Expression of a Functional Anaphylatoxin C3a Receptor by Astrocytes

Abstract

Human astrocyte cell lines reportedly contain a specific receptor for the complement anaphylatoxin C3a based on ligand‐binding studies, functional responses, and RNA analysis by RT‐PCR. Uptake of 125I‐C3a by astrocytes was specific and reversible. Scatchard analysis indicated the presence of two classes of binding sites. High‐affinity binding sites were abundantly expressed (20,000–80,000 sites per cell) with an estimated KD of 1–2 nM. Low‐affinity binding sites with a KD of 209 nM were largely expressed (n≥ 4 × 106 sites per cell) and probably did not reflect a receptor‐mediated binding, but rather an ionic interaction between C3a and the membrane. Analysis of astrocyte mRNA by RT‐PCR with three different sets of primers covering 60% of the C3a receptor (C3aR) mRNA sequence indicated that glial C3aR was identical to the leukocytic one. Western blot analysis using a specific anti‐C3aR evidenced a C3aR with a molecular mass of 60,000 Da. C3a and a superagonist peptide, E7, induced a transient increase of intracellular [Ca2+] in primary culture of astrocytes. Treatment of the ligands by carboxypeptidase B to eliminate the C‐terminus Arg considerably decreased the [Ca2+] response. Moreover, flow cytometry experiments demonstrated the expression of C3aR on normal rat astrocyte membrane. This report brings new insight for the role of the complement system in the brain inflammation response.

Characterization of C3a and C5a Receptors in Rat Cerebellar Granule Neurons during Maturation

“…In vitro studies on cultured cells confirmed the expression of C3aR and C5aR by cerebellar granule neurons; (i) measurement of C3aR and C5aR mRNAs revealed maximum expression of both receptor genes at DIV 2, and (ii) flow cytometry and immunocytochemical experiments showed that the C3aR and C5aR proteins occurred in the majority of granule cells from DIV 4 to DIV 9. Up to now the presence of C3aR and C5aR has been documented in the adult rodent brain, where the two receptors are expressed by glial cells, astrocyte (11)(12)(13), and microglia (14,15). C3aR and C5aR are present both in vitro (11)(12)(13)(14) and in vivo (15) and are upregulated in these cells after inflammation (15)(16)(17).…”

“…Up to now the presence of C3aR and C5aR has been documented in the adult rodent brain, where the two receptors are expressed by glial cells, astrocyte (11)(12)(13), and microglia (14,15). C3aR and C5aR are present both in vitro (11)(12)(13)(14) and in vivo (15) and are upregulated in these cells after inflammation (15)(16)(17). In situ hybridization and immunohistochemistry analysis also revealed a constitutive expression of mouse and rat C3aR in cortical and hippocampal neurons (17,19) as well as in cerebellar Purkinje cells (19).…”

“…C3aR and C5aR are expressed in myeloid cells (4 -8) and, more surprisingly, in non-myeloid cells and tissues (7)(8)(9)(10). In the central nervous system it has been shown that C3aR and C5aR are constitutively expressed by glial cells in vitro (11)(12)(13)(14) and in vivo (15) and that the expression of C3aR and C5aR is increased in inflammatory conditions (15)(16)(17).…”

Complement and its role in innate and adaptive immune responses

“…Binding of anaphylatoxins to extracellular, N-terminal regions of the anaphylatoxin receptors allows conformational changes to the intracellular, C-terminal regions of the receptors, resulting in coupling to G-proteins, predominantly pertussis toxin-sensitive G iα , to induce downstream signaling cascades [61][62][63][64][65]. Activities of anaphylatoxins are confined by cell types that express their receptors, predominantly thought to be cells of myeloid origin, including granulocytes (basophils, eosinophils, and neutrophils), monocytes/macrophages, mast cells, and some dendritic cells, although there are numerous reports of receptor expression in a number of nonmyeloid cell types [54,59,60,[66][67][68][69][70][71][72][73]. Inactivation of anaphylatoxin molecules is an important determinant of the duration and extent of their potent functions and represents an alternative mechanism to control complement activation.…”

Anaphylatoxins: From Supposed Toxin Anaphylactics to Effective Mediators of the Early Events of Inflammation

“…This receptor is expressed on a large variety of cells as of myeloid origin like mast cells, basophils, neutrophils, eosinophils, monocytes-macrophages, dendritic cells and microglia [55][56][57][58][59][60], as well as in non-myeloid cells like astrocytes [61,62], endothelial cells [63], and epithelioid cells, smooth muscle cells and parenchymal cells [64]. The presence of C3aR on activated but not non-activated T lymphocytes has also been reported [65].…”