HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 16/8/887 02-0033fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by RIEDEMANN, N. C. Articles by WARD, P. A. Search for Related Content PubMed PubMed Citation Articles by RIEDEMANN, N. C. Articles by WARD, P. A.

C5a receptor and thymocyte apoptosis in sepsis¹

Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, USA

²Correspondence: Department of Pathology, University of Michigan Medical School, 1301 Catherine Rd., Ann Arbor, MI 48109-0602, USA. E-mail: pward{at}umich.edu

SPECIFIC AIMS

Thymocyte- and lymphocyte-induced apoptosis during experimental sepsis is well documented and thought to be part of the immunosuppressive outcome of sepsis. Since we recently showed that blockade of C5a in rats with sepsis (after cecal ligation/puncture) greatly reduces thymocyte apoptosis but were unable to show direct induction of in vitro apoptosis in thymocytes by C5a, the aim of the current study was to investigate the presence and regulation of the C5a receptor (C5aR) on thymocytes during sepsis and the ability of C5a to directly induce apoptosis under conditions in which C5aR is up-regulated during sepsis.

PRINCIPAL FINDINGS

1. ¹²⁵I-labeled recombinant rat C5a (rrC5a) binds specifically to rat thymocytes
We used binding experiments with ¹²⁵I-rrC5a to detect C5aR in rat thymocytes from normal rats. Saturation was reached at 500 pM C5a. With increasing amounts of unlabeled rrC5a, binding of ¹²⁵I-rrC5a to thymocytes could be competed against. Between 10^-14 and 10^-11 M unlabeled rrC5a, the binding signal achieved with 200 pmol ¹²⁵I-rrC5a could be reduced by 50%. Therefore, binding of ¹²⁵I-rrC5a appeared to be highly specific and of high affinity.

2. Binding of ¹²⁵I-rrC5a to rat thymocytes from CLP rats and from rats treated with lipopolysaccharide (LPS) or interleukin 6 (IL-6) is increased
To determine whether enhanced binding of C5a occurred in rat thymocytes during the onset of sepsis, thymocytes were isolated 3 h after CLP and compared to normal thymocytes. Significantly increased binding of ¹²⁵I-rrC5a to CLP thymocytes was seen. To further investigate mediators possibly responsible for these results, thymocytes from normal rats were isolated and stimulated in vitro with LPS and IL-6 at various concentrations for 3 h at 37°C. Each mediator was capable of significantly increasing ¹²⁵I-rrC5a binding to rat thymocytes in a dose-dependent manner. At concentrations of 100 ng/ml, IL-6 and 500 ng/ml LPS, binding appeared to be maximally increased.

3. mRNA expression for C5aR is increased in rat thymocytes during sepsis and after stimulation in vitro with LPS and IL-6
To further confirm findings of the binding experiments, we conducted RT-PCR experiments to detect mRNA for C5aR in rat thymocytes. In vitro stimulation of rat thymocytes with LPS or IL-6 increased mRNA expression for C5aR in a dose-dependent manner (Fig. 1 A). At 3, 6, and 12 h after CLP, mRNA expression was clearly enhanced compared with that from normal thymocytes (Fig. 1B ). Therefore, enhanced binding of ¹²⁵I-labeled rrC5a during CLP-induced sepsis and after in vitro stimulation with LPS or IL-6 correlated with an increased expression of mRNA for C5aR.

View larger version (40K): [in this window] [in a new window]   Figure 1. Semiquantitative RT-PCR analysis for C5aR mRNA in rat thymocytes. Thymocytes from normal rats were stimulated with LPS and IL-6 in vitro at different concentrations (A). Induction of C5aR mRNA in vivo as early as 3 h and as late as 6 and 12 h after CLP (B). Equal loading conditions were demonstrated with bands for GAPDH-mRNA production (lower panels in both frames). Data are representative of results from 2 or 3 independent experiments.

4. C5a directly induces apoptosis in thymocytes from septic rats
In our recent study (mentioned above), we were not able to demonstrate that C5a directly induces apoptosis in normal thymocytes in vitro. Therefore, we conducted flow cytometry experiments where we stimulated thymocytes from CLP rats with C5a in vitro under conditions in which C5aR was up-regulated (as described above). Experiments showed the ability of rrC5a and human C5a to induce thymocyte apoptosis (as measured by annexin-V binding) in vitro in thymocytes isolated 2 and 3 h after induction of sepsis by CLP (Fig. 2 ). Figure 2A summarizes the increase of apoptosis achieved by in vitro stimulation with either human C5a or rrC5a in thymocytes 2 and 3 h after induction of CLP. Shown is the relative increase of apoptosis compared to the matching control for each experiment (no C5a addition). Figure 2B shows a typical flow cytometry experiment demonstrating direct induction of apoptosis by either human (100 nM) or rrC5a (30 nM) in thymocytes from rats 2 h after CLP. Such an effect could not be detected in thymocytes from normal rats (data not shown).

View larger version (54K): [in this window] [in a new window]   Figure 2. Flow cytometry analysis for apoptosis (annexin-V binding) in rat thymocytes. Rat thymocytes were isolated 2 and 3 h after CLP and not otherwise stimulated or incubated with huC5a (100 nM) or rrC5a (30 nM) for 30 min at 37°C. Flow cytometry was performed after staining with annexin-V for 15 min (A). Data are presented as increases in apoptosis relative to the corresponding control value in each experiment. Data are from 3–9 independent experiments per bar graph. Flow cytometric data from thymocytes obtained from rats 2 h after CLP and not otherwise stimulated or after exposure to hu C5a or rrC5a (30 min at 37°C) (B).

5. C5a-induced caspase-3, -6, and -8 activity in thymocytes from CLP rats
We conducted experiments in thymocytes obtained 3 h after CLP and measured caspase-3, -6, and -8 activity enzymatically after in vitro stimulation with human or rrC5a. Under such conditions, both mediators induced activities of the three caspases; 30 nM rrC5a appeared to be more potent than 100 nM human C5a. Under these conditions, rrC5a increased caspase-3, -6, and -8 activities between 27 and 40%.

CONCLUSIONS

Our findings suggest that C5aR is present in thymocytes from rats and up-regulated early after the onset of CLP. We demonstrated that such an increase in C5aR is accompanied by increased susceptibility of rat thymocytes to C5a-induced in vitro apoptosis. Such direct evidence for C5a-induced apoptosis in freshly isolated cells from septic animals has not yet been reported, and data showing increased C5aR expression in thymocytes during the onset of sepsis represent a new finding. C5a has been reported to have harmful effects during the onset of sepsis, but the role of C5aR expression in sepsis has not been closely studied. The evidence for a direct induction of apoptosis in thymocytes by C5a represents another step in the overall understanding of its potentially harmful effects during the onset of sepsis by suggesting a mechanism leading to rapid immunosuppression. Our findings suggest that suppressing the harmful effects of C5a during sepsis may be accomplished by interception of C5a or blockade of C5aR as a target for new drugs like antibodies or receptor antagonists.

View larger version (32K): [in this window] [in a new window]   Figure 3. Schematic diagram showing the increased C5aR expression during the onset of sepsis and the correlated susceptibility to C5a-induced apoptosis.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0033fje; to cite this article, use FASEB J. (April 23, 2002) 10.1096/fj.02-0033fje.

This article has been cited by other articles:

				M. A. Flierl, D. Rittirsch, B. A. Nadeau, D. E. Day, F. S. Zetoune, J. V. Sarma, M. S. Huber-Lang, and P. A. Ward Functions of the complement components C3 and C5 during sepsis FASEB J, October 1, 2008; 22(10): 3483 - 3490. [Abstract] [Full Text] [PDF]

				P. A. Ward Role of the complement in experimental sepsis J. Leukoc. Biol., March 1, 2008; 83(3): 467 - 470. [Abstract] [Full Text] [PDF]

				L. Bao, Y. Wang, A. Chang, A. W. Minto, J. Zhou, H. Kang, M. Haas, and R. J. Quigg Unrestricted C3 Activation Occurs in Crry-Deficient Kidneys and Rapidly Leads to Chronic Renal Failure J. Am. Soc. Nephrol., March 1, 2007; 18(3): 811 - 822. [Abstract] [Full Text] [PDF]

				A. D. Niederbichler, L. M. Hoesel, M. V. Westfall, H. Gao, K. R. Ipaktchi, L. Sun, F. S. Zetoune, G. L. Su, S. Arbabi, J. V. Sarma, et al. An essential role for complement C5a in the pathogenesis of septic cardiac dysfunction J. Exp. Med., January 23, 2006; 203(1): 53 - 61. [Abstract] [Full Text] [PDF]

				J. J. Alexander, A. Jacob, L. Bao, R. L. Macdonald, and R. J. Quigg Complement-Dependent Apoptosis and Inflammatory Gene Changes in Murine Lupus Cerebritis J. Immunol., December 15, 2005; 175(12): 8312 - 8319. [Abstract] [Full Text] [PDF]

				G. Sturfelt and L. Truedsson Complement and its breakdown products in SLE Rheumatology, October 1, 2005; 44(10): 1227 - 1232. [Abstract] [Full Text] [PDF]

				P. N. Rocha, T. J. Plumb, L. A. Robinson, R. Spurney, D. Pisetsky, B. H. Koller, and T. M. Coffman Role of Thromboxane A2 in the Induction of Apoptosis of Immature Thymocytes by Lipopolysaccharide Clin. Vaccine Immunol., August 1, 2005; 12(8): 896 - 903. [Abstract] [Full Text] [PDF]

				L. Bao, I. Osawe, M. Haas, and R. J. Quigg Signaling through Up-Regulated C3a Receptor Is Key to the Development of Experimental Lupus Nephritis J. Immunol., August 1, 2005; 175(3): 1947 - 1955. [Abstract] [Full Text] [PDF]

				M. Daveau, M. Benard, M. Scotte, M.-T. Schouft, M. Hiron, A. Francois, J.-P. Salier, and M. Fontaine Expression of a Functional C5a Receptor in Regenerating Hepatocytes and Its Involvement in a Proliferative Signaling Pathway in Rat J. Immunol., September 1, 2004; 173(5): 3418 - 3424. [Abstract] [Full Text] [PDF]

				N. C. Riedemann, R.-F. Guo, and P. A. Ward A key role of C5a/C5aR activation for the development of sepsis J. Leukoc. Biol., December 1, 2003; 74(6): 966 - 970. [Abstract] [Full Text]

				N. C. Riedemann, T. A. Neff, R.-F. Guo, K. D. Bernacki, I. J. Laudes, J. V. Sarma, J. D. Lambris, and P. A. Ward Protective Effects of IL-6 Blockade in Sepsis Are Linked to Reduced C5a Receptor Expression J. Immunol., January 1, 2003; 170(1): 503 - 507. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 16/8/887 02-0033fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by RIEDEMANN, N. C. Articles by WARD, P. A. Search for Related Content PubMed PubMed Citation Articles by RIEDEMANN, N. C. Articles by WARD, P. A.