Biomedical subjects

Stephanie R McGuire

Publications and source records attributed to Stephanie R McGuire.

6 recordsLinked to original sources

PubMed2006

Generation of C5a in the absence of C3: a new complement activation pathway.

Complement-mediated tissue injury in humans occurs upon deposition of immune complexes, such as in autoimmune diseases and acute respiratory distress syndrome. Acute lung inflammatory injury in wild-type and C3-/- mice after deposition of IgG immune complexes was of equivalent intensity and was C5a dependent, but injury was greatly attenuated in Hc-/- mice (Hc encodes C5). Injury in lungs of C3-/- mice and C5a levels in bronchoalveolar lavage (BAL) fluids from these mice were greatly reduced in the presence of antithrombin III (ATIII) or hirudin but were not reduced in similarly treated C3+/+ mice. Plasma from C3-/- mice contained threefold higher levels of thrombin activity compared to plasma from C3+/+ mice. There were higher levels of F2 mRNA (encoding prothrombin) as well as prothrombin and thrombin protein in liver of C3-/- mice compared to C3+/+ mice. A potent solid-phase C5 convertase was generated using plasma from either C3+/+ or C3-/- mice. Human C5 incubated with thrombin generated C5a that was biologically active. These data suggest that, in the genetic absence of C3, thrombin substitutes for the C3-dependent C5 convertase. This linkage between the complement and coagulation pathways may represent a new pathway of complement activation.

Animals↗

PubMed2003

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 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.

Amino Acid Sequence↗

PubMed2002

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 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.

Animals↗

PubMed2002

Activator protein-1 activation in acute lung injury.

The role of activator protein-1 (AP-1) in inflammation is primarily unknown. AP-1 was evaluated in nuclear extracts from alveolar macrophages and whole lung nuclear extracts during acute lung injury after deposition of IgG immune complexes. AP-1 activation occurred in macrophages and in whole lung extracts, but with distinctly different time courses. Low levels of constitutive AP-1 were observed in normal rat lung as determined by the electrophoretic mobility shift assay. Increased AP-1 was detected 2 hours after initiation of the inflammatory response in lung with a further increase by 4 hours, while AP-1 activation was found in alveolar macrophages 0.5 hour after onset of the inflammatory response. mRNAs and proteins for c-fos, c-jun, jun-B, and jun-D were all up-regulated in whole lung tissues and in alveolar macrophages during acute lung injury induced by IgG immune complex deposition. Depletion of lung macrophages sharply reduced AP-1 activation, as did anti-tumor necrosis factor-alpha, whereas complement depletion showed no effect on lung AP-1 activation. The data suggest that activation of AP-1 occurs in both alveolar macrophages and in the lung, and this activation process is macrophage- and tumor necrosis factor-alpha-dependent.

Acute Disease↗

PubMed2002

Generation of C5a by phagocytic cells.

The complement activation product, C5a, is a powerful phlogistic factor. Using antibodies to detect human or rat C5a, incubation at pH 7.4 of human blood neutrophils or rat alveolar macrophages (AMs) with C5 in the presence of phorbol 12-myristate 13-acetate (PMA) led to generation of C5a. Rat AMs activated with lipopolysaccharide also generated C5a from C5. With activated neutrophils, extensive cleavage of C5 occurred, whereas activated macrophages had much more selective proteolytic activity for C5. Peripheral blood human or rat mononuclear cells and rat alveolar epithelial cells when stimulated with phorbol ester all failed to demonstrate an ability to cleave C5, suggesting a specificity of C5 cleavage by phagocytic cells. With rat AMs, C5a generation was time-dependent and was blocked if AMs were pretreated with inhibitors of transcription or protein synthesis (actinomycin D or cycloheximide). Similar treatment of activated human polymorphonuclear leukocytes only partially reduced C5a generation after addition of C5. C5a generated by activated AMs was biologically (chemotactically) active. This generation was sensitive to serine protease inhibitors but not to other classes of inhibitors. These data indicate that phagocytic cells, especially lung macrophages, can generate C5a from C5. In the context of the lung, this may represent an important C5a-generating pathway that is independent of the plasma complement system.

Animals↗

PubMed2002

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.

Animals↗