1 attracts and activates monocytes and T cells (19, 20, 21, 22, 25). Although RANTES is known to attract and activate monocytes, a number of studies indicate that RANTES is a potent attractant and activator of T cells (23, 24). Infiltration of neutrophils and macrophages into xenografts has been observed at early time points, consistent with potential involvement of IL-8 and MCP-1 (49). The notable accumulation of T cells during the 2 to 3 days following transplantation of xenogeneic organs may reflect the expression of RANTES brought about by complement activation (50). A slower pattern of expression of these genes and cellular infiltration was recently observed in vivo during skin allograft rejection (51), perhaps reflecting the relative absence of active complement products.
Using IL-1-nonexpressing cells, the present study allowed a separation of primary and secondary mechanisms of induction of MCP-1 and IL-8 genes by MAC. Our investigation with IL-1-nonexpressing cells revealed that early induction of MCP-1 and IL-8 mRNAs required no de novo protein synthesis and appeared to occur at the transcriptional level in primary response to activated complement components. It appears from these observations that MAC may modulate the activity of already existing transcriptional factors and thereby up-regulate the expression of MCP-1 and IL-8 genes.
The signaling pathway by which MAC specifically promotes the expression
of MCP-1 and IL-8 genes in endothelial cells has yet to be determined.
Previous studies have shown the ability of MAC to induce a number of
different cell signaling pathways that could possibly regulate the
expression of these genes. Studies from other laboratories demonstrated
that the complement components C5b-8 and C5b-9 up-regulate
intracellular levels of diacylglycerol and activate protein kinase C
(52, 53). MAC may also influence cell function by interacting with
guanine nucleotide-binding proteins (G proteins). It is possible that
by these pathways deposition of the MAC may modulate the expression or
activity of transcriptional factors, in turn leading to a selective
up-regulation of MCP-1 and IL-8 genes. A recent study has demonstrated
that the translocation of NF-
B is necessary for endothelial
activation with the induction of IL-8 and MCP-1 genes (54). Activation
of NF-B has been shown to play a crucial role in the induction of
many different genes by a number of stimuli in the endothelial cells
(55). In this context, the IL-1-nonexpressing cells might constitute a
unique system to understand the complement-mediated primary response of
MCP-1 and IL-8 genes in endothelial cells. Additional studies are
underway to understand the precise pathways by which MAC modulates the
expression of these chemokine genes in endothelial cells.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Surgery, Box 3555 Medical Center, Duke University, Durham, NC 27710. E-mail address:
3 Address correspondence and reprint requests to: Dr. Jeffrey L. Platt, Department of Surgery, Mayo Clinic, Medical Sciences Building, Rochester, MN 55905.
4 Abbreviations used in this paper: MCP-1, monocyte chemotactic protein-1; C8, complement component-8; IL-1RA, interleukin-1 receptor antagonist; sCR1, soluble complement receptor-1; MAC, membrane attack complex.
Received for publication October 6, 1997. Accepted for publication June 22, 1998.
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M. C. Bosco, A. Rapisarda, S. Massazza, G. Melillo, H. Young, and L. Varesio The Tryptophan Catabolite Picolinic Acid Selectively Induces the Chemokines Macrophage Inflammatory Protein-1{alpha} and -1{beta} in Macrophages J. Immunol., March 15, 2000; 164(6): 3283 - 3291. [Abstract] [Full Text] [PDF]
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