ABSTRACT
Using peptides that represent linear regions of the powerful complement activation product, C5a, or loops that connect the four alpha helices of C5a, we have defined the ability of these peptides to reduce binding of (125)I-C5a to human neutrophils, inhibit chemotactic responses of neutrophils to C5a, and reduce H(2)O(2) 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.
Subject(s)
Antigens, CD/chemistry , Antigens, CD/physiology , Complement C5a/chemistry , Complement C5a/physiology , Receptors, Complement/chemistry , Receptors, Complement/physiology , Amino Acid Sequence , Antigens, CD/metabolism , Binding, Competitive/immunology , Cell Migration Inhibition , Chemotaxis, Leukocyte , Complement C5a/antagonists & inhibitors , Complement C5a/metabolism , Dose-Response Relationship, Immunologic , Humans , Hydrogen Peroxide/antagonists & inhibitors , Hydrogen Peroxide/metabolism , Iodine Radioisotopes/metabolism , Molecular Sequence Data , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/drug effects , Neutrophils/immunology , Neutrophils/metabolism , Peptide Fragments/antagonists & inhibitors , Peptide Fragments/chemical synthesis , Peptide Fragments/metabolism , Peptide Fragments/physiology , Receptor, Anaphylatoxin C5a , Receptors, Complement/metabolism , Structure-Activity Relationship , Tetradecanoylphorbol Acetate/antagonists & inhibitors , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
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.
Subject(s)
Immunity, Innate/drug effects , Oligopeptides/pharmacology , Receptors, Complement/antagonists & inhibitors , Sepsis/prevention & control , Animals , Antigens, CD , Chemotaxis, Leukocyte/drug effects , Complement C5a/metabolism , Complement C5a/pharmacology , Dose-Response Relationship, Drug , Inflammation/immunology , Inflammation/prevention & control , Lung Diseases/immunology , Lung Diseases/prevention & control , Male , Mice , Neutrophils/cytology , Neutrophils/metabolism , Oligopeptides/blood , Oligopeptides/chemical synthesis , Oxygen Consumption/drug effects , Peritoneal Cavity/cytology , Protein Binding/drug effects , Receptor, Anaphylatoxin C5a , Sepsis/immunology , Sepsis/mortality , Survival RateABSTRACT
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 p47(phox) 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 p47(phox) 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.
