Skip to main content
NCBI home page
British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 1999 Feb;126(3):551–554. doi: 10.1038/sj.bjp.0702338

Effects of a new C5a receptor antagonist on C5a- and endotoxin- induced neutropenia in the rat

Anna Short 1, Allan K Wong 2, Angela M Finch 1, Gerald Haaima 2, Ian A Shiels 1, David P Fairlie 2, Stephen M Taylor 1,*
PMCID: PMC1565845  PMID: 10188960

Abstract

A new C5a receptor antagonist, the cyclic peptide Phe-[Orn-Pro-D-cyclohexylalanine-Trp-Arg], (F-[OPdChaWR]), was tested for its ability to antagonize the neutropenic effects of both C5a and endotoxin in rats. Human recombinant C5a (2 μg kg−1 i.v.) caused rapid neutropenia, characterized by an 83% decrease in circulating polymorphonuclear leukocytes (PMNs) at 5 min. Administration of F-[OPdChaWR] (0.3–3 mg kg−1 i.v.), did not affect the levels of circulating PMNs but, when given 10 min prior to C5a, it inhibited the C5a-induced neutropenia by up to 70%. Administration of E. Coli lipopolysaccharide (LPS, 1 mg kg−1 i.v.) also caused neutropenia with an 88% decrease in circulating PMNs after 30 min. When rats were pretreated with F-[OPdChaWR] (0.3–10 mg kg−1 i.v.) 10 min prior to LPS, there was a dose-dependent antagonism of the neutropenia caused by LPS, with up to 69% reversal of neutropenia observed 30 min after LPS administration. These findings suggest that C5a receptor antagonists may have therapeutic potential in the many diseases known to involve either endotoxin or C5a.

Keywords: C5a, C5a antagonist, lipopolysaccharide, endotoxic shock, neutropenia

Introduction

Activation of the complement system has been implicated in a wide range of inflammatory disease states, including septic shock and adult respiratory distress syndrome (ARDS), which can occur as a consequence of bacterial infection (Martin & Silverman, 1992). The 74 amino acid peptide complement factor 5a (C5a) is a potent anaphylatoxin with key roles in the inflammatory and immune response, and is thought to be a major pathogenic factor in sepsis (Stevens et al., 1986) and ARDS (Mulligan et al., 1996). A drug which blocks the effects of C5a may be a useful therapeutic agent in these and other immunoinflammatory diseases. As yet, there are no C5a receptor antagonists available clinically.

Endotoxic shock is an acute disease state characterized by systemic hypotension (Lundberg et al., 1987), pulmonary hypertension (Marceau et al., 1987), depletion of circulating leukocytes and platelets, activation of the complement system (Smedegard et al., 1989), and can lead to the development of multiple organ failure (Seidenfeld et al., 1986). The tissue damage and organ dysfunction is associated with the migration of PMNs from the blood into the interstitium (Ahmed et al., 1996). The first stage of this process is adherence of the PMNs to vascular endothelium, and this results in a drop in circulating PMNs, or neutropenia (Smedegard et al., 1989). An agent which inhibited this process would have the potential to reduce tissue damage in inflammatory diseases such as sepsis.

Lipopolysaccharide (LPS) is a major component of the cell wall of gram-negative bacteria, and induces rapid endotoxic shock when injected intravenously in the rat (Smith et al., 1985). The hypotension caused by intravenous administration of LPS can be blocked by inhibitors of the inducible form of nitric oxide synthase (Szabo et al., 1996) as well as by a thromboxane A2 receptor antagonist (Altavilla et al., 1994), while the neutropenia has been reported to be inhibited by a platelet activating factor (PAF) receptor antagonist in the early stages (Coughlan et al., 1994) or by lipid A analogues (Soejima et al., 1996). The effects of a specific C5a receptor antagonist have not been previously described in this, or any other animal model. In this report we describe the effects of a new C5a antagonist, the cyclic peptide Phe-[Orn-Pro-dCha-Trp-Arg], or F-[OPdChaWR], on C5a- and LPS-induced neutropenia in the rat.

Methods

Female Wistar rats (250–350 g) were anaesthetized with i.p. ketamine (80 mg kg−1) and xylazine (12 mg kg−1). A polyethylene catheter was inserted in the femoral vein and rats were infused i.v. over 2 min with 0.2 ml of either F-[OPdChaWR] (0.3–10 mg kg−1) or sterile, pyrogen-free saline for controls. Rats were dosed i.v. 10 min later with 0.2 ml of either C5a (2 μg kg−1) or LPS (1 mg kg−1) or the appropriate vehicle control, each infused over a 1 min period. Blood samples (0.2 ml) were collected periodically over a 150 min period, layered on to an equal volume of Histopaque-Ficoll solution, then centrifuged at 400×g for 30 min at room temperature. The supernatant was discarded, and distilled water was added to the remaining pellet and shaken for 40 s to lyse the red blood cells. Dulbecco's phosphate buffered saline (10×concentrate) was added to restore isotonicity before being centrifuged at 400×g for 10 min at 4°C. This process lysed the red blood cells leaving a pellet of PMNs, which were washed and resuspended in 0.1 ml saline, and cell number was counted on a haemocytometer. Staining of cells with Diff Quik showed purity of PMNs was 95–98% by this method. PMN counts were expressed as a per cent of the blood concentrations obtained immediately prior to C5a or LPS challenge. The apparent binding affinity of F-[OPdChaWR] on isolated rat PMNs was determined using a com-petition binding assay with [125I]-C5a as described previously (Finch et al., 1997).

F-[OPdChaWR] was synthesized as follows. The linear peptide FOPdChaWR was synthesized using butoxycarbonyl (Boc)-Arg(Tosyl)-Pam resin employing in situ neutralization protocols for Boc chemistry (Schnolzer et al., 1992). The terminal Phe was introduced as fluorenylmethoxycarbonyl (Fmoc)-Phe to protect the N terminus during cyclisation. The peptide was cleaved and deprotected using 10% 1 : 1 p-cresol-thiocresol in anhydrous HF (0°C, 60 min). Cyclisation was effected using 5 eq benzotriazole1-yl-oxy-tris-(dimethylamino)-phosphoniumhexa-fluorophosphate and 10 eq diisopropylethylamine in dimethyl-formamide (10−4 M, 15 h). After cyclisation the Fmoc group was removed (1 : 1 piperidine-DMF, 15 min) and the crude peptide was purified by HPLC. The cyclised product was characterized by electrospray mass spectroscopy (M+H+=854.5) giving Mr 853.5 (calculated for C45H65N11O6=853.5 monoisotopic). Details of the synthetic methods for other antagonists of C5a receptors have been recently described (Wong et al., 1998).

Human recombinant C5a, lipopolysaccharide (serotype 055 : B5), Histopaque Ficoll (density 1077) solution and phosphate buffered saline were purchased from Sigma (St Louis, MO, U.S.A.). [125I]-C5a was purchased from New England Nuclear (MA, U.S.A.). Dilutions of drugs were made in 0.2 ml sterile pyrogen-free saline on the day of experimentation and brought to 37°C before intravenous infusion.

Statistical significance of results was determined using a non-parametric ANOVA with a Dunn's post-test at all time points. Receptor binding values were expressed as a per cent of the maximal response. Nonlinear regression was performed on these values to calculate the concentration of peptide that caused a 50% inhibition of [125I]-C5a binding (IC50) to isolated rat PMNs. All data are expressed as mean±s.e. mean.

Results

The chemical structure of F-[OPdChaWR] is shown in Figure 1. Cyclisation of the molecule was achieved via the sidechain of ornithine and the carboxyterminus of arginine. Human recombinant C5a and F-[OPdChaWR] demonstrated competitive inhibition of [125I]-C5a binding to isolated rat PMNs, with −log IC50 values of 9.78±0.12 and 7.57±0.29 respectively (Figure 2).

Figure 1.

Figure 1

Open in a new tab

Structure of F-[OPdChaWR].

Figure 2.

Figure 2

Open in a new tab

Inhibition of [125I]-C5a binding to rat PMNs by increasing concentrations of human recombinant C5a or F-[OPdChaWR]. Data are expressed as a per cent of maximal binding of [125I]-C5a. Results shown from PMNs from three animals.

The baseline level of circulating PMNs in rats was 2.04±0.25×106 cells ml−1 (n=25). Intravenous injection of 2 μg kg−1 C5a caused a rapid, transient neutropenia, which reached a maximum at 5 min, and returned to baseline by 30 min (Figure 3). Intravenous administration of F-[OPdChaWR], 10 min prior to the C5a injection, resulted in dose-dependent reductions in C5a-induced neutropenia over the 30 min observation period. The maximal inhibition (70±8%; n=3) of C5a-induced neutropenia was observed at 5 min with 3 mg kg−1 F-[OPdChaWR]. Intravenous administration of F-[OPdChaWR] (0.3–3 mg kg−1) alone had no effect on PMN levels prior to C5a (Figure 3), and a dose of 1 mg kg−1 did not affect PMN levels throughout a 150 min time period (data not shown).

Figure 3.

Figure 3

Open in a new tab

Inhibition of C5a- and LPS-induced neutropenia in the rat by F-[OPdChaWR]. Neutropenia was induced by i.v. 2 μg kg−1 C5a or 1 mg kg−1 LPS 10 min after i.v. F-[OPdChaWR] (0.3–10 mg kg−1). PMN concentrations are expressed as per cent of the zero time value. *indicates P<0.05 compared to C5a- or LPS-control value. (n=3–4 rats for each experiment).

Intravenous injection of 1 mg kg−1 LPS caused a decrease in blood PMN levels which reached a maximum at 30 min and returned to baseline at 150 min (Figure 3). Intravenous administration of F-[OPdChaWR], (0.3–10 mg kg−1) 10 min prior to LPS, resulted in a dose-dependent inhibition of LPS-induced neutropenia. A dose of 10 mg kg−1 F-[OPdChaWR] inhibited LPS-induced neutropenia by 69±4% (n=3) from the maximum value observed at 30 min.

Receptor selectivity of the C5a antagonist was ascertained by examining the activity against the degranulating peptide, formylmethionyl-leucyl-phenylalanine (fMLP) in human PMNs. Concentrations of F-[OPdChaWR] up to 1 mM were ineffective in altering the response of the PMNs to a maximally effective concentration of fMLP (1 μM), while the concentration of F-[OPdChaWR] reducing the secretory reponse to a maximally effective concentration of C5a (100 nM) (Wong et al., 1998) by 50% was 40 nM (Finch & Taylor, unpublished data).

Discussion

Acute endotoxaemia is characterized by a number of prominent haemodynamic disturbances, such as hypotension (Lundberg et al., 1987) and neutropenia (Till et al., 1982). Hypotension is mediated by nitric oxide, and inhibitors of the inducible form of nitric oxide synthetase were effective in blocking the decreased blood pressure (Szabo et al., 1996) as was a thromboxane A2 receptor antagonist (Altavilla et al., 1994). The neutropenia which occurs represents the first stage of diapedesis, and this extravasation of activated PMNs can lead to organ dysfunction and failure (Seidenfeld et al., 1986). Any agent which could prevent the adherence and subsequent migration of PMNs into the interstitium has therapeutic potential to reduce tissue damage in shock syndromes such as ARDS or endotoxemia. Of the synthetic agents produced to date, only an inhibitor of PAF receptors (Coughlan et al., 1994) and a lipid A analogue (Soejima et al., 1996) have been demonstrated to attenuate neutropenia caused by LPS.

The serum complement system is activated by LPS, and this leads to the generation of the anaphylatoxin C5a (Smedegard et al., 1989). Intravenous administration of C5a in animals results in haemodynamic changes, such as hypotension and neutropenia (Smedegard et al., 1989). The hypotension is blocked by inhibitors of cyclo-oxygenase, suggesting that vasodilator prostanoids mediate the decreased blood pressure, while neutropenia is not affected by these agents (Drapeau et al., 1993). The neutropenic effects of C5a are due to the rapid and transient expression of intracellular adhesion molecules (ICAMs) on neutrophils (Foreman et al., 1996) as well as P-selectin on vascular endothelial cells (Coughlan et al., 1994), resulting in the adherence of PMNs to the endothelium and a subsequent decrease in circulating levels of PMNs. This expression of ICAMs and P-selectin is caused by the activity of C5a on its receptors.

In the rat, administration of a high dose of LPS (50 mg kg−1 i.v.) led within 5 min to the generation of high circulating concentrations of C5a (0.6 μg ml−1) which returned to baseline levels within 30 min (Smedegard et al., 1989). The neutropenia caused by C5a was rapid and transient and paralleled the circulating levels of C5a. This report prompted our present study, to determine if the neutropenia to LPS could be inhibited by a C5a receptor antagonist. We used a lower dose of LPS (1 mg kg−1) in the present study to reduce the risk of mortality during the experiment, and our results demonstrate a similar level of neutropenia (circa 90%) as for the higher dose of LPS used by the earlier investigators.

In the present experiments, the neutropenia to i.v. C5a was inhibited, in a dose-dependent fashion, by a new C5a receptor antagonist designed and synthesized in our laboratories. When administered prior to LPS, a dose-dependent inhibition of the LPS-induced neutropenia was also seen. The highest dose of F-[OPdChaWR] tested in this study, 10 mg kg−1, did not entirely block the neutropenia to LPS at the early time points, although complete blockade was produced 60 min after LPS administration. Coughlan et al. (1994) have reported that a PAF receptor antagonist, CV-3988, inhibited the neutropenia to i.v. LPS in the first 20 min but was ineffective after this time. The incomplete inhibition of neutropenia in the first 15–30 min in the present experiments may reflect a PAF-induced component, and further experiments are necessary to determine if this is the case. The lipid A analogue, B464, has been reported to attenuate the neutropenia to LPS in the guinea-pig and reduce lung injury to LPS (Soejima et al., 1996) but its activity in the rat model has not yet been reported.

The first C5a receptor antagonist developed by modification of the carboxyterminal region of C5a was reported by Konteatis et al. (1994). To date, there have been no reports of in vivo activity for this, or any other antagonist derived from C5a. We have developed a series of analogues, involving cyclisation and modification of C-terminal analogues of C5a, and some of these have increased binding affinity and antagonist potency (Wong et al., 1998). We used human recombinant C5a in our experiments to determine receptor binding parameters and to cause neutropenia. Human C5a bound effectively to rat PMNs and a dose of 2 μg kg−1 caused a similar level of neutropenia as did 12 μg kg−1 rat C5a des arg (Smedegard et al., 1989). The new compound tested in the present study, a cyclised peptide, has an apparent binding affinity of 27 nM against [125I]-C5a in intact rat PMNs. Administration of the compound prior to C5a or LPS did not cause neutropenia, indicating that the compound does not activate PMN C5a receptors in vivo, and no agonist activities have been discerned for this compound in human foetal artery or human PMNs in vitro to date (unpublished data). Drapeau et al. (1993) have reported that neutropenia is more sensitive to C5a and C5a agonist peptides than are alterations in blood pressure. The present study did not involve blood pressure measurements, and the effects of new C5a antagonists on this and on other effects of C5a in the rat remain to be determined.

In summary, the present study has demonstrated the in vivo effectiveness of a new C5a receptor antagonist which inhibited the adherence of PMNs to the vascular endothelium induced by either C5a or LPS. Our results also suggest that C5a is a major mediator inducing neutropenia following LPS. Current studies are underway to determine the pharmacological activities of new C5a receptor antagonists in this and other models of immunoinflammatory diseases where C5a is implicated as a major pathogenic factor.

Acknowledgments

This work was supported by a URG grant from the University of Queensland.

Abbreviations

ARDS

adult respiratory distress syndrome

Fmoc

fluorenylmethoxycarbonyl

fMLP

formylmethionyl-leucyl-phenylalanine

HPLC

high performance liquid chromatography

LPS

lipopolysaccharide

PAF

platelet activating factor

PMNs

polymorphonuclear leukocytes

References

  1. AHMED N., THORLEY R., XIA D., SAMOLS D., WEBSTER R.O. Transgenic mice expressing rabbit C-reactive protein exhibit diminished chemotactic factor-induced alveolitis. Am. J. Respir. Crit. Care Med. 1996;153:1141–1147. doi: 10.1164/ajrccm.153.3.8630558. [DOI] [PubMed] [Google Scholar]
  2. ALTAVILLA D., CANALE P., SQUADRITO F., SARDELLA A., AMMENDOLIA L., URNA G., IOCULANO M., SQUADRITO G., CAPUTI A.P. Protective effects of BAY U 3405, a thromboxane A2 receptor antagonist, in endotoxin shock. Pharmacol. Res. 1994;30:137–151. doi: 10.1016/1043-6618(94)80005-7. [DOI] [PubMed] [Google Scholar]
  3. COUGHLAN A.F., HAU H., DUNLOP L.C., BERNDT M.C., HANCOCK W.W. P-selectin and platelet-activating factor mediate initial endotoxin-induced neutropenia. J. Exp. Med. 1994;179:329–334. doi: 10.1084/jem.179.1.329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. DRAPEAU G., BROCHU S., GODIN D., LEVESQUE L., RIOUX F., MARCEAU F. Synthetic C5a receptor agonists. Pharmacology, metabolism and in vivo cardiovascular and hematologic effects. Biochem. Pharmacol. 1993;45:1289–1299. doi: 10.1016/0006-2952(93)90282-2. [DOI] [PubMed] [Google Scholar]
  5. FINCH A.M., VOGEN S.M., SHERMAN S.A., KIRNARSKY L., TAYLOR S.M., SANDERSON S.D. Biologically active conformer of the effector region of human C5a and modulatory effects of N-terminal receptor binding determinants on activity. J. Med. Chem. 1997;40:877–884. doi: 10.1021/jm960727r. [DOI] [PubMed] [Google Scholar]
  6. FOREMAN K.E., GLOVSKY M.M., WARNER R.L., HORVATH S.J., WARD P.A. Comparative effect of C3a and C5a on adhesion molecule expression on neutrophils and endothelial cells. Inflammation. 1996;20:1–9. doi: 10.1007/BF01487740. [DOI] [PubMed] [Google Scholar]
  7. KONTEATIS Z.D., SICILIANO S.J., VAN RIPER G., MOLINEAUX C.J., PANDYA S., FISCHER P., ROSEN H., MUMFORD R.A., SPRINGER M.S. Development of C5a receptor antagonists. Differential loss of functional responses. J. Immunol. 1994;153:4200–4205. [PubMed] [Google Scholar]
  8. LUNDBERG C., MARCEAU F., HUGLI T.E. C5a-induced hemodynamic and hematologic changes in the rabbit. Role of cyclooxygenase products and polymorphonuclear leukocytes. Am. J. Pathol. 1987;128:471–483. [PMC free article] [PubMed] [Google Scholar]
  9. MARCEAU F., LUNDBERG C., HUGLI T.E. Effects of the anaphylatoxins on circulation. Immunopharmacology. 1987;14:67–84. doi: 10.1016/0162-3109(87)90031-2. [DOI] [PubMed] [Google Scholar]
  10. MARTIN M.A., SILVERMAN H.J. Gram-negative sepsis and the adult respiratory distress syndrome. Clin. Infect. Dis. 1992;14:1213–1228. doi: 10.1093/clinids/14.6.1213. [DOI] [PubMed] [Google Scholar]
  11. MULLIGAN M.S., SCHMID E., BECK SCHIMMER B., TILL G.O., FRIEDL H.P., BRAUER R.B., HUGLI T.E., MIYASAKA M., WARNER R.L., JOHNSON K.J., WARD P. A. Requirement and role of C5a in acute lung inflammatory injury in rats. J. Clin. Invest. 1996;98:503–512. doi: 10.1172/JCI118818. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. SCHNOLZER M., ALEWOOD P., JONES A., ALEWOOD D., KENT S.B. In situ neutralization in Boc-chemistry solid phase peptide synthesis. Rapid, high yield assembly of difficult sequences. Int. J. Pept. Protein. Res. 1992;40:180–193. doi: 10.1111/j.1399-3011.1992.tb00291.x. [DOI] [PubMed] [Google Scholar]
  13. SEIDENFELD J.J., POHL D.F., BELL R.C., HARRIS G.D., JOHANSON W.G., JR Incidence, site, and outcome of infections in patients with the adult respiratory distress syndrome. Am. Rev. Respir. Dis. 1986;134:12–16. doi: 10.1164/arrd.1986.134.1.12. [DOI] [PubMed] [Google Scholar]
  14. SMEDEGARD G., CUI L.X., HUGLI T.E. Endotoxin-induced shock in the rat. A role for C5a. Am. J. Pathol. 1989;135:489–497. [PMC free article] [PubMed] [Google Scholar]
  15. SMITH E.F.D., TEMPEL G.E., WISE W.C., HALUSHKA P.V., COOK J.A. Experimental endotoxemia in the rat: efficacy of prostacyclin or the prostacyclin analog iloprost. Circ. Shock. 1985;16:1–7. [PubMed] [Google Scholar]
  16. SOEJIMA K., ISHIZAKA A., URANO T., SAYAMA K., SAKAMAKI F., NAKAMURA H., TERASHIMA T., WAKI Y., TASAKA S., FUJISHIMA S., KAWATA T., CHRIST W.J., KANAZAWA M. Protective effect of B464, a lipid A analog, on endotoxin-induced cellular responses and acute lung injury. Am. J. Respir. Crit. Care. Med. 1996;154:900–906. doi: 10.1164/ajrccm.154.4.8887583. [DOI] [PubMed] [Google Scholar]
  17. STEVENS J.H., P O.H., SHAPIRO J.M., MIHM F.G., SATOH P.S., COLLINS J.A., RAFFIN T.A. Effects of anti-C5a antibodies on the adult respiratory distress syndrome in septic primates. J. Clin. Invest. 1986;77:1812–1816. doi: 10.1172/JCI112506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. SZABO C., BRYK R., ZINGARELLI B., SOUTHAN G.J., GAHMAN T.C., BHAT V., SALZMAN A.L., WOLFF D.J. Pharmacological characterization of guanidinoethyldisulphide (GED), a novel inhibitor of nitric oxide synthase with selectivity towards the inducible isoform. Br. J. Pharmacol. 1996;118:1659–1668. doi: 10.1111/j.1476-5381.1996.tb15589.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. TILL G.O., JOHNSON K.J., KUNKEL R., WARD P.A. Intravascular activation of complement and acute lung injury. Dependency on neutrophils and toxic oxygen metabolites. J. Clin. Invest. 1982;6:1126–1135. doi: 10.1172/JCI110548. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. WONG A.K., FINCH A.M., PIERENS G.K., CRAIK D.J., TAYLOR S.M., FAIRLIE D.P. Small Molecular probes for G protein-coupled C5a receptors. Conformationally constrained antagonists derived from the C-terminus of the human plasma protein C5a. J. Med. Chem. 1998;41:3417–3425. doi: 10.1021/jm9800651. [DOI] [PubMed] [Google Scholar]

Articles from British Journal of Pharmacology are provided here courtesy of The British Pharmacological Society

RESOURCES