C5a receptor and interleukin-6 are expressed in tissue macrophages and stimulated keratinocytes but not in pulmonary and intestinal epithelial cells.
(1/1307)
The anaphylatoxin derived from the fifth component of the human complement system (C5a) mediates its effects by binding to a single high-affinity receptor (C5aR/CD88), the expression of which has been traditionally thought to be restricted to granulocytes, monocytes, macrophages (Mphi), and cell lines of myeloid origin. Recent immunohistochemical data suggested that human bronchial and alveolar cells express C5aR as well. To reexamine the tissue distribution of human C5aR expression, transcription of the C5aR gene was investigated in normal and pathologically affected human lung (bronchopneumonia, tuberculosis), large intestine (acute appendicitis, Crohn's disease), and skin (pyogenic granuloma, lichen planus) using in situ hybridization. In contrast to previous evidence, C5aR mRNA could not be detected in pulmonary or intestinal epithelial cells, whereas keratinocytes in inflamed but not in normal skin revealed detectable levels of C5aR transcripts. Additionally, it could be documented that only migrating Mphi express C5aR mRNA, whereas sessile Mphi in normal tissues and epithelioid/multinucleated Mphi found in granulomatous lesions do not. Because C5a has been demonstrated to upregulate the expression of interleukin (IL)-6 in human monocytes, we also studied IL-6 gene transcription in parallel to the C5aR. IL-6 mRNA was detectable in many tissue Mphi. Surprisingly, a tight co-expression of C5aR and IL-6 mRNA was observed in keratinocytes from lesions of pyogenic granuloma and lichen planus. These results point to an as yet unknown role for C5a in the pathogenesis of skin disorders beyond its well-defined function as a chemoattractant and activator of leukocytes. (+info)
Chimeric receptors of the human C3a receptor and C5a receptor (CD88).
(2/1307)
Chimeras were generated between the human anaphylatoxin C3a and C5a receptors (C3aR and C5aR, respectively) to define the structural requirements for ligand binding and discrimination. Chimeric receptors were generated by systematically exchanging between the two receptors four receptor modules (the N terminus, transmembrane regions 1 to 4, the second extracellular loop, and transmembrane region 5 to the C terminus). The mutants were transiently expressed in HEK-293 cells (with or without Galpha-16) and analyzed for cell surface expression, binding of C3a and C5a, and functional responsiveness (calcium mobilization) toward C3a, C5a, and a C3a as well as a C5a analogue peptide. The data indicate that in both anaphylatoxin receptors the transmembrane regions and the second extracellular loop act as a functional unit that is disrupted by any reciprocal exchange. N-terminal substitution confirmed the two-binding site model for the human C5aR, in which the receptor N terminus is required for high affinity binding of the native ligand but not a C5a analogue peptide. In contrast, the human C3a receptor did not require the original N terminus for high affinity binding of and activation by C3a, a result that was confirmed by N-terminal deletion mutants. This indicates a completely different binding mode of the anaphylatoxins to their corresponding receptors. The C5a analogue peptide, but not C5a, was an agonist of the C3aR. Replacement of the C3aR N terminus by the C5aR sequence, however, lead to the generation of a true hybrid C3a/C5a receptor, which bound and functionally responded to both ligands, C3a and C5a. (+info)
Role of the second extracellular loop of human C3a receptor in agonist binding and receptor function.
(3/1307)
The C3a anaphylatoxin receptor (C3aR) is a G protein-coupled receptor with an unusually large second extracellular loop (e2 loop, approximately 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 of 125I-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. (+info)
C1qRP is a heavily O-glycosylated cell surface protein involved in the regulation of phagocytic activity.
(4/1307)
C1q, mannose-binding lectin (MBL), and pulmonary surfactant protein A (SPA) interact with human monocytes and macrophages, resulting in the enhancement of phagocytosis of suboptimally opsonized targets. mAbs that recognize a cell surface molecule of 126,000 Mr, designated C1qRP, have been shown to inhibit C1q- and MBL-mediated enhancement of phagocytosis. Similar inhibition of the SPA-mediated enhancement of phagocytosis by these mAbs now suggests that C1qRP is a common component of a receptor for these macromolecules. Ligation of human monocytes with immobilized R3, a IgM mAb recognizing C1qRP, also triggers enhanced phagocytic capacity of these cells in the absence of ligand, verifying the direct involvement of this polypeptide in the regulation of phagocytosis. While the cDNA for C1qRP encodes a 631 amino acid membrane protein, Chinese hamster ovary cells transfected with the cDNA of the C1qRP coding region express a surface glycoprotein with the identical 126,000 Mr in SDS-PAGE as the native C1qRP. Use of glycosylation inhibitors, cleavage of the mature C1qRP with specific glycosidases, and in vitro translation of C1qRP cDNA demonstrated that both posttranslational glycosylation and the nature of the amino acid sequence of the protein contribute to the difference between its predicted m.w. and its migration on SDS-PAGE. These results verify that the cDNA cloned codes for the mature C1qRP, that C1qRP contains a relatively high degree of O-linked glycoslyation, and that C1qRP cross-linked directly by monoclonal anti-C1qRP or engaged as a result of cell surface ligation of SPA, as well as C1q and MBL, enhances phagocytosis. (+info)
Crystal structure of human p32, a doughnut-shaped acidic mitochondrial matrix protein.
(5/1307)
Human p32 (also known as SF2-associated p32, p32/TAP, and gC1qR) is a conserved eukaryotic protein that localizes predominantly in the mitochondrial matrix. It is thought to be involved in mitochondrial oxidative phosphorylation and in nucleus-mitochondrion interactions. We report the crystal structure of p32 determined at 2.25 A resolution. The structure reveals that p32 adopts a novel fold with seven consecutive antiparallel beta-strands flanked by one N-terminal and two C-terminal alpha-helices. Three monomers form a doughnut-shaped quaternary structure with an unusually asymmetric charge distribution on the surface. The implications of the structure on previously proposed functions of p32 are discussed and new specific functional properties are suggested. (+info)
Is the oocyte a non-professional phagocyte?
(6/1307)
Although fertilization has been described as a series of events during which the spermatozoon penetrates the oocyte, introducing its nuclear contents, there is strong evidence that either gamete can be the active partner at different stages of this process. Indeed, while sperm motility is essential for its penetration of the egg vestments, immotile spermatozoa are capable of entering the ooplasm once they adhere to the oolemma. Entry of the spermatozoon into the oocyte appears to require two distinct but perhaps related events, namely gamete cell membrane fusion, at the level of the equatorial segment of the sperm acrosome with the oolemma, and a quasi-phagocytic event involving the incorporation by the oocyte of the rostral portion of the acrosome-reacted spermatozoon head within an oolemmal-derived vesicle. This review explores the biology of phagocytosis by macrophages and non-professional phagocytes, and in particular the roles played by phagocytosis-promoting receptors (FcgR, complement receptors and integrins), in both signal transduction and their linkage with the cytoskeleton. It asks whether the oocyte might not utilize similar mechanisms during its incorporation of the spermatozoon. (+info)
Effects of a new C5a receptor antagonist on C5a- and endotoxin-induced neutropenia in the rat.
(7/1307)
A new C5a receptor antagonist, the cyclic peptide Phe-[Orn-Pro-D-cyclohexylalanine-Trp-Arg], (F-[OPdChaWR]), was tested for its ability to antagonize the neutropenic effects of both C5a and endotoxin in rats. Human recombinant C5a (2 microg kg(-1) i.v.) caused rapid neutropenia, characterized by an 83% decrease in circulating polymorphonuclear leukocytes (PMNs) at 5 min. Administration of F-[OPdChaWR] (0.3-3 mg kg(-1) i.v.), did not affect the levels of circulating PMNs but, when given 10 min prior to C5a, it inhibited the C5a-induced neutropenia by up to 70%. Administration of E. Coli lipopolysaccharide (LPS, 1 mg kg(-1) i.v.) also caused neutropenia with an 88% decrease in circulating PMNs after 30 min. When rats were pretreated with F-[OPdChaWR] (0.3 - 10 mg kg(-1) i.v.) 10 min prior to LPS, there was a dose-dependent antagonism of the neutropenia caused by LPS, with up to 69% reversal of neutropenia observed 30 min after LPS administration. These findings suggest that C5a receptor antagonists may have therapeutic potential in the many diseases known to involve either endotoxin or C5a. (+info)
Monocyte activation in rheumatoid arthritis (RA): increased integrin, Fc gamma and complement receptor expression and the effect of glucocorticoids.
(8/1307)
The aim of this work was to study the expression of beta 1- and beta 2-integrins, CR1, CD44 and Fc gamma receptors on peripheral blood monocytes in RA. The expression of these receptors was measured by flow cytometry, before and after treatment with low-dose prednisolone. Expression of the same receptors was also measured before and after treatment with metyrapone, a substance that inhibits the synthesis of cortisol in the adrenals. The expression of the beta 2-integrins CD11a, CD11b and CD18, of CD35 (CR1), and of Fc gamma RII and Fc gamma RI (CD32 and CD64) on monocytes was elevated in the RA patients compared with healthy controls, while the expression of the beta 1-integrins (CD29, CD49d, CD49f) was unaffected. A significant correlation between monocyte expression of CD64 and C-reactive protein (CRP), and blood platelet count, respectively, was found in the group of patients with RA. After 4-6 weeks of treatment with low-dose prednisolone, the expression on the monocytes of CD11a, CD11b, CD18, CD35, CD32 and CD64 was normalized. A significant correlation (r = 0.64, P = 0.02) was found between the decrease in expression of CD11b and clinical improvement after prednisolone treatment. Two days of metyrapone treatment, which significantly lowered the serum cortisol levels, elevated the expression of CD35 and CD49f. Priming of peripheral monocytes seems to be one of the mechanisms behind the recruitment of monocytes to the rheumatoid synovium. One reason for the good clinical effects of prednisolone in RA could be a down-regulation of adhesion and phagocytosis receptors on monocytes. (+info)