The aim of our study was to investigate the roles played by sphingosine kinase (SPHK) in the anaphylatoxin C5a-triggered responses in vivo. Our data show that i.v. administration of C5a triggers a rapid neutropenic response, but pretreating mice with the SPHK inhibitor, N,N-dimethylsphingosine (DMS), 10 min before the C5a i.v. administration substantially inhibited the C5a-triggered neutropenia. Similarly the i.v. administration of C5a caused a rapid increase in the serum levels of TNF-α and IL-6, and this increase in cytokine levels was blocked by DMS. We then induced acute peritonitis with C5a. The C5a i.p. injection triggered a fast recruitment of neutrophils, later followed by monocytes, into the peritoneal cavity. Vascular permeability was also observed: when we i.v. injected Evans blue before C5a i.p. injection, we could observe a continued influx of the dye into the peritoneum. In mice pretreated with DMS, there was a significant reduction on the C5a-triggered neutrophil and monocyte infiltration, as well as a marked reduction on the Evans blue influx. Our data also show that the i.p. administration of C5a caused a rapid increase in TNF-α and IL-6 levels in the peritoneal cavity, and this increase in cytokine levels was substantially inhibited in mice pretreated with the SPHK inhibitor. Taken together, these observations suggest a potential role for SPHK in the C5a-triggered inflammatory responses in vivo.
Activation of the complement cascade plays a key role in host defense. However, activation of the complement system leads to the generation of the potent proinflammatory anaphylatoxin, C5a. Significant amounts of C5a, as well as other complement products in the blood, can lead to a series of adverse effects associated with a variety of pathologies, including septic shock, adult respiratory distress syndrome, and immune complex-dependent autoimmune diseases such as rheumatoid arthritis (1, 2, 3).
Recently, the anaphylatoxin C5a has been shown to have an immune-regulatory role able to stimulate mediators of both acute and chronic inflammation (4, 5, 6, 7, 8). The significance of C5a in several inflammatory diseases is demonstrated by the fact that agents that blocked the action of C5a also suppressed inflammation in several animal models (9, 10, 11, 12, 13).
In primates, Escherichia coli-induced septic shock and adult respiratory distress syndrome can be attenuated by blocking C5a with specific Abs (1, 4). Studies in rats showed that LPS-induced septic shock can be mimicked by injection of C5a, and Ab blocking of C5a substantially reduces the LPS-induced shock (2). Moreover, blockade of C5a after infusion of LPS or induction of cecal ligation puncture in rats has been found to be protective (2, 12). Furthermore, C5a blockade after cecal ligation puncture in rats attenuates the development of multiorgan failure (13).
Most of these studies used blocking Abs raised against C5a (10, 12) or recombinant proteins that are receptor antagonists or analogues of C5a (9, 14). However, there are many problems associated with the use of such proteins to treat human patients. Immunogenicity is a problem, and proteins are expensive to manufacture, very susceptible to degradation by proteases in serum or the gastrointestinal track, and generally display poor pharmacokinetic properties. More recently attempts have been made to make smaller molecules that are more stable, cheaper to make, have better bioavailability, and are more attractive as drug candidates for treating human diseases mediated by C5a (15, 16). However, very little is known about the intracellular signaling pathways activated by C5a in immune-effector cells.
During the last few years, it has become clear that sphingolipids, in addition to being structural constituents of cell membranes, are sources of important signaling molecules. Particularly, the sphingolipid metabolites, ceramide and sphingosine-1-phosphate (SPP), 3 have emerged as a new class of potent bioactive molecules, implicated in a variety of cellular processes such as cell differentiation, apoptosis, and proliferation (17, 18, 19, 20). Interest in SPP focused recently on two distinct cellular actions of this lipid: namely, its function as an extracellular ligand activating specific G protein-coupled receptors, and its role as an intracellular second messenger (21). Several findings enforced the notion of SPP as an important intracellular second messenger. First, activation of various plasma membrane receptors, such as the platelet-derived growth factor receptor (22, 23), the FcεRI and FcγRI Ag receptors (24, 25, 26), as well as the fMLP receptor (27), was found to rapidly increase intracellular SPP production through the stimulation of sphingosine kinase (SPHK). Second, inhibition of SPHK stimulation strongly reduced or even prevented cellular events triggered by these receptors, such as receptor-stimulated DNA synthesis, Ca2+ mobilization and vesicular trafficking (22, 23, 24, 25, 26, 27).
Very recently, we have shown that in human neutrophils and macrophages, C5a activates the intracellular signaling molecule SPHK and that inhibition of SPHK activity, by N,N-dimethylsphingosine (DMS), in primary human neutrophils, neutrophil-differentiated HL-60 cells, as well as in monocyte-derived macrophages, largely inhibits C5a-stimulated Ca2+ mobilization, enzyme release, chemotaxis, and cytokine production, suggesting a potential role for SPHK in the C5a-triggered inflammatory responses (28, 29).
Here we show for the first time that the C5a-triggered neutropenia and peritonitis can be attenuated by inhibition of SPHK. We show here that the C5a-triggered neutrophil and monocyte infiltration into the peritoneal cavity is inhibited in mice pretreated with the SPHK inhibitor, as well as the cytokine levels found in serum and in the peritoneal lavage. Thus, our data supports a critical role for SPHK in anaphylatoxin-induced inflammatory responses.
Materials and Methods
All materials unless stated otherwise were bought from Sigma-Aldrich.
Male BALB/c mice (8–10 wk old) were obtained from the National University of Singapore, Sembawang Laboratory Animals Centre. All animal experiments conducted in this study were performed in accordance with Animal Experimentation Ethics Committee ethical guidelines.
Mice were anesthetized and a catheter was placed in the femoral vein. Mice were i.v. injected with DMS (150 μM in a final volume of 200 μl of PBS) or 200 μl of PBS control injection 10 min before C5a challenge. Mice were then given a bolus i.v. dose of recombinant human C5a (2 μg/kg in a final volume of 200 μl), and blood samples were collected into heparinized microfuge tubes at regular intervals over a 2-h observation period. Polymorphonuclear neutrophils (PMNs) were isolated and counted as previously reported (11) and expressed as a percentage of the PMN concentration before C5a challenge. Additionally, whole blood was collected and allowed to clot spontaneously on ice, and serum samples were used for cytokine measurements. Five mice were used for each group per experiment, and the experiments were conducted three times.
Peritoneal inflammatory reaction
Acute inflammation in the peritoneal cavity was induced by an i.p. injection of recombinant human C5a (2 μg/kg in a final volume of 200 μl). The SPHK inhibitor DMS (150 μM/200 μl of PBS) was i.v. injected 10 min before C5a injection. Control mice were first i.v. injected with 200 μl of PBS; then after 10 min, 200 μl of PBS were i.p. injected. At the indicated times, mice were sacrificed, and their peritoneal cavity was washed with 2 ml of ice-cold PBS, 0.1% BSA. The recovered peritoneal lavage fluid was analyzed for different cell infiltrates and the level of cytokines was measured. Five mice were used for each group per experiment, and the experiments were conducted three times.
For permeability analysis, the Evans blue dye, 6.25 mg/ml in 200 μl of PBS with or without the abovementioned amount of DMS, was i.v. administered 10 min before the C5a or PBS i.p. administration. At the indicated times, mice were sacrificed, and their peritoneal cavity was washed with 2 ml of ice-cold PBS, 0.1% BSA. The cells were spun down and the OD of the supernatant at 620 nm was measured as an indicator of Evans blue leakage into the peritoneal cavity. Five mice were used for each group per experiment, and the experiments were conducted three times.
Cytokine detection from serum and peritoneal lavage samples
Levels of TNF-α and IL-6 from serum and peritoneal lavage fluid were analyzed using ELISA kits (R&D Systems) following the manufacturer’s instructions. Five mice were used for each group per experiment, and the experiments were conducted three times.
Differences in responses in the various groups of mice were tested for significance by the unpaired Student’s t test. After determining that responses of individual groups of mice of specific challenge protocol did not differ significantly in replicate experiments, the results were pooled for statistical analyses and for presentation.
Agents that trigger acute inflammation or endotoxic shock induce neutropenia. Anaphylatoxins, such as C5a, can induce neutropenia in animal models (30). We have recently demonstrated that in human neutrophils and macrophages the intracellular signaling molecule SPHK plays a key role in the inflammatory responses triggered by C5a (28, 29). Here we show that administration of a bolus i.v. dose of recombinant human C5a in mice resulted in the rapid decrease of circulating PMNs, dropping to 20 ± 5%, of the levels observed in the unstimulated/controls by 5 min after the C5a i.v., then returning to normal/control values by 1 h (Fig. 1,A). However, in mice pretreated with DMS 10 min before C5a administration, the PMN levels were similar to that of the unstimulated controls (Fig. 1,A). Fig. 1 B shows a dose-response curve for the DMS inhibition of the C5a-triggered neutropenia.
TNF-α and IL-6 serum levels
TNF-α is one of the most important proinflammatory cytokines and a key mediator of inflammatory responses well known to be released by a wide variety of stimuli. Here we show that administration of a bolus i.v. dose of recombinant human C5a in mice resulted in the elevation of TNF-α in the serum, reaching a peak of 35 ± 5 ng/ml at 1 h (Fig. 2). However, in mice pretreated with DMS 10 min before C5a administration no significant elevation of TNF-α levels was observed (Fig. 2,A). IL-6 is also a key proinflammatory cytokine, which is released early in an inflammatory response. Here we show that administration of a bolus i.v. dose of recombinant human C5a in mice resulted in the elevation of IL-6 in the serum, reaching a peak of 115 ± 5 ng/ml at 2 h (Fig. 2,B). However, in mice pretreated with DMS 10 min before C5a administration no significant elevation of IL-6 levels was observed (Fig. 2 B).
C5a-mediated neutrophil and monocyte infiltration into the peritoneal cavity
It has previously been shown that C5a plays a key role in leukocyte infiltration and activation in the peritoneal Arthus reaction, triggered by immunocomplexes or by bacterial LPSs (1, 9, 11, 12, 13). These events have been shown to be inhibited by C5a antagonists or in mice lacking the C5a receptor (11, 12, 13). We have previously demonstrated that, in the C5a-triggered chemotactic responses in human neutrophils and macrophages, SPHK plays a key role (28, 29). In this study, we investigated the role of SPHK in the recruitment and activation of acute inflammatory leukocytes in vivo in a C5a-induced peritonitis model. Injection of recombinant hC5a into the peritoneal cavity caused a rapid influx of neutrophils into the peritoneal cavity, reaching a peak at 2 h, but then dropping rapidly by 6 h (Fig. 3,A); this was later followed by monocyte infiltration into the cavity, observed only after 6 h, and continuing to increase by 12 h (Fig. 3,B). However, in mice pretreated with the SPHK inhibitor DMS, there was a significant reduction of neutrophil and monocyte infiltration at all time points (Fig. 3).
Vascular permeability triggered by C5a
Alterations in vascular permeability were determined by i.v. injection of Evans blue dye and quantifying the amount of Evans blue in the peritoneal lavage. The Evans blue dye binds to serum proteins and thus can be used to quantify alterations in vascular permeability. Injection of recombinant hC5a into the peritoneal cavity caused a steady influx of Evans blue into the peritoneal cavity with a continued increase from 2 to 12 h (Fig. 4). However, in mice pretreated with the SPHK inhibitor DMS, there was no significant increase of Evans blue influx into the peritoneal cavity (Fig. 4).
Peritoneal TNF-α and IL-6 levels
We have recently shown that SPHK plays a key role in inflammatory relevant events in human neutrophils and macrophages, including the generation of cytokines triggered by C5a stimulation (28, 29). Thus, we compared the release of TNF-α and IL-6 in our C5a-induced peritonitis model in mice pretreated or not with DMS. Following C5a injection (i.p.), there was a substantial increase in the TNF-α that at 6 h reached 8 ± 0.5 ng/ml (Fig. 5,A) in the peritoneal lavage fluid. However, in mice pretreated with DMS, the C5a-triggered TNF-α levels were substantially reduced (1 ± 0.05 ng/ml) (Fig. 5,A) in the peritoneal lavage fluid. The C5a-triggered increase in TNF-α levels measured in the peritoneal lavage fluid returned to unstimulated/control values by 12 h of the C5a-induced peritonitis (Fig. 5,A). C5a triggered a substantial increase in the IL-6 levels in the peritoneal lavage. At 6 h, the IL-6 levels reached 18 ± 2 ng/ml; however, in mice pretreated with the SPHK inhibitor, the C5a-triggered IL-6 levels were similar to levels observed in the unstimulated control (1.5 ± 0.5 ng/ml) (Fig. 5 B).
In this study, we have attempted to elucidate some of the molecular mechanisms used by the anaphylatoxin C5a, during the inflammatory response. This is an important area of research, because C5a has been shown to be linked to a wide range of pathologies. It is well known that in inflammatory and autoimmune diseases, such as rheumatoid arthritis, as well as in endotoxic shock triggered by bacteria-derived products, there is activation of the serum complement system and a substantial elevation of circulating C5a (1, 2, 3, 4). Elevated levels of C5a trigger a variety of physiological responses including the up-regulation of cell adhesion molecules on endothelial cells, as well as on neutrophils (1, 4), resulting in the rapid adhesion of neutrophils to the vascular endothelium, and the rapid decrease in levels of circulating neutrophils (1, 2, 4).
We have recently reported that, on human neutrophils and macrophages, C5a signaling uses the intracellular phospholipid-modifying enzyme, SPHK, to trigger various physiological responses including calcium signals, degranulation, NADPH-oxidative burst, cytokine production, and chemotaxis (24, 25, 26, 27, 28, 29). Recently, we showed that the SPHK inhibitor, DMS, blocks the C5a-triggered responses in neutrophils and monocytes (28, 29). However, very little is known about the role of these intracellular signaling molecules triggered by C5a in vivo. Here we show, for the first time, that pretreatment of mice with DMS significantly inhibited the C5a-triggered inflammatory responses in vivo. Our data shows that i.v. administration of C5a triggers a rapid neutropenic response, but pretreating mice with DMS 10 min before the C5a i.v. administration substantially inhibited the C5a-triggered neutropenia.
C5a can trigger proinflammatory cytokines and chemokines, such as TNF-α, IL-6, and IL8, production (5, 6, 7, 8). These molecules share many activities, including the ability to induce fever and shock syndrome in animal models (31). In our previous cell-based studies, we showed that C5a triggered the generation of TNF-α, IL-6, and IL-8 to different levels in a SPHK-dependent manner (29). However, this effect has not yet been described in vivo. Here we show that the i.v. administration of C5a caused a rapid increase in the serum levels of TNF-α and IL-6 and that this increase in cytokine levels was blocked by the SPHK inhibitor, DMS. Thus, taking these observations together suggests a pivotal role for SPHK in C5a-triggered neutropenia and the systemic release of TNF-α and IL-6.
Where local inflammation is triggered by infection, trauma, or immunocomplex deposition, C5a is likely to be an important chemotactic peptide. C5a has been shown to trigger chemotaxis in cell suspensions (32), and C5a-triggered cell migration has been used as a sensitive test for measuring the activation of the cell’s internal motile apparatus (33). Moreover, these local inflammatory reactions have previously been shown to be C5a dependent in a mice model in which the C5a receptor has been genetically deleted where the inflammatory responses triggered by immunocomplexes were severely reduced when compared with control mice (34). Similarly, utilization of C5a receptor antagonists inhibited a wide range of proinflammatory events triggered by immunocomplexes in animal models (30, 35). Thus, we decided to investigate the potential role played by SPHK in the inflammatory response triggered by C5a, for this we triggered peritonitis in mice.
The peritoneal Arthus reaction is characterized by acute inflammation that involves the migration of PMN, vascular leakage, and cytokine production in the peritoneal cavity. We report here that the C5a i.p. administration triggered an inflammatory response that was inhibited by the SPHK inhibitor DMS. We observed that the C5a i.p. injection triggered a fast recruitment of neutrophils, later followed by monocytes, into the peritoneal cavity. Vascular permeability was also observed: when we i.v. injected Evans blue before C5a i.p. injection, we could observe a continued influx of the dye into the peritoneum. However, in mice pretreated with DMS, there was a significant reduction in the C5a-triggered neutrophil and monocyte infiltration, as well as a marked reduction in the Evans blue influx. We also show here that the i.p. administration of C5a caused a rapid increase in TNF-α and IL-6 levels in the peritoneal cavity and that this increase in cytokine levels was substantially inhibited in mice pretreated with DMS.
It is well established that phagocytic cell infiltration and proinflammatory cytokine production are universal components of a wide range of diseases, including immunocomplex-mediated conditions such as nephritis (36), arthritis (37), and acute graft rejection (38). Thus, agents that can inhibit phagocyte infiltration and/or the production of cytokines, such as TNF-α and IL-6, may have wide therapeutic applications in the prevention and treatment of these and other diseases. The present study indicates that the SPHK inhibitor, DMS, very effectively blocked the cytokine production and chemotactic responses triggered by the anaphylatoxin C5a in vivo. These observations suggest a potential role for SPHK in the C5a-triggered proinflammatory responses in vivo. However, it is possible that DMS has an effect not only on the C5a receptor-mediated signaling but also on other receptors that may be stimulated as secondary events following C5a-triggered responses.
The results presented here are relative to changes in mice during i.v. or i.p. injection of C5a with respect to mice that have been injected with saline alone. Whether the observed changes in the physiological responses triggered by C5a under these experimental conditions are representative of a pathological state is not currently known. However, these observations into the molecular basis of the inflammatory response are likely to improve our knowledge on the mechanisms by which C5a may contribute to the overall activation of the immune response; thus, having potential clinical implications for improving not only acute inflammatory conditions but also other inflammatory diseases where anaphylatoxins may play a role.
We thank A.-K. Fraser-Andrews for proofreading the manuscript.
The authors have no financial conflict of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work was supported by a grant from the NMRC (R-185-000-052-213).
Abbreviations used in this paper: SPP, sphingosine-1-phosphate; DMS, N,N-dimethylsphingosine; SPHK, sphingosine kinase; PMN, polymorphonuclear neutrophil.