Julia A. Ember

Complement factors and their receptors.

In summary, recent advances in molecular cloning of anaphylatoxins and the anaphylatoxin receptors add new dimensions to our investigations and understanding of the molecular mechanisms involved in anaphylatoxin action. Combining knowledge accumulated from peptide modeling of the ligands with mutagenesis studies of these ligands and their receptors makes it possible to more accurately model interactive sites and understand the sequence of molecular interactions required for cellular activation. In addition, these new developments provide valuable tools for investigating, yet unknown, activities and cellular targets of the anaphylatoxin molecules.

Neuronal Expression of a Functional Receptor for the C5a Complement Activation Fragment

The present studies were undertaken to determine whether neuronal subsets in normal brains constitutively express functionally competent C5a receptors. In situ hybridization studies coupled with immunohistochemical approaches revealed that most neurons in the hippocampal formation, many pyramidal cortical neurons, and cerebellar Purkinje neurons in normal human and murine brains constitutively express C5a receptors. Neuronal C5a receptors bound C5a-coated fluorescent microspheres, and primary rodent hippocampal neurons responded to C5a with increased calcium fluxes via a pertussis-sensitive, presumably Gi-coupled protein. Additional studies with human neuroblastoma cells conducted to address the functional role of C5a receptors revealed that C5a triggered rapid activation of protein kinase C and activation and nuclear translocation of the NF-κB transcription factor. In addition, C5a was found to be mitogenic for undifferentiated human neuroblastoma cells, a novel action for the C5aR. In contrast, C5a protected terminally differentiated human neuroblastoma cells from toxicity mediated by the amyloid Aβ peptide. Thus, normal rodent hippocampal neurons as well as undifferentiated and differentiated human neuroblastoma cells express functional C5a receptors. These results have implications for understanding the role of neuronal C5aR receptors in normal neuronal development, neuronal homeostasis, and neuroinflammatory conditions such as Alzheimer’s disease.

Role of the Second Extracellular Loop of Human C3a Receptor in Agonist Binding and Receptor Function

The C3a anaphylatoxin receptor (C3aR) is a G protein-coupled receptor with an unusually large second extracellular loop (e2 loop, ∼172 amino acids). To determine the function of this unique structure, chimeric and deletion mutants were prepared and analyzed in transfected RBL-2H3 cells. Whereas replacement of the C3aR N-terminal segment with that from the human C5a receptor had minimal effect on C3a binding, substitution of the e2 loop with a smaller e2 loop from the C5a receptor (C5aR) abolished binding of125I-C3a and C3a-stimulated calcium mobilization. However, as much as 65% of the e2 loop sequence (amino acids 198–308) may be removed without affecting C3a binding or calcium responses. The e2 loop sequences adjacent to the transmembrane domains contain multiple aspartate residues and are found to play an important role in C3a binding based on deletion mutagenesis. Replacement of five aspartate residues in the e2 loop with lysyl residues significantly compromised both the binding and functional capabilities of the C3a receptor mediated by intact C3a or by two C3a analog peptides. These data suggest a two-site C3a-C3aR interaction model similar to that established for C5a/C5aR. The anionic residues near the N and C termini of the C3aR e2 loop constitute a non-effector secondary interaction site with cationic residues in the C-terminal helical region of C3a, whereas the C3a C-terminal sequence LGLAR engages the primary effector site in C3aR.

Cloning and functional characterization of the mouse C3a anaphylatoxin receptor gene.

Abstract The mouse anaphylatoxin C3a receptor (mC3aR) gene was isolated using a human C3aR cDNA probe. The genomic fragment contains an open reading frame of 1431 base pairs that encodes a peptide of 477 amino acids. A cDNA with identical sequence was subsequently obtained from the mouse pre-B cell line 70Z/3 by reverse transcriptase polymerase chain reaction based on sequence of the mC3aR gene. Northern blot analysis suggested expression of the mC3aR in lung and heart, and to a lesser extent, in brain, liver, muscle, kidney, and testis. The deduced amino acid sequence of the mouse C3aR is 65% identical to that of the human C3aR. Like the human receptor, mouse C3aR contains a predicted large extracellular loop of ∼165 amino acids (residues 161–325) between the fourth and fifth transmembrane domains. This loop, however, is the least conserved structure (45% identical sequence) of all the extracellular and intracellular domains between the mouse and human C3aRs. The mouse gene product bound 125I-labeled human C3a with a Kd of 2.54 nM when expressed in the stably transfected rat basophilic leukemic cell line RBL-2H3. Bound C3a could be effectively displaced by excess quantities of unlabeled C3a, but not by C4a or C5a. C3a induced dose-dependent calcium mobilization in the transfected cells, which could be blocked by pertussis toxin treatment. These results confirm that the cloned gene encodes a functional C3aR capable of coupling to a pertussis toxin-sensitive G protein. The sequence divergence of the large extracellular loop does not appear to affect C3a binding and transmembrane signaling.

Effect of Tirilazad Mesylate on Middle Cerebral Artery Occlusion/Reperfusion in Nonhuman Primates

Agents with lipid membrane protective properties may theoretically reduce the zone of ischemic damage during cerebral arterial recanalization in the early hours of focal cerebral ischemia. The effect of the putative lipid peroxidation inhibitor tirilazad mesylate (U74006F) on infarction volume and neurological outcome following 3-hour middle cerebral artery occlusion and subsequent reperfusion in an awake baboon model was examined in a blinded, randomized placebo-controlled study. Awake subjects (8 each) were assigned randomly to receive either tirilazad mesylate (3 mg/kg) or matched vehicle given 15 min prior to reperfusion, and again at 2, 4, 12, and 24 h after reperfusion. Neurological status was serially assessed according to a quantitative scale and infarction volumes were computed on perfusion-fixed specimens at 14 days. A 40% reduction in mean total infarction volume index, normalized for basal ganglia volume, was seen in the tirilazad group. Persistent and increased improvement in mean neurological score with tirilazad infusion paralleled that of the placebo. Parallel studies on polymorphonuclear leukocyte responses indicated no significant changes in polymorphonuclear leukocyte reactivity. A reduction in infarction volume, predominantly in the cortex/subcortical white matter, and an improvement in neurological outcome were observed when tirilazad mesylate was given prior to reperfusion, but after the onset of focal cerebral ischemia.

Cleavage of the Human C5A Receptor by Protienases Derived from Porphyromonas Gingivalis

The anaerobic bacteria P. gingivalis has been implicated as a primary causative agent in adult periodontitis. Several proteinases are produced by this bacteria and it is suggested that they contribute to virulence and to local tissue injury resulting from infection by P. gingivalis. Collagenases and cysteine proteinases (i.e., the gingipains)* have been characterized as the predominant vesicular enzymes produced by this bacterium. It has been shown that an arginine-specific cysteine proteinase from P. gingivalis, called gingipain-1 or Arg-gingipain, can selectively cleave complement components C3 and C5. In the case of C5, cleavage by Arg-gingipain results in the generation of C5a, a potent chemotactic factor for PMNs. Since these bacterial proteinases are capable of generating pro-inflammatory factors at sites of infection, we examined the possibility that gingipains or other proteinases from this bacterium might attack or destroy cell surface proteins, such as receptor molecules. Using an affinity-purified rabbit antibody raised against residues 9-29 of the C5a receptor (i.e., C5aR; CD88), the signal transmitting element for the pro-inflammatory mediator C5a, we demonstrated that the mixture of proteinases in P. gingivalis vesicles cleaves the C5a receptor on human neutrophils. This vesicular proteinase activity did not require cysteine activation which indicates that proteinases other than the gingipains may be responsible for cleavage of the C5aR molecule. In addition, the purified Lys-gingipain, but not Arg-gingipain, also cleaved C5aR on the human neutrophils. The N-terminal region of C5aR (residues 9-29, PDYGHYDDKDTLDLNTPVDKT) was readily cleaved by chy- motrypsin, but not by trypsin, despite the presence of potential trypsin (i.e., lysyl-X) cleavage sites. The specific sites of C5aR 9-29 peptide cleavage were determined by mass spectroscopy for both chymotrypsin and Lys-gingipain. These studies suggest that the proteolytic activity in the bacterial vesicles that is responsible for cleaving C5aR is primarily a non-tryptic proteinase, distinct from either Arg- or Lys-gingipain. Consequently, there appear to be additional proteinase(s) in the vesicles that attacks the cell surface molecule C5aR which are not the same (i.e., Arg- and Lys-gingipain) as were shown to generate pro-inflammatory activity from complement components C3 and C5. Evidence that the proteinases which attack the inflammatory precursor molecules (i.e., C3 and C5) exhibit different specificities than those that attack receptors to these bioactive complement products makes a particularly interesting story of how this bacteria avoids major host defense mechanisms.

Polymorphonuclear Leukocyte Behavior in a Nonhuman Primate Focal Ischemia Model

There is increasing interest in the role of polymorphonuclear (PMN) leukocytes in the evolution of focal cerebral infarction. Surgical preparation of focal cerebral ischemia models may alter leukocyte reactivity and thereby make interpretation of leukocyte function following ischemia/reperfusion difficult. The effects of surgical preparation and of experimental ischemia/reperfusion on granulocyte function have been examined prospectively in a baboon model. Twenty-six adolescent male baboons underwent surgical preparation, of which 21 underwent middle cerebral artery occlusion/reperfusion. Four additional animals served as nonsurgical controls. Peripheral venous blood specimens were taken for performing assays of leukocyte function at defined intervals before and after both the surgical preparation (i.e., the overall procedure for implantation of the middle cerebral artery occlusion device) and occlusion/reperfusion. A stress-related elevation in total leukocyte number was attributed mainly to an increase in the number of circulating PMN leukocytes. Values rose from 13.9 ± 4.9 × 103 to 27.8 ± 5.8 × 103/μl, (±SD; n = 21) for total leukocyte number, with p < 0.001, and from 4.3 ± 2.1 × 103 to 15.9 ± 4.7 × 103/μl (n = 21) for PMN leukocytes, with p < 0.001. Surgical preparation had no effect (p ≥ 0.4) on the ability of PMN leukocytes, isolated 24 h after the implantation procedure, to display polarization, O2– production, or β-glucuronidase release when stimulated with human C5a. A moderate decrease in the chemotactic response to C5a resolved within the 7-day postsurgery (preocclusion) period. Three-hour middle cerebral artery occlusion and 1-h reperfusion resulted in a significant reduction in C5a-induced polarization. The preocclusion value of 82 ± 9.7 (n = 7) was compared with the occlusion/reperfusion value at 58.8 ± 13.7 (n = 6; p < 0.05). A moderate decrease was observed in C5a-induced O2– and β-glucuronidase release, as well as a decrease in the chemotactic response. In the nonhuman primate model, the reversible alterations (i.e., chemotaxis) in granulocyte function that were noted following surgical preparation resolved within 7 days. In contrast, middle cerebral artery occlusion/reperfusion was associated with a more dramatic and significant reduction in multiple granulocyte functions elicited by the endogenous mediator C5a as observed 1 h postreperfusion.

cDNA Cloning and Characterization of Rat C5a Anaphylatoxin Receptor

Activation of the complement cascade plays an esssential role in the early stages of inflammation. C5a and its receptor are particularly active in anaphylaxis. To determine the pathological roles played by C5a and C5a receptor (C5aR) in rats, we cloned C5aR cDNA and analyzed distribution of its mRNA in various organs including lung from an LPS‐stimulated rat. Furthermore, we generated a polyclonal antiserum which specifically recognizes rat C5aR, as confirmed by its specific interaction with cells transfected with rat C5aR cDNA.

The Bacterial Peptide N-Formyl-Met-Leu-Phe Inhibits Killing of Staphylococcus epidermidis by Human Neutrophils in Fibrin Gels

To study human neutrophil (polymorphonuclear leukocyte (PMN)) migration and killing of bacteria in an environment similar to that found in inflamed tissues in vivo, we have used fibrin gels. Fibrin gels (1500 μm thick) containing Staphylococcus epidermidis were formed in Boyden-type chemotaxis chambers. PMN migrated <300 μm into these gels in 6 h and did not kill S. epidermidis when the gels contained heat-inactivated serum, C5-deficient serum, a streptococcal peptidase specific for a fragment of cleaved C5 (C5a), or anti-C5aR IgG. In contrast, in gels containing normal human serum, PMN migrated ∼1000 μm into the gels in 4 h and into the full thickness of the gels in 6 h, and killed 90% of S. epidermidis in 6 h. fMLP reduced PMN migration into fibrin gels and allowed S. epidermidis to increase by ∼300% in 4 h, whereas leukotriene B4 stimulated PMN to migrate the full thickness of the gels and to kill 80% of S. epidermidis in 4 h. We conclude that both complement opsonization and C5a-stimulated chemotaxis are required for PMN bacterial killing in fibrin gels, and that fMLP inhibits PMN bactericidal activity in fibrin gels. The latter finding is surprising and suggests that in the presence of fibrin fMLP promotes bacterial virulence.

Cloning and preliminary pharmacological characterization of the anaphylatoxin C5a receptor in the rabbit

The rabbit receptor for C5a was cloned from a genomic library and found to be 79.5% identical to the human homologue, the highest degree of similarity found so far in nonprimate laboratory animals. The rabbit C5a receptor stably expressed in RBL cells binds human 125I‐C5a (2 nM). Unlabelled C5a and the C‐terminal analogue N‐acetyl‐Tyr‐Ser‐Phe‐Lys‐Pro‐Met‐Pro‐Leu‐D‐Ala‐Arg (Ac‐YSFKPMPLaR) were found to be competitors of that binding, the peptide analogue retaining approximately 0.1% of the affinity of human C5a. The order of potency human C5a>Ac‐YSFKPMPLaR was conserved in bioassays based on rabbits (relaxation of the isolated portal vein and pulmonary artery; acute in vivo neutropenia), but with a decreasing potency gap between the two compounds, a likely consequence of the resistance to peptidases of the analogue. The molecular definition of the rabbit C5a receptor evidenced a high preservation degree of sequence and pharmacologic properties relative to the human ortholog receptor, thus defining a set of molecular tools for the investigation of the role of C5a in physiologic and pathologic models based on the rabbit (e.g. atherosclerosis, inflammation).