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*
Department of Physiology and Pharmacology, and
Drug, Design and Development Centre, University of Queensland, St. Lucia, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, as
well as polymorphonuclear leukocyte migration, increased TNF- levels
and vascular leakage at the site of immune complex deposition. These
results indicate potent anti-inflammatory activities of a new C5a
receptor antagonist and provide more evidence for a key early role for
C5a in sepsis and the reverse Arthus reaction. The results support a
role for antagonists of C5a receptors in the therapeutic intervention
of immunoinflammatory disease states such as sepsis and immune complex
disease. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Specific C5a receptor antagonists can provide evidence of the selective effects of the anaphylatoxin C5a in animal models of immunoinflammatory disease, since the antagonist spares the actions of C3a and the membrane attack complex (C5b-9). To date, only a few recent in vivo studies have been performed with specific C5a receptor antagonists (6, 7, 8, 9), and only two of these reports involve agents of low (<1000 Da) m.w. (8, 9). In these studies, the compounds were shown to block the neutropenic (8) and hypotensive (9) effects of endotoxin in rats.
In the present study, we report for the first time some in vivo
anti-inflammatory activities of the potent, low m.w. cyclic
antagonist
AcPhe[L-ornithine-Pro-D-cyclohexylalanine-Trp-Arg]
(AcF-[OPdChaWR])3 of
the C5a receptor (10), recently developed in our
laboratories, against C5a- and LPS-induced neutropenia, as well as in
the reverse-passive Arthus model in the rat peritoneum. This molecule
is an analogue of another C5aR antagonist, F-[OPdChaWR], recently
reported to show in vivo activity (8), which now has been
acetylated to improve its metabolic stability. We demonstrate that
AcF-[OPdChaWR] not only inhibits the cellular influx, vascular
leakage, and neutropenia in these models, but also suppresses evoked
levels of the proinflammatory cytokines TNF- and IL-6. These results
confirm C5a as a key mediator in certain inflammatory reactions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The C5a receptor antagonist AcF-[OPdChaWR] was synthesized previously as described (10). The compound was purified by reversed-phase HPLC and fully characterized by mass spectrometry and proton nuclear magnetic resonance spectroscopy, and the receptor affinity and antagonist potency were determined on intact human polymorphonuclear leukocytes (PMNs) (10).
Receptor-binding assay
The apparent binding affinity of AcF-[OPdChaWR] on isolated rat PMNs was determined using a competition binding assay with 125I-C5a as described previously (11).
Neutropenia assay
Female Wistar rats (200–300 g) were anesthetized and a catheter was placed in the femoral vein. (All animal experimentation conducted in this study was performed in accordance with Animal Experimentation Ethics Committee ethical guidelines.) Rats were treated with antagonist (1 mg/kg i.v. in a final volume of 200 µl with 5% ethanol) or 5% ethanol control injection 10 min before C5a or LPS challenge. Rats were given a bolus i.v. dose of either human recombinant C5a (2 µg/kg; Sigma, St. Louis, MO) or LPS (1 mg/kg; from Escherichia coli serotype 055:B5; Sigma), and blood samples were collected into heparinized Eppendorf tubes at regular intervals over a 150-min observation period. PMNs were isolated and counted as previously reported (8) and expressed as a percentage of the PMN concentration before complement challenge. Additionally, whole blood was collected and allowed to clot spontaneously on ice, and serum samples were stored at -20°C until evaluation of cytokine concentrations.
Reverse-passive Arthus reaction
Female Wistar rats (200–250g) were anaesthetized with ketamine (80 mg/kg) and xylazine (12 mg/kg) and a polyethylene catheter was placed in the left femoral vein. A reverse-passive Arthus reaction was induced in the peritoneal cavity by injecting Evans blue (10 mg/kg i.v.), chicken OVA (20 mg/kg i.v.), and rabbit anti-chicken OVA (10 mg/kg i.p.). A group of rats was pretreated with a C5a receptor antagonist, AcF-[OPdChaWR] (1 mg/kg i.v.) 10 min before induction of the Arthus reaction. Rats treated with Evans blue plus antagonist only served as antagonist controls, Evans blue plus chicken OVA only as Ag controls, and Evans blue plus i.p. rabbit anti-chicken OVA served as Ab controls. Rats were placed on a heating pad, and anesthetic was maintained over a 4-h treatment period with periodic collection of blood samples. Blood was allowed to spontaneously clot on ice, and serum samples were collected and stored at -20°C. Four hours after induction of the peritoneal Arthus reaction, the peritoneal cavity was lavaged with 25 ml of saline, and the lavage fluid was collected for assessment of the total cell number with a hemocytometer, the differential cell count of a smear was determined using DiffQuick stain, and the OD of the supernatant at 650 nm was measured as an indicator of Evans blue leakage into the peritoneal cavity.
TNF- measurement
Serum and peritoneal lavage TNF- concentrations were measured
using an ELISA. A 96-well plate (Nunc Maxisorb; Nunc, Roskilde,
Denmark) was coated with rabbit anti-rat TNF- Ab (50 µl of 1
µg/ml in phosphate buffer, pH 6.8; Research Diagnostics), sealed, and
incubated for 2 h at 37°C. The plate was washed three times with
PBS-Tween 20, and 100 µl of PBS-Tween 20 containing 0.1% BSA was
added to each well, sealed, and incubated for 2 h at 37°C. The
plate was washed three times with PBS-Tween 20, and samples were
diluted 1:100–1:10,000 in PBS-Tween 20-BSA added in duplicate.
Recombinant rat TNF- (PharMingen, San Diego, CA) was diluted from 1
µg/ml to 1 pg/ml in PBS-Tween 20-BSA and added in duplicate to each
plate as a standard curve. The plate was sealed and incubated for
2 h at 37°C. The plate was again washed three times with
PBS-Tween 20, and 50 µl of murine anti-rat TNF- Ab (2 µg/ml
in PBS-Tween 20-BSA; PharMingen) was added to each well and incubated
at room temperature for 30 min. The plate was washed, and 50 µl of
biotinylated sheep anti-rat IgG (1 µg/ml; Amersham, Arlington
Heights, IL) was added to each well and incubated at room temperature
for 30 min. The plate was washed six times with PBS-Tween 20, and 100
µl of streptavidin-peroxidase conjugate (100 ng/ml; Sigma) was added
to each well and incubated at room temperature for 30 min. The plate
was again washed six times with PBS-Tween 20, and 100 µl of substrate
was added to each well (tetramethylbenzidine substrate system; Sigma).
Color development occurred over 30 min at room temperature, followed by
the addition of 100 µl of 0.5 M
H2SO4 to each well.
Absorbance was read at 450 nm using a 96-well plate reader (Tecan).
Concentrations of TNF- in serum samples were determined by linear
regression analysis from the standard curve.
IL-6 measurement
An ELISA method as described above also measured serum and peritoneal lavage fluid IL-6 concentrations. Briefly, a 96-well plate was coated with goat anti-rat IL-6 (1 µg/ml; Research Diagnostics), followed by the addition of PBS-Tween 20-BSA to block nonspecific binding sites. Serum samples and recombinant rat IL-6 (1 µg/ml-1 pg/ml; Research Diagnostics) were diluted in PBS-Tween 20-BSA and added to the plate in duplicate and incubated for 2 h. The plate was washed, and biotinylated goat anti-rat IL-6 Ab (1 µg/ml; Research Diagnostics) was added to each well. Streptavidin-peroxidase conjugate was added to each well, followed by substrate, and color development was allowed to occur for 30 min before addition of 0.5 M H2SO4. Absorbance was read at 450 nm.
Data and statistical analyses
Receptor-binding affinities were determined for C5a and
AcF-[OPdChaWR] to isolated human and rat PMNs. Analysis of
dose-response curves was performed using nonlinear regression analysis
(GraphPad Prism 2.0; GraphPad, San Diego, CA).
IC50 values (molar concentration of peptide
required to cause 50% inhibition of maximal response) were determined
from these dose-response curves, and the mean -log
IC50 values calculated from individual curves was
used for statistical analysis. PMN concentrations following C5a or LPS
challenge were expressed as means ± SEM for each treatment group
(n = 3–6). Results derived from the Arthus model were
expressed as means ± SEM (n = 3–5) for each
treatment group. Mean values for each group were compared with Ab
control values using Student’s t test, and statistical
significance was assessed at p 
0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human recombinant C5a demonstrated competitive inhibition of
125I-C5a binding with comparable affinity to both
isolated human and rat PMNs, with -log IC50
values of 9.25 ± 0.09 (IC50 = 0.5 nM) and
9.75 ± 0.17 (IC50 = 0.2 nM), respectively.
The antagonist peptide AcF-[OPdChaWR] similarly demonstrated
competitive inhibition of 125I-C5a binding to
isolated rat PMNs, with a -log IC50 of value
7.11 ± 0.33 (IC50 = 70 nM) (Fig. 1
). The receptor-binding affinity of
AcF-[OPdChaWR] in human PMNs has been previously reported with a
-log IC50 value of 6.57 ± 0.05
(IC50 = 20 nM) (10) and was not
significantly different from that obtained for rat PMNs (Fig. 1) .
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Intravenous treatment of rats with human C5a resulted in a rapid
and transient decrease in circulating PMNs (neutropenia), with the
concentration of PMNs reaching a minimum of 18 ± 6%
prestimulation controls levels at 5 min, then returning to pretreatment
values within 60 min (Fig. 2). Similarly,
i.v. LPS (1 mg/kg) resulted in a rapid decrease in circulating PMNs
which reached a minimum value of 31 ± 4% at 30 min and returned
to pretreatment values by 150 min (Fig. 2). Pretreatment of rats with a
single dose (1 mg/kg i.v.) of AcF-[OPdChaWR] 10 min before C5a or LPS
challenge significantly inhibited both C5a- and LPS-induced neutropenia
(Fig. 2). Intravenous administration of the same doses of C5a and LPS
produced significant elevations in serum TNF- levels, reaching a
maximum value of 30 ± 3 ng/ml (n = 6) at 30 min
for C5a and 54 ± 11 ng/ml (n = 6) at 90 min for
LPS (Fig. 2). Pretreatment of rats with a single dose of the C5a
receptor antagonist (1 mg/kg i.v. 10 min before challenge) completely
blocked both C5a- and LPS-induced elevation of serum TNF- (Fig. 2).
To eliminate the possibility that the recombinant human C5a used in
these experiments may have contained trace levels of LPS affecting the
interpretation of the results, the C5a stock solution was boiled at
110°C for 30 min and then injected into a rat (2 µg/kg i.v.)
(12). The neutropenic activity was lost (data not shown),
demonstrating that the recombinant human C5a used in this study was
free of pharmacologically active levels of contaminating LPS.
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The injection of Ab into the peritoneum resulted in a large
cellular influx after 4 h, 46% of the cells being PMNs, which
represented about a 10-fold increase in the peritoneal PMN cell content
over controls (Fig. 3). Accompanying this
PMN influx was vascular leakage of serum, as demonstrated by the
increased levels of Evans blue in the peritoneal lavage fluid (Fig. 3).
Neither the administration of Ag alone, Ab alone, nor C5a antagonist
alone affected cellular influx or leakage of Evans blue into the
peritoneal cavity. Pretreatment of rats with a single i.v. dose (1
mg/kg) of the C5a receptor antagonist AcF-[OPdChaWR] at 10 min before
induction of the Arthus reaction completely blocked both the PMN influx
and the increased leakage of serum, as monitored by Evans
blue.
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and IL-6 by
AcF-[OPdChaWR]
Treatment of rats with Evans blue and either the C5a antagonist,
Ag, or Ab alone caused no significant change in baseline serum or
peritoneal lavage, TNF-, or IL-6 concentrations. Induction of the
reverse-passive Arthus reaction in the peritoneal cavity caused a
significant increase in circulating TNF- concentrations at 3 h,
which returned to pretreatment values within 4 h of the Arthus
reaction induction (Fig. 4). The
peritoneal Arthus reaction also caused an increase in IL-6
concentrations in the serum, which reached statistical significance at
1–3 h and returned to pretreatment values within 4 h of induction
(Fig. 4). In addition, the peritoneal lavage fluid of rats undergoing
an Arthus reaction demonstrated significantly elevated TNF- and IL-6
levels 4 h after induction (Fig. 5).
Pretreatment of rats with the C5a receptor antagonist at 1 mg/kg i.v.
10 min before induction of the Arthus reaction blocked the elevation in
cellular influx and cytokine expression at the site of inflammation, as
well as the elevation of circulating cytokine levels when compared with
Ab control values (
Figs. 3–5
). This result demonstrates complete
suppression of cytokine formation levels by the C5a antagonist with the
dose and route of administration used.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C5a causes the release of TNF- and ILs from monocytes in vitro
(17, 18), but this effect in vivo has not been described
previously. The i.v. administration of C5a caused an increase in
circulating levels of TNF-, and this increase was blocked by
pretreatment with the C5a antagonist. We have recently shown that an
analogue of AcF-[OPdChaWR] is effective in inhibiting the neutropenia
induced by endotoxin and C5a in a model of acute endotoxic shock
(8). In the present study, the new compound
AcF-[OPdChaWR] blocks both neutropenia as well as the elevation of
TNF- levels following LPS, and this strongly suggests that C5a is a
key mediator for both of these effects.
The peritoneal Arthus reaction involves an acute localized inflammatory
reaction that is also characterized by PMN migration to the
inflammatory locus, vascular leakage, and cytokine production at the
site of inflammation (19, 20, 21). The acute inflammatory
events in this model of immune complex disease can be inhibited by
intervention in the complement system (22, 23).
Recombinant soluble human complement receptor type 1, which blocks
activation of the complement cascade, inhibits neutrophil accumulation
and margination, tissue immunoreactivity to C3b, and formation of the
membrane attack complex in a dermal Arthus reaction (24).
The Arthus reaction can also be limited by cobra venom factor depletion
of systemic complement activity (25), supporting the
requirement for complement in this inflammatory reaction. Furthermore,
these inflammatory effects have previously been demonstrated to be C5a
dependent, in that genetic deletion of the C5a receptor in mice showed
impaired inflammatory responses to immune complex formation in both the
peritoneum and skin (23). Mice deficient in C5a receptors
showed significantly reduced neutrophil influx, TNF- and IL-6
expression, and vascular leakage at the site of inflammation when
compared with wild-type littermates. The Arthus reaction appears to be
also dependent on adhesion and migration of PMNs to the
site of immune complex formation (26). Pretreatment with a
P-selectin Ab inhibits Arthus-induced swelling, neutrophil
accumulation, and endothelial P-selectin expression, despite complement
deposition at the site of dermal immune complement formation
(27). The present study now demonstrates that a small
molecule C5a receptor antagonist inhibits a wide range of the
immunoinflammatory events associated with immune complex formation and
is in agreement with a preliminary finding utilizing a large m.w. C5a
analogue antagonist (6).
Although it is widely accepted that immune complex deposition leads to
increased cytokine expression at the local site of inflammation
(20, 23), the systemic expression of these proinflammatory
cytokines has not previously been described. In the present study,
serum samples were collected at regular intervals following induction
of the peritoneal Arthus reaction, and the circulating levels of
TNF- and IL-6 were measured. Both of these proinflammatory cytokines
were significantly elevated in circulation, indicating that the Arthus
reaction is not necessarily limited to the site of inflammation, but
can also involve a systemic component. Both the local and systemic
induction of cytokines were significantly inhibited by pretreatment of
rats with the C5a receptor antagonist, again indicating a requirement
for C5a in cytokine production in the Arthus reaction.
Proinflammatory cytokine production is a universal component of a wide
range of disease states including immune complex-mediated conditions
such as nephritis (28), arthritis (29), and
acute graft rejection (30). An agent which can inhibit the
production of cytokines such as TNF- and IL-6 both locally and
systemically may have wide therapeutic applications in the prevention
and treatment of these and other disease states. The present study
indicates that a C5a receptor antagonist is very effective in
inhibiting cytokine production when administered before direct
challenge with exogenous or endogenous C5a (by LPS administration), as
well as in an acute immune complex disease model. The application of a
C5a receptor antagonist in other disease states involving complement
activation and up-regulation of local and systemic cytokines needs to
be further explored both as a preventative and therapeutic agent. The
availability of small molecule C5a receptor antagonists now allows for
the elucidation of the role of C5a in immunoinflammatory diseases as
well as the development of orally active agents as prospective drugs
for treating inflammatory diseases.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Stephen M. Taylor, Department of Physiology and Pharmacology, University of Queensland, St. Lucia 4072, Australia.
3 Abbreviations used in this paper: AcF-[OPdChaWR], AcPhe[L-ornithine-Pro-D-cyclohexylalanine-Trp-Arg]; PMN, polymorphonuclear leukocyte.
Received for publication December 14, 1999. Accepted for publication April 6, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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on graft-versus-host disease and graft versus leukemia. J. Clin. Invest. 104:459.[Medline]
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