| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 19, 2001 as doi:10.1096/fj.00-0653fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
* Department of Pathology, and
Department of Emergency Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA
2Correspondence: Department of Pathology, The University of Michigan Medical School, 1301 Catherine Road, Ann Arbor, MI 48109-0602, USA. E-mail: pward{at}umich.edu
SPECIFIC AIMS
Our recent studies of experimental sepsis, induced by cecal ligation and puncture (CLP) in rats, showed that anti-C5a antibody protects against the lethal effects of sepsis. The main aim of the current study was to obtain maximally protective antibodies to rat C5a that would protect in the CLP model and to determine to what extent delayed treatment with anti-C5a would still afford protection against sepsis-induced lethality.
PRINCIPAL FINDINGS
1. Antibodies to different peptide regions of rat C5a have
differential inhibitory activities on the functional (chemotactic)
activity of rat C5a
Rabbit polyclonal antibodies were developed to the following
peptide regions of rat C5a: amino-terminal region (A), residues 1–16;
middle region (M), residues 17–36; and the carboxyl-terminal region
(C), residues 58–77 (Fig. 1
). With the use of rat neutrophils, the chemotactic activity of rat C5a
was significantly inhibited by antibodies with the following descending
rank order: anti-C > anti-M >> anti-A. The antibodies by themselves
did not evoke a significant response in rat neutrophils.
|
2. The anti-C5a antibodies do not inhibit hemolytic activity of rat
serum
To assess to what extent anti-C5a preparations (anti-C5a M or C)
might interfere with the hemolytic activity of rat serum (implying a
blocking effect on the parent molecule, C5) and assembly of C5b-9
(membrane attack complex), whole hemolytic activity was measured in
presence or absence of these antibodies. There were no significant
differences in hemolysis of sensitized sheep erythrocytes in the
presence of fresh rat serum containing preimmune IgG or anti-C5a M or C
antibodies, which suggests that these protective antibodies do not
block activation of the complement system.
3. Differential protective effects of C5a antibodies during
experimental sepsis
Our original studies of experimental sepsis (cecal ligation and
puncture in rats) showed that anti-C5a antibodies could protect against
the lethal effects of sepsis (Czermak et al. Nature Medicine
5, 788–92, 1999). To determine the targets on rat C5a that
are most effective for blockade, antibodies against A, M, and C
peptides of rat C5a (Fig. 1)
or preimmune IgG were administered
immediately after the CLP procedure. The relative protective efficacies
(10-day survival rates of animals) of anti-C5a preparations (in
descending order of efficacy) were anti-C > anti-M >> anti-A.
Dose-dependent protective effects against the lethal complications of
sepsis in CLP animals was found when antibodies to the M or C regions
was used.
4. Anti-C5a M or C antibodies confer protective effects during
sepsis even after onset of symptoms of sepsis
To determine if delayed infusion of antibodies could still confer
protective effects in vivo after CLP, 600 µg of the most
protective antibodies (anti-C5a M or C antibodies) were intravenously
infused 6, 12, or 24 h after onset of CLP. When infused
immediately after CLP (0 h, Fig. 2A
), anti-C5a M or C-treated groups exhibited highly
protective effects (leading to 80% survival). When infusion was
delayed until 6 h after CLP (Fig. 2B
), a time when
clinical symptoms of sepsis were evident, both antibodies still
conferred significant protection. When infused 12 h after CLP, the
majority of animals exhibited typical clinical signs of sepsis:
decreased physical activity, piloerection, cessation of grooming
behavior, glazed eyes with crusting exudates, tachypnea, and reduced
urinary output. Under these conditions, infusion of anti-C5a M and
anti-C5a C still significantly improved survival at 10 days (37% and
46%, respectively) as compared with the preimmune IgG-treated group
(12%) (Fig. 2C
). The protective effects of the anti-C5a M
and C peptide antibodies were lost, if infusion was delayed until
24 h after CLP (data not shown). Although the anti-C5a protective
effects were diminished when delayed until late symptoms of sepsis,
these data suggest that delayed infusion of anti-C5a, even in the
presence of overt clinical signs of sepsis, confers protective effects.
|
CONCLUSIONS
Abundant evidence suggests that homeostatic control of the
inflammatory system has been lost during sepsis, as is reflected by
cytokine and chemokine appearance and unremitting data of complement
activation. In experimental sepsis induced by CLP, we have recently
demonstrated in rats that extensive complement activation occurs, which
leads to C5a deposition on blood neutrophils and a loss of the
respiratory burst in these cells and results in defective
oxygen-dependent bacterial killing and a high mortality rate. A rabbit
polyclonal antibody to the middle peptide region (M) of rat C5a caused
protective effects during sepsis. To establish the most protective
anti-C5a antibody preparation as well as the dose of anti-C5a that
provides optimal protection against the lethal effects of sepsis, we
raised polyclonal antibodies against different peptide regions (A,M,C)
of the rat C5a molecule (Fig. 1)
. IgG antibodies targeting the
amino-terminal residues 1–16 (A region of C5a) did not prevent the
lethal consequences of experimental sepsis, whereas antibodies against
the middle core residues 17–36 of the M region or against the
carboxyl-terminal residues 58–77 (C region) greatly improved survival
of CLP-rats in a dose dependent manner. Although the amino-terminal
region of C5a contributes to the full potency of the intact C5a
molecule and serves to stabilize a biologically favorable C5a
conformation, blocking antibody to the amino terminal region (residues
1–16) was not an effective target, as defined by lack of protective
effects in CLP-induced sepsis.
In the present study, antibodies against different peptide regions of C5a suppressed the chemotactic activity of rat neutrophils to rat C5a in a descending order of potency: CMA, which mirrors the protective in vivo effects of these antibodies. Because both recruitment and activation of neutrophils during sepsis are essential for local (e.g., peritoneal) remote organ (e.g., lung, liver, kidney) injury, effective blockade of anti-C5a antibodies preserves vital protective functional responses of neutrophils, especially the ability of these cells to activate NADPH oxidase, result in less organ injury and improved survival.
We have shown previously that injection of anti-C5a immediately after
sepsis-induction provides protection against its detrimental effects.
However, the present data suggest that even after a considerable delay
(6–12 h) in administering antibodies, which were targeted against the
M or C terminal regions of C5a, there was still a significant
improvement of survival (Fig. 2
and Fig. 3
). In the CLP model, relatively rapid development of polymicrobial
sepsis occurs within the first 12 h resulting in lethal
complications. The early events are characterized by a hyperdynamic
phase (2–10 h after CLP), followed by a hypodynamic phase (after
16 h). The hyperdynamic phase of sepsis is compressed in the CLP
model in rats into a rather brief period (16 h) relative to that
observed in humans, which often appears to extend over a three-day
period. The data in the rat model suggest that anti-C5a intervention is
effective during the hyperdynamic phase, although the protective
effects diminish with time (Fig. 2)
. These data suggest that, in humans
with sepsis, there may be a ‘window’ of time of several days during
which such an intervention may be useful.
|
FOOTNOTES
1 To read the full text of this article, go to
http://www.fasebj.org/cgi/doi/10.1096/fj.00-0653fje ; to cite
this article, use FASEB J. (January 19, 2001)
10.1096/fj.00-0653fje 
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]
|
|
|
|
 |
|
 D. Rittirsch, M. A. Flierl, D. E. Day, B. A. Nadeau, S. R. McGuire, L. M. Hoesel, K. Ipaktchi, F. S. Zetoune, J. V. Sarma, L. Leng, et al. Acute Lung Injury Induced by Lipopolysaccharide Is Independent of Complement Activation J. Immunol., June 1, 2008; 180(11): 7664 - 7672. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. N. Patel, J. Berghout, F. E. Lovegrove, K. Ayi, A. Conroy, L. Serghides, G. Min-oo, D. C. Gowda, J. V. Sarma, D. Rittirsch, et al. C5 deficiency and C5a or C5aR blockade protects against cerebral malaria J. Exp. Med., May 12, 2008; 205(5): 1133 - 1143. [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]
|
|
|
|
|
|
R.-F. Guo, L. Sun, H. Gao, K. X. Shi, D. Rittirsch, V. J. Sarma, F. S. Zetoune, and P. A. Ward In vivo regulation of neutrophil apoptosis by C5a during sepsis J. Leukoc. Biol., December 1, 2006; 80(6): 1575 - 1583. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R.-F. Guo, N. C. Riedemann, L. Sun, H. Gao, K. X. Shi, J. S. Reuben, V. J. Sarma, F. S. Zetoune, and P. A. Ward Divergent Signaling Pathways in Phagocytic Cells during Sepsis J. Immunol., July 15, 2006; 177(2): 1306 - 1313. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Nozaki, B. J. Raisler, E. Sakurai, J. V. Sarma, S. R. Barnum, J. D. Lambris, Y. Chen, K. Zhang, B. K. Ambati, J. Z. Baffi, et al. Drusen complement components C3a and C5a promote choroidal neovascularization PNAS, February 14, 2006; 103(7): 2328 - 2333. [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]
|
|
|
|
 |
|
 S. M. Waters, R. M. Brodbeck, J. Steflik, J. Yu, C. Baltazar, A. E. Peck, D. Severance, L. Y. Zhang, K. Currie, B. L. Chenard, et al. Molecular Characterization of the Gerbil C5a Receptor and Identification of a Transmembrane Domain V Amino Acid That Is Crucial for Small Molecule Antagonist Interaction J. Biol. Chem., December 9, 2005; 280(49): 40617 - 40623. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Huber-Lang, J. V. Sarma, D. Rittirsch, H. Schreiber, M. Weiss, M. Flierl, E. Younkin, M. Schneider, H. Suger-Wiedeck, F. Gebhard, et al. Changes in the Novel Orphan, C5a Receptor (C5L2), during Experimental Sepsis and Sepsis in Humans J. Immunol., January 15, 2005; 174(2): 1104 - 1110. [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]
|
|
|
|
 |
|
 R. S. Hotchkiss and I. E. Karl The Pathophysiology and Treatment of Sepsis N. Engl. J. Med., January 9, 2003; 348(2): 138 - 150. [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
H. Sumichika, K. Sakata, N. Sato, S. Takeshita, S. Ishibuchi, M. Nakamura, T. Kamahori, S. Ehara, K. Itoh, T. Ohtsuka, et al. Identification of a Potent and Orally Active Non-peptide C5a Receptor Antagonist J. Biol. Chem., December 13, 2002; 277(51): 49403 - 49407. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. S. HUBER-LANG, N. C. RIEDEMAN, J. V. SARMA, E. M. YOUNKIN, S. R. McGUIRE, I. J. LAUDES, K. T. LU, R.-F. GUO, T. A. NEFF, V. A. PADGAONKAR, et al. Protection of innate immunity by C5aR antagonist in septic mice FASEB J, October 1, 2002; 16(12): 1567 - 1574. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
