Peritonitis, and Cytokine Production in Vivo Anaphylatoxin-Induced Neutropenia, a Critical Role for Sphingosine Kinase In

A Critical Role for Sphingosine Kinase in Anaphylatoxin-Induced Neutropenia, Peritonitis, and Cytokine Production in Vivo

This information is current as Liudmila Pietrovna Vlasenko and Alirio J. Melendez of September 23, 2021. J Immunol 2005; 174:6456-6461; ; doi: 10.4049/jimmunol.174.10.6456 http://www.jimmunol.org/content/174/10/6456 Downloaded from

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A Critical Role for Sphingosine Kinase in Anaphylatoxin-Induced Neutropenia, Peritonitis, and Cytokine Production in Vivo1

Liudmila Pietrovna Vlasenko and Alirio J. Melendez2

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-␣ 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: Downloaded from 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-␣ 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. The Journal of Immunology, 2005, 174: 6456–6461. http://www.jimmunol.org/

ctivation of the complement cascade plays a key role in ated with the use of such proteins to treat human patients. Immu- host defense. However, activation of the complement nogenicity is a problem, and proteins are expensive to A system leads to the generation of the potent proinflam- manufacture, very susceptible to degradation by proteases in serum matory anaphylatoxin, C5a. Significant amounts of C5a, as well as or the gastrointestinal track, and generally display poor pharma- other complement products in the blood, can lead to a series of ad- cokinetic properties. More recently attempts have been made to verse effects associated with a variety of pathologies, including septic make smaller molecules that are more stable, cheaper to make, shock, adult respiratory distress syndrome, and immune complex-de- have better bioavailability, and are more attractive as drug candi- pendent autoimmune diseases such as rheumatoid arthritis (1–3). dates for treating human diseases mediated by C5a (15, 16). How- by guest on September 23, 2021 Recently, the anaphylatoxin C5a has been shown to have an ever, very little is known about the intracellular signaling pathways immune-regulatory role able to stimulate mediators of both acute activated by C5a in immune-effector cells. and chronic inflammation (4–8). The significance of C5a in sev- During the last few years, it has become clear that sphingolipids, eral inflammatory diseases is demonstrated by the fact that agents in addition to being structural constituents of cell membranes, are that blocked the action of C5a also suppressed inflammation in sources of important signaling molecules. Particularly, the sphin- several animal models (9–13). golipid metabolites, ceramide and sphingosine-1-phosphate In primates, Escherichia coli-induced septic shock and adult re- (SPP),3 have emerged as a new class of potent bioactive mole- spiratory distress syndrome can be attenuated by blocking C5a cules, implicated in a variety of cellular processes such as cell with specific Abs (1, 4). Studies in rats showed that LPS-induced differentiation, apoptosis, and proliferation (17–20). Interest in septic shock can be mimicked by injection of C5a, and Ab block- SPP focused recently on two distinct cellular actions of this lipid: ing of C5a substantially reduces the LPS-induced shock (2). More- namely, its function as an extracellular ligand activating specific G over, blockade of C5a after infusion of LPS or induction of cecal protein-coupled receptors, and its role as an intracellular second ligation puncture in rats has been found to be protective (2, 12). messenger (21). Several findings enforced the notion of SPP as an Furthermore, C5a blockade after cecal ligation puncture in rats important intracellular second messenger. First, activation of var- attenuates the development of multiorgan failure (13). ious plasma membrane receptors, such as the platelet-derived Most of these studies used blocking Abs raised against C5a (10, growth factor receptor (22, 23), the Fc⑀RI and Fc␥RI Ag receptors 12) or recombinant proteins that are receptor antagonists or ana- (24–26), as well as the fMLP receptor (27), was found to rapidly logues of C5a (9, 14). However, there are many problems associ- increase intracellular SPP production through the stimulation of sphingosine kinase (SPHK). Second, inhibition of SPHK stimulation strongly reduced or even prevented cellular events triggered Department of Physiology, National University of Singapore, Singapore by these receptors, such as receptor-stimulated DNA synthesis, 2ϩ Received for publication November 5, 2004. Accepted for publication March 3, 2005. Ca mobilization and vesicular trafficking (22–27). The costs of publication of this article were defrayed in part by the payment of page Very recently, we have shown that in human neutrophils and charges. This article must therefore be hereby marked advertisement in accordance macrophages, C5a activates the intracellular signaling molecule with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by a grant from the NMRC (R-185-000-052-213). 2 Address correspondence and reprint requests to Dr. Alirio J. Melendez, Department of Physiology, 2 Medical Drive, MD9 #01-05, Singapore 117597. E-mail address: 3 Abbreviations used in this paper: SPP, sphingosine-1-phosphate; DMS, N,N-di- [email protected] methylsphingosine; SPHK, sphingosine kinase; PMN, polymorphonuclear neutrophil.

SPHK and that inhibition of SPHK activity, by N,N-dimethyl- differ significantly in replicate experiments, the results were pooled for sphingosine (DMS), in primary human neutrophils, neutrophil-dif- statistical analyses and for presentation. ferentiated HL-60 cells, as well as in monocyte-derived macrophages, largely inhibits C5a-stimulated Ca2ϩ mobilization, Results enzyme release, chemotaxis, and cytokine production, suggesting C5a-triggered neutropenia a potential role for SPHK in the C5a-triggered inflammatory re- Agents that trigger acute inflammation or endotoxic shock induce sponses (28, 29). neutropenia. Anaphylatoxins, such as C5a, can induce neutropenia Here we show for the first time that the C5a-triggered neutro- in animal models (30). We have recently demonstrated that in hu- penia and peritonitis can be attenuated by inhibition of SPHK. We man neutrophils and macrophages the intracellular signaling mol- show here that the C5a-triggered neutrophil and monocyte infil- ecule SPHK plays a key role in the inflammatory responses trig- tration into the peritoneal cavity is inhibited in mice pretreated gered by C5a (28, 29). Here we show that administration of a bolus with the SPHK inhibitor, as well as the cytokine levels found in i.v. dose of recombinant human C5a in mice resulted in the rapid serum and in the peritoneal lavage. Thus, our data supports a crit- decrease of circulating PMNs, dropping to 20 Ϯ 5%, of the levels ical role for SPHK in anaphylatoxin-induced inflammatory observed in the unstimulated/controls by 5 min after the C5a i.v., responses. then returning to normal/control values by 1 h (Fig. 1A). However, in mice pretreated with DMS 10 min before C5a administration, the PMN levels were similar to that of the unstimulated controls Materials and Methods

All materials unless stated otherwise were bought from Sigma-Aldrich. Downloaded from Mice Male BALB/c mice (8–10 wk old) were obtained from the National Uni- versity of Singapore, Sembawang Laboratory Animals Centre. All animal experiments conducted in this study were performed in accordance with Animal Experimentation Ethics Committee ethical guidelines. Neutropenia assay http://www.jimmunol.org/ Mice were anesthetized and a catheter was placed in the femoral vein. Mice were i.v. injected with DMS (150 ␮M in a ﬁnal volume of 200 ␮l of PBS) or 200 ␮l of PBS control injection 10 min before C5a challenge. Mice were then given a bolus i.v. dose of recombinant human C5a (2 ␮g/kg in a ﬁnal volume of 200 ␮l), and blood samples were collected into heparinized microfuge tubes at regular intervals over a 2-h observation period. Poly- morphonuclear neutrophils (PMNs) were isolated and counted as previously reported (11) and expressed as a percentage of the PMN concentra-

tion before C5a challenge. Additionally, whole blood was collected and by guest on September 23, 2021 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 ␮g/kg in a final volume of 200 ␮l). The SPHK inhibitor DMS (150 ␮M/200 ␮l of PBS) was i.v. injected 10 min before C5a injection. Control mice were first i.v. injected with 200 ␮lof PBS; then after 10 min, 200 ␮l 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. Permeability changes For permeability analysis, the Evans blue dye, 6.25 mg/ml in 200 ␮lof PBS with or without the abovementioned amount of DMS, was i.v. ad- ministered 10 min before the C5a or PBS i.p. administration. At the indi- FIGURE 1. C5a-induced neutropenia is inhibited by the SPHK inhibi- cated times, mice were sacrificed, and their peritoneal cavity was washed tor, DMS. A, Blood levels of neutrophils following the i.v. injection of C5a. with 2 ml of ice-cold PBS, 0.1% BSA. The cells were spun down and the Blood levels of neutrophils in mice pretreated with DMS for 10 min before OD of the supernatant at 620 nm was measured as an indicator of Evans the i.v. C5a injection (C5a ϩ DMS). Control, blood levels of neutrophils blue leakage into the peritoneal cavity. Five mice were used for each group following the i.v. injection of PBS. The blood was drawn at the times per experiment, and the experiments were conducted three times. indicated in the figure. Data shown as means Ϯ SD of three different .(p Ͻ 0.05 ,ء p Ͻ 0.01; and ,ءء) Cytokine detection from serum and peritoneal lavage samples experiments, and Student’s t test p values Five mice were used per treatment group per experiment. B, Dose response Levels of TNF-␣ and IL-6 from serum and peritoneal lavage fluid were analyzed using ELISA kits (R&D Systems) following the manufacturer’s for DMS in the inhibition of the neutropenia triggered by C5a. Blood levels instructions. Five mice were used for each group per experiment, and the of neutrophils following the i.v. injection of C5a. Blood levels of neutro- experiments were conducted three times. phils in mice pretreated with increasing concentrations of DMS for 10 min before the i.v. C5a injection (C5a ϩ 50 ␮M DMS), (C5a ϩ 100 ␮M DMS), Statistical analyses (C5a ϩ 150 ␮M DMS), (C5a ϩ 200 ␮M DMS). Control, blood levels of Differences in responses in the various groups of mice were tested for neutrophils following the i.v. injection of PBS. The blood was drawn at the significance by the unpaired Student’s t test. After determining that re- times indicated in the figure. Data shown as means Ϯ SD of three different sponses of individual groups of mice of specific challenge protocol did not experiments, 5 mice were used per treatment group per experiment. 6458 ROLE OF SPHK IN C5a-TRIGGERED NEUTROPENIA AND PERITONITIS

(Fig. 1A). Fig. 1B shows a dose-response curve for the DMS in- C5a-mediated neutrophil and monocyte inﬁltration into the hibition of the C5a-triggered neutropenia. peritoneal cavity

TNF-␣ and IL-6 serum levels It has previously been shown that C5a plays a key role in leukocyte infiltration and activation in the peritoneal Arthus reaction, trig- ␣ TNF- is one of the most important proinflammatory cytokines gered by immunocomplexes or by bacterial LPSs (1, 9, 11–13). and a key mediator of inflammatory responses well known to be These events have been shown to be inhibited by C5a antagonists released by a wide variety of stimuli. Here we show that admin- or in mice lacking the C5a receptor (11–13). We have previously istration of a bolus i.v. dose of recombinant human C5a in mice demonstrated that, in the C5a-triggered chemotactic responses in ␣ resulted in the elevation of TNF- in the serum, reaching a peak of human neutrophils and macrophages, SPHK plays a key role (28, Ϯ 35 5 ng/ml at 1 h (Fig. 2). However, in mice pretreated with 29). In this study, we investigated the role of SPHK in the recruit- DMS 10 min before C5a administration no significant elevation of ment and activation of acute inflammatory leukocytes in vivo in a ␣ TNF- levels was observed (Fig. 2A). IL-6 is also a key proin- C5a-induced peritonitis model. Injection of recombinant hC5a into flammatory cytokine, which is released early in an inflammatory the peritoneal cavity caused a rapid influx of neutrophils into the response. Here we show that administration of a bolus i.v. dose of peritoneal cavity, reaching a peak at 2 h, but then dropping rapidly recombinant human C5a in mice resulted in the elevation of IL-6 by 6 h (Fig. 3A); this was later followed by monocyte infiltration in the serum, reaching a peak of 115 Ϯ 5 ng/ml at 2 h (Fig. 2B). However, in mice pretreated with DMS 10 min before C5a administration no significant elevation of IL-6 levels was observed (Fig. 2B). Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 3. C5a-induced cellular infiltration into the peritoneal cavity is FIGURE 2. C5a-triggered increase in serum levels of TNF-␣ and IL-6 inhibited by the SPHK inhibitor, DMS. A, Neutrophil infiltration. Neutro- is inhibited by the SPHK inhibitor, DMS. A, Serum levels of TNF-␣ fol- phil numbers in the peritoneum following the i.p. injection of C5a. Neu- lowing the i.v. injection of C5a. Serum levels of TNF-␣ in mice pretreated trophil numbers in the peritoneum in mice pretreated with DMS for 10 min with DMS for 10 min before the i.v. C5a injection (C5a ϩ DMS). Control, before the i.p. C5a injection (C5a ϩ DMS). Control, neutrophil numbers in serum levels of TNF-␣ following the i.v. injection of PBS. The blood was the peritoneum following the i.p. injection of PBS. The lavage was per- drawn at the times indicated in the figure, and serum was extracted imme- formed at the times indicated in the figure. Data shown as means Ϯ SD of p Ͻ 0.01; and ,ءء) diately after. Data shown as means Ϯ SD of three different experiments, three different experiments, and Student’s t test p values ,p Ͻ 0.05). Five mice were used per treatment group per experiment. B ,ء p Ͻ 0.05). Five mice ,ء p Ͻ 0.01; and ,ءء) and Student’s t test p values were used per treatment group per experiment. B, Serum levels of IL-6 Monocyte infiltration. Monocyte numbers in the peritoneum following the following the i.v. injection of C5a. Serum levels of TNF-␣ in mice pre- i.p. injection of C5a. Monocyte numbers in the peritoneum in mice pretreated with DMS for 10 min before the i.v. C5a injection (C5a ϩ DMS). treated with DMS for 10 min before the i.p. C5a injection (C5a ϩ DMS). Control, serum levels of TNF-␣ following the i.v. injection of PBS. The Control, monocyte numbers in the peritoneum following the i.p. injection blood was drawn at the times indicated in the figure, and serum was ex- of PBS (PBS). The lavage was performed at the times indicated in the tracted immediately after. Data shown as means Ϯ SD of three different figure. Data shown as means Ϯ SD of three different experiments, and p Ͻ 0.05). Five mice were ,ء p Ͻ 0.01; and ,ءء) p Ͻ 0.05). Student’s t test p values ,ء p Ͻ 0.01; and ,ءء) experiments, and Student’s t test p values Five mice were used per treatment group per experiment. used per treatment group per experiment. The Journal of Immunology 6459

FIGURE 4. C5a-induced vascular permeability is inhibited by the SPHK inhibitor, DMS. Peritoneal lavage OD. Following the i.p. injection of C5a, the OD was measured at the indicated times (C5a). Peritoneal lavage OD in mice pretreated with DMS for 10 min before the i.p. C5a injection measured at the indicated times (C5a ϩ DMS). Control, peritoneal lavage OD following the i.p. injection of PBS measured at the indicated times, (PBS). Data shown as means Ϯ SD of three different exper- Downloaded from p Ͻ 0.05). Five ,ء p Ͻ 0.01; and ,ءء) iments, and Student’s t test p values mice were used per treatment group per experiment.

into the cavity, observed only after 6 h, and continuing to increase by 12 h (Fig. 3B). However, in mice pretreated with the SPHK http://www.jimmunol.org/ 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 FIGURE 5. C5a-induced elevated levels of cytokines in the peritoneal proteins and thus can be used to quantify alterations in vascular cavity is inhibited by the SPHK inhibitor, DMS. A, Peritoneal lavage permeability. Injection of recombinant hC5a into the peritoneal TNF-␣ levels measured following the i.p. injection of C5a at the indicated by guest on September 23, 2021 cavity caused a steady influx of Evans blue into the peritoneal times (C5a). Peritoneal lavage TNF-␣ levels in mice pretreated with DMS cavity with a continued increase from 2 to 12 h (Fig. 4). However, for 10 min before the i.p. C5a injection measured at the indicated times in mice pretreated with the SPHK inhibitor DMS, there was no (C5a ϩ DMS). Control, peritoneal lavage TNF-␣ levels following the i.p. significant increase of Evans blue influx into the peritoneal cavity injection of PBS measured at the indicated times (PBS). Data shown as Ϯ (Fig. 4). means SD of three different experiments, and Student’s t test p values p Ͻ 0.05). Five mice were used per treatment group ,ء p Ͻ 0.01; and ,ءء) Peritoneal TNF-␣ and IL-6 levels per experiment. B, Peritoneal lavage IL-6 levels measured following the i.p. injection of C5a at the indicated times (C5a). Peritoneal lavage IL-6 We have recently shown that SPHK plays a key role in inflam- levels in mice pretreated with DMS, for 10 min, before the i.p. C5a injec- matory relevant events in human neutrophils and macrophages, tion measured at the indicated times (C5a ϩ DMS). Control, peritoneal including the generation of cytokines triggered by C5a stimulation lavage IL-6 levels following the i.p. injection of PBS measured at the (28, 29). Thus, we compared the release of TNF-␣ and IL-6 in our indicated times (PBS). Data shown as means Ϯ SD of three different ex- .(p Ͻ 0.05 ,ء p Ͻ 0.01; and ,ءء) C5a-induced peritonitis model in mice pretreated or not with DMS. periments, and Student’s t test p values Following C5a injection (i.p.), there was a substantial increase in Five mice were used per treatment group per experiment. the TNF-␣ that at 6 h reached 8 Ϯ 0.5 ng/ml (Fig. 5A)inthe peritoneal lavage fluid. However, in mice pretreated with DMS, the C5a-triggered TNF-␣ levels were substantially reduced (1 Ϯ well known that in inflammatory and autoimmune diseases, such 0.05 ng/ml) (Fig. 5A) in the peritoneal lavage fluid. The C5a-trig- as rheumatoid arthritis, as well as in endotoxic shock triggered by gered increase in TNF-␣ levels measured in the peritoneal lavage bacteria-derived products, there is activation of the serum comple- fluid returned to unstimulated/control values by 12 h of the C5a- ment system and a substantial elevation of circulating C5a (1–4). induced peritonitis (Fig. 5A). C5a triggered a substantial increase Elevated levels of C5a trigger a variety of physiological responses in the IL-6 levels in the peritoneal lavage. At 6 h, the IL-6 levels including the up-regulation of cell adhesion molecules on endo- reached 18 Ϯ 2 ng/ml; however, in mice pretreated with the SPHK thelial cells, as well as on neutrophils (1, 4), resulting in the rapid inhibitor, the C5a-triggered IL-6 levels were similar to levels ob- adhesion of neutrophils to the vascular endothelium, and the rapid served in the unstimulated control (1.5 Ϯ 0.5 ng/ml) (Fig. 5B). decrease in levels of circulating neutrophils (1, 2, 4). We have recently reported that, on human neutrophils and mac- Discussion rophages, C5a signaling uses the intracellular phospholipid-mod- In this study, we have attempted to elucidate some of the molecular ifying enzyme, SPHK, to trigger various physiological responses mechanisms used by the anaphylatoxin C5a, during the inflamma- including calcium signals, degranulation, NADPH-oxidative burst, tory response. This is an important area of research, because C5a cytokine production, and chemotaxis (24–29). Recently, we has been shown to be linked to a wide range of pathologies. It is showed that the SPHK inhibitor, DMS, blocks the C5a-triggered 6460 ROLE OF SPHK IN C5a-TRIGGERED NEUTROPENIA AND PERITONITIS responses in neutrophils and monocytes (28, 29). However, very it is possible that DMS has an effect not only on the C5a receptor- little is known about the role of these intracellular signaling mol- mediated signaling but also on other receptors that may be stim- ecules triggered by C5a in vivo. Here we show, for the first time, ulated as secondary events following C5a-triggered responses. that pretreatment of mice with DMS significantly inhibited the The results presented here are relative to changes in mice during C5a-triggered inflammatory responses in vivo. Our data shows that i.v. or i.p. injection of C5a with respect to mice that have been i.v. administration of C5a triggers a rapid neutropenic response, injected with saline alone. Whether the observed changes in the but pretreating mice with DMS 10 min before the C5a i.v. admin- physiological responses triggered by C5a under these experimental istration substantially inhibited the C5a-triggered neutropenia. conditions are representative of a pathological state is not currently C5a can trigger proinflammatory cytokines and chemokines, known. However, these observations into the molecular basis of such as TNF-␣, IL-6, and IL8, production (5–8). These molecules the inflammatory response are likely to improve our knowledge on share many activities, including the ability to induce fever and the mechanisms by which C5a may contribute to the overall acti- shock syndrome in animal models (31). In our previous cell-based vation of the immune response; thus, having potential clinical im- studies, we showed that C5a triggered the generation of TNF-␣, plications for improving not only acute inflammatory conditions IL-6, and IL-8 to different levels in a SPHK-dependent manner but also other inflammatory diseases where anaphylatoxins may (29). However, this effect has not yet been described in vivo. Here play a role. we show that the i.v. administration of C5a caused a rapid increase in the serum levels of TNF-␣ and IL-6 and that this increase in Acknowledgments cytokine levels was blocked by the SPHK inhibitor, DMS. Thus, We thank A.-K. Fraser-Andrews for proofreading the manuscript. taking these observations together suggests a pivotal role for Downloaded from SPHK in C5a-triggered neutropenia and the systemic release of Disclosures TNF-␣ and IL-6. The authors have no financial conflict of interest. Where local inflammation is triggered by infection, trauma, or immunocomplex deposition, C5a is likely to be an important che- References motactic peptide. C5a has been shown to trigger chemotaxis in cell 1. Smedegard, G., L. X. Cui, and T. E. Hugli. 1989. Endotoxin-induced shock in the rat: a role for C5a. Am. J. Pathol. 135: 489–497. suspensions (32), and C5a-triggered cell migration has been used http://www.jimmunol.org/ 2. Stevens, J. H., P. O’Hanley, J. M. Shapiro, F.G. Mihm, P. S. Satoh, J. A. Collins, as a sensitive test for measuring the activation of the cell’s internal and T. A. Raffin. 1986. Effects of anti-C5a antibodies on the adult respiratory motile apparatus (33). Moreover, these local inflammatory reac- distress syndrome in septic primates. J. Clin. Invest. 77: 1812–1816. tions have previously been shown to be C5a dependent in a mice 3. Wang, Y., S. A. Rollins, J. A. Madri, and L. A. Matis. 1995. Anti-C5 monoclonal antibody therapy prevents collagen-induced arthritis and ameliorates established model in which the C5a receptor has been genetically deleted disease. Proc. Natl. Acad. Sci. USA 92: 8955–8959. where the inflammatory responses triggered by immunocomplexes 4. Hangen, D. H., J. H. Stevens, P. S. Satoh, E. W. Hall, P. T. O’Hanley, and T. A. Raffin. 1989. Complement levels in septic primates treated with anti-C5a were severely reduced when compared with control mice (34). antibodies. J. Surg. Res. 46: 195–197. Similarly, utilization of C5a receptor antagonists inhibited a wide 5. Okusawa, S., K. B. Yancey, J. W. van der Meer, S. Endres, G. Lonnemann, range of proinflammatory events triggered by immunocomplexes K. Hefter, M. M. Frank, J. F. Burke, C. A. Dinarello, and J. A. Gelfand. 1988.

C5a stimulates secretion of tumor necrosis factor from human mononuclear cells by guest on September 23, 2021 in animal models (30, 35). Thus, we decided to investigate the in vitro: comparison with secretion of interleukin 1␤ and interleukin 1␣. J. Exp. potential role played by SPHK in the inflammatory response trig- Med. 168: 443–448. gered by C5a, for this we triggered peritonitis in mice. 6. Arend, W. P., R. J. Massoni, M. A. Niemann, and P. C. Giclas. 1989. Absence of induction of IL-1 production in human monocytes by complement fragments. The peritoneal Arthus reaction is characterized by acute inflam- J. Immunol. 142: 173–178. mation that involves the migration of PMN, vascular leakage, and 7. Okusawa, S., C. A. Dinarello, K. B. Yancey, S. Endres, T. J. Lawley, M. M. Frank, J. F. Burke, and J. A. Gelfand. 1987. C5a induction of human cytokine production in the peritoneal cavity. We report here that interleukin 1. J. Immunol. 139: 2635–2640. the C5a i.p. administration triggered an inflammatory response that 8. Cavaillon, J.-M., C. Fitting, and N. Haeffner-Cavaillon. 1990. Recombinant C5a was inhibited by the SPHK inhibitor DMS. We observed that the enhances interleukin 1 and tumor necrosis factor release by lipopolysaccharide- stimulated monocytes and macrophages. Eur. J. Immunol. 20: 253–257. C5a i.p. injection triggered a fast recruitment of neutrophils, later 9. Pellas, T. C., W. Boyar, J. van Oostrum, J. Wasvary, L. R. Fryer, G. Pastor, followed by monocytes, into the peritoneal cavity. Vascular per- M. Sills, A. Braunwalder, D. R. Yarwood, R. Kramer, et al. 1998. Novel C5a meability was also observed: when we i.v. injected Evans blue antagonists regulate neutrophil functions in vitro and in vivo. J. Immunol. 160: 5616–5621. before C5a i.p. injection, we could observe a continued influx of 10. Mohr, M., U. Hopken, M. Oppermann, C. Mathes, K. Goldmann, S. Siever, the dye into the peritoneum. However, in mice pretreated with O. Gotze, and H. Burchardi. 1998. Effects of anti-C5a monoclonal antibodies on oxygen use in a porcine model of severe sepsis. Eur. J. Clin. Invest. 28: 227–234. DMS, there was a significant reduction in the C5a-triggered neu- 11. Short, A., A. K. Wong, A. M. Finch, G. Haaima, I. A. Shiels, D. P. Fairlie, and trophil and monocyte infiltration, as well as a marked reduction in S. M. Taylor. 1999. Effects of a new C5a receptor antagonist on C5a- and en- the Evans blue influx. We also show here that the i.p. administra- dotoxin-induced neutropenia in the rat. Br. J. Pharmacol. 126: 551–554. ␣ 12. Czermak, B. J., V. Sarma, C. L. Pierson, R. L. Warner, M. Huber-Lang, tion of C5a caused a rapid increase in TNF- and IL-6 levels in the N. M. Bless, H. Schmal, H. P. Friedl, and P. A. Ward. 1999. Protective effects of peritoneal cavity and that this increase in cytokine levels was sub- C5a blockade in sepsis. Nat. Med. 5: 788–792. stantially inhibited in mice pretreated with DMS. 13. Huber-Lang, M., V. J. Sarma, K. T. Lu, S. R. McGuire, V. A. Padgaonkar, R. F. Guo, E. M. Younkin, R. G. Kunkel, J. Ding, R. Erickson, et al. 2001. Role It is well established that phagocytic cell infiltration and proin- of C5a in multiorgan failure during sepsis. J. Immunol. 166: 1193–1199. flammatory cytokine production are universal components of a 14. Heller, T., M. Hennecke, U. Baumann, J. E. Gessner, A. M. zu Vilsendorf, M. Baensch, F. Boulay, F. Kola, A. Klos, W. Bautsch, and J. Kohl, 1999. Se- wide range of diseases, including immunocomplex-mediated con- lection of a C5a receptor antagonist from phage libraries attenuating the inflam- ditions such as nephritis (36), arthritis (37), and acute graft rejec- matory response in immune complex disease and ischemia/reperfusion injury. tion (38). Thus, agents that can inhibit phagocyte infiltration and/or J. Immunol. 163: 985–994. ␣ 15. Wong, A. K., A. M. Finch, G. K. Pierens, D. J. Craik, S. M. Taylor, and P. Fairlie. the production of cytokines, such as TNF- and IL-6, may have 1998. Small molecular probes for the G-protein-coupled C5a receptors: confor- wide therapeutic applications in the prevention and treatment of mationally constrained antagonists derived from the C terminus of the human these and other diseases. The present study indicates that the plasma protein C5a. J. Med. Chem. 41: 3417–3425. 16. Finch, A. M., A. K. Wong, N. J. Paczkowski, S. K. Wadi, D. J. Craik, SPHK inhibitor, DMS, very effectively blocked the cytokine pro- D. P. Fairlie, and S. M. Taylor. 1999. Low-molecular-weight peptidic and cyclic duction and chemotactic responses triggered by the anaphylatoxin antagonists of the receptor for the complement factor C5a. J. Med. Chem. 42: 1965–1974. C5a in vivo. These observations suggest a potential role for SPHK 17. Hannun, Y. A. 1994. The sphingomyelin cycle and the second messenger func- in the C5a-triggered proinflammatory responses in vivo. However, tions of ceramide. J. Biol. Chem. 269: 3125–3128. The Journal of Immunology 6461

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