Inhibition of C5a-induced neutrophil chemotaxis and macrophage cytokine production in vitro by a new C5a receptor antagonist

doi:10.1016/S0006-2952(00)00361-0

Biochemical Pharmacology

Volume 60, Issue 5, 1 September 2000, Pages 729-733

https://doi.org/10.1016/S0006-2952(00)00361-0 Get rights and content

Abstract

A cyclic peptide, Phe-[Orn-Pro-d-Cyclohexylalanine-Trp-Arg] (F-[OPdChaWR]), was recently shown in vitro to antagonise the binding of C5a to its receptor (CD88) on human polymorphonuclear leukocytes (PMNs) and in vivo to inhibit the neutropenia associated with septic shock induced by lipopolysaccharide (LPS) in rats. The aim of this study was to investigate whether F-[OPdChaWR] inhibits C5a-mediated chemotaxis of human PMNs using a modified Boyden chamber and C5a-stimulated release of cytokines from human monocytes in vitro. Approximately 50% of the chemotactic activity induced by 10 nM C5a was inhibited by 76 nM F-[OPdChaWR]. This correlated with inhibition of C5a-induced polarisation of PMNs by F-[OPdChaWR]. C5a alone failed to induce release of the inflammatory cytokines interleukin(IL)-1β, tumour necrosis factor (TNF)-α, and IL-6 from human monocytes at concentrations up to 100 nM. However, in the presence of low concentrations of LPS (50 ng/mL), both IL-1β and TNF-α were stimulated by 1 nM C5a. This co-stimulation was inhibited by F-[OPdChaWR] with ic₅₀s of 0.8 and 6.9 nM for release of TNF-α and IL-1β, respectively. No agonist activity was detected for F-[OPdChaWR] in either the chemotaxis or cytokine release assays at concentrations up to 50 μM. These results show that F-[OPdChaWR] inhibits several important inflammatory activities of C5a and suggest that C5a receptor antagonists may be effective in the treatment of inflammatory diseases mediated by C5a.

Section snippets

Materials

C5a was purchased from Sigma Chemical Co. F-[OPdChaWR] was synthesised as previously described [8]. The water-soluble receptor antagonist features six amino acids (l-phenylalanine, l-ornithine, l-proline, d-cyclohexylalanine, l-tryptophan, l-arginine) connected in the sequence F-O-P-dCha-W-R with the C-terminus of the arginine residue condensed onto the side chain amine of the ornithine (Fig. 1). The key receptor-binding residues of the resulting cyclic peptide F-[OPdChaWR] (F, dCha, W, R) are

Chemotaxis

C5a strongly stimulated PMN chemotaxis (see Methods). The C5a antagonist was tested over a range of concentrations from 0.5 to 5000 nM with ∼90% of the inhibition of chemotactic activity observed at a concentration of 5000 nM. The ic₅₀ for the inhibition of 10 nM C5a was 75 nM of the antagonist (Fig. 2). Agonist activity of F-[OPdChaWR] was tested over a wide range of concentrations (0.5–50 μM), but no agonist activity was detected at any concentration examined.

Polarisation

The effects of F-[OPdChaWR] on

Discussion

Many inflammatory responses are characterised by the accumulation of PMNs at an inflammatory site. Where local inflammation is triggered by infection, trauma, or immune complex deposition, C5a is likely to be an important chemokine. The locomotory response of PMN to C5a, like those to other chemotactic factors, begins with a rapid change in cell morphology that is most clearly studied in cell suspensions [15]. Changes in cell shape correlate with those required for optimal migration across a

Acknowledgements

We are grateful to the National Health and Medical Research Council of Australia for partial financial support.

References (22)

L.P. Bignold
Use of sparse-pore polycarbonate (Nuclepore) membrane for the measurement of chemotaxis of polymorphonuclear leucocytes
J Immunol Methods
(1989)
D.G. Harkin et al.
Neutrophil polarisation in plasma differs to that induced by endogenous chemoattractants with regard to frequency of uropod formation and requirement for divalent cations
Cell Biol Int
(1994)
R. Schindler et al.
Recombinant C5a stimulates transcription rather than translation of interleukin-1 (IL-1) and tumor necrosis factorTranslational signal provided by lipopolysaccharide or IL-1 itself
Blood
(1990)
S.V. Boyden
The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leukocytes
J Exp Med
(1962)
S. Okusawa et al.
C5a stimulates secretion of tumor necrosis factor from human mononuclear cells in vitro. Comparison with secretion of interleukin 1 beta and interleukin 1 alpha
J Exp Med
(1988)
W.P. Arend et al.
Absence of induction of IL-1 production in human monocytes by complement fragments
J Immunol
(1989)
S. Okusawa et al.
C5a induction of human interleukin 1
J Immunol
(1987)
J.-M. Cavaillon et al.
Recombinant C5a enhances interleukin 1 and tumor necrosis factor release by lipopolysaccharide-stimulated monocytes and macrophages
Eur J Immunol
(1990)
T.C. Pellas et al.
Novel C5a antagonists regulate neutrophil functions in vitro and in vivo
J Immunol
(1998)
M. Mohr et al.
Effects of anti-C5a monoclonal antibodies on oxygen use in a porcine model of severe sepsis
Eur J Clin Invest
(1998)

A. Short et al.

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

Br J Pharmacol

(1999)

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The complement system is an essential component of the innate immune response and plays a vital role in host defense and inflammation. Dysregulation of the complement system, particularly involving the anaphylatoxin C5a and its receptors (C5aR1 and C5aR2), has been linked to several autoimmune diseases, indicating the potential for targeted therapies. C5aR1 and C5aR2 are seven-transmembrane receptors with distinct signaling mechanisms that play both partially overlapping and opposing roles in immunity. Both receptors are expressed on a broad spectrum of immune and non-immune cells and are involved in cellular functions and physiological processes during homeostasis and inflammation. Dysregulated C5a-mediated inflammation contributes to autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, epidermolysis bullosa acquisita, antiphospholipid syndrome, and others. Therefore, targeting C5a or its receptors may yield therapeutic innovations in these autoimmune diseases by reducing the recruitment and activation of immune cells that lead to tissue inflammation and injury, thereby exacerbating the autoimmune response. Clinical trials focused on the inhibition of C5 cleavage or the C5a/C5aR1-axis using small molecules or monoclonal antibodies hold promise for bringing novel treatments for autoimmune diseases into practice. However, given the heterogeneous nature of (systemic) autoimmune diseases, there are still several challenges, such as patient selection, optimal dosing, and treatment duration, that require further investigation and development to realize the full therapeutic potential of C5a receptor inhibition, ideally in the context of a personalized medicine approach. Here, we aim to provide a brief overview of the current knowledge on the function of C5a receptors, the involvement of C5a receptors in autoimmune disorders, the molecular mechanisms underlying C5a receptor-mediated autoimmunity, and the potential for targeted therapies to modulate their activity.
Contact-dependent inhibition of HIV-1 replication in ex vivo human tonsil cultures by polymorphonuclear neutrophils
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Citation Excerpt :
This included inhibition of serine protease activity involved in PMN trogocytosis required for killing T. vaginalis6 with 4-(2-aminoethyl) benzenesulfonylfluoride (AEBSF) (Figure 5J). In addition, because it has been shown to be involved in PMN chemotaxis and degranulation,57-59 we interfered with C5a using the C5a receptor agonist W-54011 (Figure 5K). Another study showed antibody-mediated trogoptosis of cancer cells by PMNs involving release of Ca2+,7 which was inhibited by the calcium chelator BAPTA-AM (Figure 5L).
Polymorphonuclear neutrophils (PMNs), the most abundant white blood cells, are recruited rapidly to sites of infection to exert potent anti-microbial activity. Information regarding their role in infection with human immunodeficiency virus (HIV) is limited. Here we report that addition of PMNs to HIV-infected cultures of human tonsil tissue or peripheral blood mononuclear cells causes immediate and long-lasting suppression of HIV-1 spread and virus-induced depletion of CD4 T cells. This inhibition of HIV-1 spread strictly requires PMN contact with infected cells and is not mediated by soluble factors. 2-Photon (2PM) imaging visualized contacts of PMNs with HIV-1-infected CD4 T cells in tonsil tissue that do not result in lysis or uptake of infected cells. The anti-HIV activity of PMNs also does not involve degranulation, formation of neutrophil extracellular traps, or integrin-dependent cell communication. These results reveal that PMNs efficiently blunt HIV-1 replication in primary target cells and tissue by an unconventional mechanism.
The critical role of C5a as an initiator of neutrophil-mediated autoimmune inflammation of the joint and skin
2018, Seminars in Immunology
The deposition of IgG autoantibodies in peripheral tissues and the subsequent activation of the complement system, which leads to the accumulation of the anaphylatoxin C5a in these tissues, is a common hallmark of diverse autoimmune diseases, including rheumatoid arthritis (RA) and pemphigoid diseases (PDs). C5a is a potent chemoattractant for granulocytes and mice deficient in its precursor C5 or its receptor C5aR1 are resistant to granulocyte recruitment and, consequently, to tissue inflammation in several models of autoimmune diseases. However, the mechanism whereby C5a/C5aR regulates granulocyte recruitment in these diseases has remained elusive. Mechanistic studies over the past five years into the role of C5a/C5aR1 in the K/BxN serum arthritis mouse model have provided novel insights into the mechanisms C5a/C5aR1 engages to initiate granulocyte recruitment into the joint. It is now established that the critical actions of C5a/C5aR1 do not proceed in the joint itself, but on the luminal endothelial surface of the joint vasculature, where C5a/C5aR1 mediate the arrest of neutrophils on the endothelium by activating β₂ integrin. Then, C5a/C5aR1 induces the release of leukotriene B₄ (LTB₄) from the arrested neutrophils. The latter, subsequently, initiates by autocrine/paracrine actions via its receptor BLT1 the egress of neutrophils from the blood vessel lumen into the interstitial. Compelling evidence suggests that this C5a/C5aR1-LTB₄/BLT1 axis driving granulocyte recruitment in arthritis may represent a more generalizable biological principle critically regulating effector cell recruitment in other IgG autoantibody-induced diseases, such as in pemphigoid diseases. Thus, dual inhibition of C5a and LTB₄, as implemented in nature by the lipocalin coversin in the soft-tick Ornithodoros moubata, may constitute a most effective therapeutic principle for the treatment of IgG autoantibody-driven diseases.
Effects of adrenomedullin and vascular endothelial growth factor on ischemia/reperfusion injury in skeletal muscle in rats
2013, Journal of Surgical Research
Citation Excerpt :
Although there are a few clinical strategies designed to reduce the detrimental effects of I/R injury, none of them is completely successful in attenuating the skeletal muscle injury [23]. These strategies include pharmacologic intervention, hyperbaric oxygen, hypothermia, and pre- and post-injury conditioning [24–27]. In this study, the effects of two pharmacologic agents, AM and VEGF, on abdominal aortic clamping-induced skeletal muscle I/R injury were investigated in an experimental model.
In this study we investigated the effects of adrenomedullin (AM) and vascular endothelial growth factor (VEGF) on skeletal muscle ischemia/reperfusion (I/R) injury in a rat model.
Thirty-six Wistar rats were randomized into six groups (n = 6). Laparotomy was performed in all groups under general anesthesia. Nothing else was done in Group S (Sham). The Group I/R underwent I/R performed by clamping and declamping of the infrarenal abdominal aorta for 120 min, respectively. Group VEGF and Group AM received intravenous infusion of VEGF (0.8 μg/kg) or AM (12 μg/kg) respectively, without I/R. Group I/R + VEGF and Group I/R + AM received intravenous infusion of VEGF (0.8 μg/kg) or AM (12 μg/kg) immediately after 2 h period of ischemia, respectively. At the end of reperfusion period, skeletal muscle samples of lower extremity were taken from all groups for biochemical and histopathologic examinations.
Tissue levels of malondialdehyde (MDA), superoxide dismutase (SOD), nitric oxide (NO), and hypoxia inducible factor 1 alpha (HIF 1α) were found to be significantly higher in Group I/R than the levels in Group S (P < 0.05). Tissue levels of MDA, SOD, NO, and HIF 1α were significantly lower in Group I/R + AM compared with the levels in Group I/R (P < 0.05). In Group I/R + VEGF, tissue levels of MDA and NO were significantly lower than the levels in Group I/R (P < 0.05). No statistically significant difference was found in the tissue levels of catalase among the groups. Histologic examination revealed a larger central muscular necrosis than the peripheral necrosis, red blood cells in the lumens of capillary vessels, and a stronger atrophy and elliptical or round shape in muscle fibers in Group I/R. Terminal deoxynucleotidyl transferase mediated dUPT nick end labeling (TUNEL)-positive cell count was significantly lower in groups I/R + AM and I/R + VEGF than Group I/R (P < 0.0001, P < 0.0001, respectively).
These results indicate that AM and VEGF have protective effects on I/R injury in skeletal muscle in a rat model.
The effect of ischemia reperfusion injury on skeletal muscle
2012, Injury
Citation Excerpt :
The administration of poloxamer 188, a copolymer surfactant, has been shown to decrease myocyte injury and improve survival in a mouse hind-limb ischemia model.47 Various complement blocking compounds are also being explored to mitigate IR damage.27,36 Hyperbaric (HBO) treatment can increase the partial pressure of oxygen in tissue.
Ischemia reperfusion (IR) injury occurs when tissue is reperfused following a period of ischemia, and results from acute inflammation involving various mechanisms. IR injury can occur following a range of circumstances, ranging from a seemingly minor condition to major trauma. The intense inflammatory response has local as well as systemic effects because of the physiological, biochemical and immunological changes that occur during the ischemic and reperfusion periods. The sequellae of the cellular injury of IR may lead to the loss of organ or limb function, or even death. There are many factors which influence the outcome of these injuries, and it is important for clinicians to understand IR injury in order to minimize patient morbidity and mortality. In this paper, we review the pathophysiology, the effects of IR injury in skeletal muscle, and the associated clinical conditions; compartment syndrome, crush syndrome, and vascular injuries.
Organ Transplant: Strategies for Prevention of Antibody-Mediated Allograft Rejection
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Organ transplantation is considered the therapy of choice for end-stage failure or congenital abnormalities. Technically, organ donation and transplantation have been highly successful with excellent peri-operative survival rates. However, graft rejection remains the major obstacle in the long term to transplant survival. Over the past 18 years, the contribution of humoral immunity and its associated involvement of alloantibody and complement to allograft rejection have been clarified to a large extent. Antibody-mediated rejection (AMR) remains the one of the major unresolved hurdles of clinical transplantation, as it is refractory to anti-T cell therapy and carries an ominous prognosis, leading to early and even late graft loss. Despite the recognition in the transplantation field of the clinical importance of AMR, more work is still needed to precisely elucidate the role of the humoral immune response during the various stages of solid organ rejection, and in particular, chronic allograft failure. A more acute identification and understanding of AMR will enable the development of novel immunosuppressive treatments specifically directed to its prevention. These therapeutics could ultimately result in the better management of high-risk transplantations such as presensitization, and crossmatch-positive and ABO-incompatible transplantations. The results from our recent studies demonstrated that several important and previously underestimated strategies for preventing AMR exist and have the potential to provide effective solutions to currently unmet medical needs. In this article, we provide an introduction to organ transplantation and rejection, as well as discuss the clinical treatment protocol and the rationale behind the design of these novel experimental approaches aimed at preventing AMR.

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Biochemical Pharmacology

Abstract

Section snippets

Materials

Chemotaxis

Polarisation

Discussion

Acknowledgements

References (22)

Use of sparse-pore polycarbonate (Nuclepore) membrane for the measurement of chemotaxis of polymorphonuclear leucocytes

J Immunol Methods

Neutrophil polarisation in plasma differs to that induced by endogenous chemoattractants with regard to frequency of uropod formation and requirement for divalent cations

Cell Biol Int

Recombinant C5a stimulates transcription rather than translation of interleukin-1 (IL-1) and tumor necrosis factorTranslational signal provided by lipopolysaccharide or IL-1 itself

Blood

The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leukocytes

J Exp Med

C5a stimulates secretion of tumor necrosis factor from human mononuclear cells in vitro. Comparison with secretion of interleukin 1 beta and interleukin 1 alpha

J Exp Med

Absence of induction of IL-1 production in human monocytes by complement fragments

J Immunol

C5a induction of human interleukin 1

J Immunol

Recombinant C5a enhances interleukin 1 and tumor necrosis factor release by lipopolysaccharide-stimulated monocytes and macrophages

Eur J Immunol

Novel C5a antagonists regulate neutrophil functions in vitro and in vivo

J Immunol

Effects of anti-C5a monoclonal antibodies on oxygen use in a porcine model of severe sepsis

Eur J Clin Invest

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

Br J Pharmacol

Cited by (56)

The role of C5a receptors in autoimmunity

Contact-dependent inhibition of HIV-1 replication in ex vivo human tonsil cultures by polymorphonuclear neutrophils

The critical role of C5a as an initiator of neutrophil-mediated autoimmune inflammation of the joint and skin

Effects of adrenomedullin and vascular endothelial growth factor on ischemia/reperfusion injury in skeletal muscle in rats

The effect of ischemia reperfusion injury on skeletal muscle

Organ Transplant: Strategies for Prevention of Antibody-Mediated Allograft Rejection

Inflammation & immunopharmacologyInhibition of C5a-induced neutrophil chemotaxis and macrophage cytokine production in vitro by a new C5a receptor antagonist

Abstract

Section snippets

Materials

Chemotaxis

Polarisation

Discussion

Acknowledgements

J Immunol Methods

Cell Biol Int

Blood

The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leukocytes

J Exp Med

C5a stimulates secretion of tumor necrosis factor from human mononuclear cells in vitro. Comparison with secretion of interleukin 1 beta and interleukin 1 alpha

J Exp Med

Absence of induction of IL-1 production in human monocytes by complement fragments

J Immunol

C5a induction of human interleukin 1

J Immunol

Recombinant C5a enhances interleukin 1 and tumor necrosis factor release by lipopolysaccharide-stimulated monocytes and macrophages

Eur J Immunol

Novel C5a antagonists regulate neutrophil functions in vitro and in vivo

J Immunol

Effects of anti-C5a monoclonal antibodies on oxygen use in a porcine model of severe sepsis

Eur J Clin Invest

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

Br J Pharmacol

Inflammation & immunopharmacology
Inhibition of C5a-induced neutrophil chemotaxis and macrophage cytokine production in vitro by a new C5a receptor antagonist