Vaishalee A. Padgaonkar

Role of C5a in Multiorgan Failure During Sepsis

In humans with sepsis, the onset of multiorgan failure (MOF), especially involving liver, lungs, and kidneys, is a well known complication that is associated with a high mortality rate. Our previous studies with the cecal ligation/puncture (CLP) model of sepsis in rats have revealed a C5a-induced defect in the respiratory burst of neutrophils. In the current CLP studies, MOF occurred during the first 48 h with development of liver dysfunction and pulmonary dysfunction (falling arterial partial pressure of O2, rising partial pressure of CO2). In this model an early respiratory alkalosis developed, followed by a metabolic acidosis with increased levels of blood lactate. During these events, blood neutrophils lost their chemotactic responsiveness both to C5a and to the bacterial chemotaxin, fMLP. Neutrophil dysfunction was associated with virtually complete loss in binding of C5a, but binding of fMLP remained normal. If CLP animals were treated with anti-C5a, indicators of MOF and lactate acidosis were greatly attenuated. Under the same conditions, C5a binding to blood neutrophils remained intact; in tandem, in vitro chemotactic responses to C5a and fMLP were retained. These data suggest that, in the CLP model of sepsis, treatment with anti-C5a prevents development of MOF and the accompanying onset of blood neutrophil dysfunction. This may explain the protective effects of anti-C5a in the CLP model of sepsis.

Complement-Induced Impairment of Innate Immunity During Sepsis

This study defines the molecular basis for defects in innate immunity involving neutrophils during cecal ligation/puncture (CLP)-induced sepsis in rats. Blood neutrophils from CLP rats demonstrated defective phagocytosis and defective assembly of NADPH oxidase, the latter being due to the inability of p47phox to translocate from the cytosol to the cell membrane of neutrophils after cell stimulation by phorbol ester (PMA). The appearance of these defects was prevented by in vivo blockade of C5a in CLP rats. In vitro exposure of neutrophils to C5a led to reduced surface expression of C5aR and defective assembly of NADPH oxidase, as defined by failure in phosphorylation of p47phox and its translocation to the cell membrane, together with failure in phosphorylation of p42/p44 mitogen-activated protein kinases. These data identify a molecular basis for defective innate immunity involving neutrophils during sepsis.

Protection of innate immunity by C5aR antagonist in septic mice

Innate immune functions are known to be compromised during sepsis, often with lethal consequences. There is also evidence in rats that sepsis is associated with excessive complement activation and generation of the potent anaphylatoxin C5a. In the presence of a cyclic peptide antagonist (C5aRa) to the C5a receptor (C5aR), the binding of murine 125I‐C5a to murine neutrophils was reduced, the in vitro chemotactic responses of mouse neutrophils to mouse C5a were markedly diminished, the acquired defect in hydrogen peroxide (H2O2) production of C5a‐exposed neutrophils was reversed, and the lung permeability index (extravascular leakage of albumin) in mice after intrapulmonary deposition of IgG immune complexes was markedly diminished. Mice that developed sepsis after cecal ligation/puncture (CLP) and were treated with C5aRa had greatly improved survival rates. These data suggest that C5aRa interferes with neutrophil responses to C5a, preventing C5a‐induced compromise of innate immunity during sepsis, with greatly improved survival rates after CLP.—Huber‐Lang, M. S., Riedeman, N. C., Sarma, J. V., Younkin, E. M., McGuire, S. R., Laudes, I. J., Lu, K. T., Guo, R.‐F., Neff, T. A., Padgaonkar, V. A., Lambris, J. D., Spruce, L., Mastellos, D., Zetoune, F. S., Ward, P. A. Protection of innate immunity by C5aR antagonist in septic mice. FASEB J. 16, 1567–1574 (2002)

Protective effects of anti-C5a in sepsis-induced thymocyte apoptosis

Multiorgan apoptosis occurs during sepsis. Following cecal ligation and puncture (CLP) in rats, thymocytes underwent apoptosis in a time-dependent manner. C5a blockade dramatically reduced thymocyte apoptosis as measured by thymic weight, binding of annexin V to thymocytes, and laddering of thymocyte DNA. When C5a was generated in vivo by infusion of purified cobra venom factor (CVF), thymocyte apoptosis was significantly increased. Similar results were found when CVF was injected in vivo during the early stages of CLP. In animals 12 hours after induction of CLP, there was an increase in the activities of caspase-3, -6, and -9, but not caspase-1 and -8. Cytosolic cytochrome c levels increased by twofold, whereas mitochondrial levels showed a 50% decrease. Western blot analysis revealed that the content of Bcl-X(L) (but not of Bcl-2, BAX, Bad, and Bim) significantly decreased in thymocytes after CLP. C5a blockade in the sepsis model almost completely inhibited caspase-3, -6, and -9 activation, significantly preserved cytochrome c in the mitochondrial fraction, and restored Bcl-X(L) expression. These data suggest that systemic activation of complement induces C5a-dependent apoptosis of thymocytes and that the blockade of C5a during sepsis rescues thymocytes from apoptosis.

Protective effects of anti-C5a peptide antibodies in experimental sepsis

We evaluated antibodies to different peptide regions of rat C5a in the sepsis model of cecal ligation and puncture (CLP) for their protective effects in rats. 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. With rat neutrophils, the chemotactic activity of rat C5a was significantly inhibited by antibodies with the following rank order: anti‐C > anti‐M ≫ anti‐A. In vivo, antibodies to the M and C (but not A) regions of C5a were protective in experimental sepsis, as determined by survival over a 10‐day period, in a dose‐dependent manner. The relative protective efficacies of anti‐C5a preparations (in descending order of efficacy) were anti‐C ≥ anti‐M ≫ anti‐A. In CLP rats, a delay in infusion of antibodies, which were injected at 6 or 12 h after CLP, still resulted in significant improvement in survival rates. These in vivo and in vitro data suggest that there are optimal targets on C5a for blockade during sepsis and that delayed infusion of anti‐C5a antibody until after onset of clinical evidence of sepsis still provides protective effects.

C5a receptor and thymocyte apoptosis in sepsis

In sepsis, apoptosis occurs in many different organs. The mediators responsible for induction of apoptosis are not clearly known, although there are some suggestions that C5a and the C5a receptor (C5aR) might be directly linked to apoptosis. In the cecal ligation/puncture (CLP) model of sepsis in rats, apoptosis occurs early in a variety of organs, especially in the thymus. We demonstrate that thymocytes from normal rats show specific, saturable, and high affinity binding of 125I‐labeled recombinant rat C5a. C5a binding to thymocytes was significantly increased 3 h after CLP and also when thymocytes from normal rats were first incubated in vitro with lipopolysaccharide (LPS) or IL‐6. The expression of C5aR mRNA in thymocytes was markedly increased 3, 6, and 12 h after CLP and increased similarly when normal thymocytes were first exposed to LPS or IL‐6 in vitro. Thymocytes obtained 2 or 3 h after CLP and exposed in vitro to C5a, but not normal thymocytes, underwent increased apoptosis, as demonstrated by annexin‐V binding, coinciding with increased activation of caspases 3, 6, and 8. These data provide the first direct evidence that in the early onset of sepsis, increased expression of C5aR occurs in thymocytes, which increases their susceptibility to C5a‐induced apoptosis.

Roles for C-X-C chemokines and C5a in lung injury after hindlimb ischemia-reperfusion

We evaluated the roles of the C-X-C chemokines cytokine-induced neutrophil chemoattractant (CINC) and macrophage inflammatory protein-2 (MIP-2) as well as the complement activation product C5a in development of lung injury after hindlimb ischemia-reperfusion in rats. During reperfusion, CD11b and CD18, but not CD11a, were upregulated on neutrophils [bronchoalveolar lavage (BAL) and blood] and lung macrophages. BAL levels of CINC and MIP-2 were increased during the ischemic and reperfusion periods. Treatment with either anti-CINC or anti-MIP-2 IgG significantly reduced lung vascular permeability and decreased lung myeloperoxidase content by 93 and 68%, respectively (P < 0.05). During the same period, there were significant increases in serum C5a-related neutrophil chemotactic activity. Treatment with anti-C5a decreased lung vascular permeability, lung myeloperoxidase, and BAL CINC by 51, 58, and 23%, respectively (P < 0.05). The data suggest that the C-X-C chemokines CINC and MIP-2 as well as the complement activation product C5a are required for lung neutrophil recruitment and full induction of lung injury after hindlimb ischemia-reperfusion in rats.We evaluated the roles of the C-X-C chemokines cytokine-induced neutrophil chemoattractant (CINC) and macrophage inflammatory protein-2 (MIP-2) as well as the complement activation product C5a in development of lung injury after hindlimb ischemia-reperfusion in rats. During reperfusion, CD11b and CD18, but not CD11a, were upregulated on neutrophils [bronchoalveolar lavage (BAL) and blood] and lung macrophages. BAL levels of CINC and MIP-2 were increased during the ischemic and reperfusion periods. Treatment with either anti-CINC or anti-MIP-2 IgG significantly reduced lung vascular permeability and decreased lung myeloperoxidase content by 93 and 68%, respectively ( P < 0.05). During the same period, there were significant increases in serum C5a-related neutrophil chemotactic activity. Treatment with anti-C5a decreased lung vascular permeability, lung myeloperoxidase, and BAL CINC by 51, 58, and 23%, respectively ( P < 0.05). The data suggest that the C-X-C chemokines CINC and MIP-2 as well as the complement activation product C5a are required for lung neutrophil recruitment and full induction of lung injury after hindlimb ischemia-reperfusion in rats.

Structure-Function Relationships of Human C5a and C5aR

Using peptides that represent linear regions of the powerful complement activation product, C5a, or loops that connect the four α helices of C5a, we have defined the ability of these peptides to reduce binding of 125I-C5a to human neutrophils, inhibit chemotactic responses of neutrophils to C5a, and reduce H2O2 production in neutrophils stimulated with PMA. The data have defined likely sites of interaction of C5a with C5aR. The peptides had no functional activity per se on neutrophils and did not interfere with neutrophil responses to the unrelated chemotactic peptide, N-formyl-Met-Leu-Phe. Although previous data have suggested that there are two separate sites on C5a reactive with C5aR, the current data suggest that C5a interacts with C5aR in a manner that engages three discontinuous regions of C5a.

Differing Patterns of P-Selectin Expression in Lung Injury

Using two models of acute lung inflammatory injury in rats (intrapulmonary deposition of immunoglobulin G immune complexes and systemic activation of complement after infusion of purified cobra venom factor), we have analyzed the requirements and patterns for upregulation of lung vascular P-selectin. In the immune complex model, upregulation of P-selectin was defined by Northern and Western blot analysis of lung homogenates, by immunostaining of lung tissue, and by vascular fixation of 125I-labeled anti-P-selectin. P-selectin protein was detected by 1 hour (long before detection of mRNA) and expression was sustained for the next 7 hours, in striking contrast to the pattern of P-selectin expression in the cobra venom factor model, in which upregulation was very transient (within the 1st hour). In the immune complex model, injury and neutrophil accumulation were P-selectin dependent. Upregulation of P-selectin was dependent on an intact complement system, and the presence of blood neutrophils was susceptible to the antioxidant dimethyl sulfoxide and required C5a but not tumor necrosis factor alpha. In contrast, in the cobra venom factor model, upregulation of P-selectin, which is C5a dependent, was also dimethyl sulfoxide sensitive but neutrophil independent. Different mechanisms that may explain why upregulation of lung vascular P-selectin is either transient or sustained are discussed.