Journal of Leukocyte Biology Volume 52, September 1992 343 Lectinophagocytosis of type 1 fimbriated (mannose-specific) Escherichia co/i in the mouse peritoneum Wolfgang Bernhard, Awni Gbarah, and Nathan Sharon Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot 76100, Israeu Abstract: Bacteria can bind specifically to phagocytic cells via lectin-carbohydrate interactions and such bind- ing is often followed by activation and degranulation of the phagocytes, as well as uptake and killing of the bac- teria, a phenomenon designated lectinophagocytosis. Al- though extensively studied in vitro, no direct evidence for the occurrence of lectinophagocytosis in vivo has been available. To obtain such evidence, we injected type 1 fimbriated (mannose-specific) or nonfimbriated Escher- ichia coli into the peritoneal cavity of mice (107_lob bacteria/animal) in the absence or presence of sugars and quantified the phagocytic activity by assaying the release of lysosomal N-acetyl-13-D-glucosaminidase into the pen- toneal fluid, up to 45 mm after injection. Following injec- tion of the type 1 fimbriated bacteria, significant release of the enzyme was observed which was time dependent and increased with the number of bacteria injected, whereas the nonfimbriated bacteria caused only little release. Methyl a-D-mannoside (50 mM), but not methyl cs-D-galactoside or sucrose, inhibited the release by 60 to 100%. No release of N-acetyl- 3-D-g1ucosaminidase was induced by bacteria injected into a peritoneal cavity from which the macrophages had been removed. Our findings show that lectinophagocytosis can occur in vivo and may contribute to the host’s defence against type 1 fimbriated bacteria. J. Leukoc. Biol. 52: 343-348; 1992. Key Words: lectinophagocytosis . N-acetyl-13-D-glucosaminidase periloneal macrophages type 1 fimbriae methyt’ ce-D-mannoside nonopsonic phagocytosis INTRODUCTION Phagocytosis, the most important defense mechanism of animals against invading microorganisms, is initiated by at- tachment of the microorganism to the phagocyte [1-3]. Until recently, it was generally believed that recognition of the prey by the phagocytic cells is mediated exclusively by opso- nins, namely antibodies and complement. However, as origi- nally demonstrated by Bar-Shavit et al. [4, 5] with type 1 fimbriated (mannose-specific) Esc/zerichia coli and Salmonella typhi, the bacteria can bind specifically by means of their fimbriae to complementary carbohydrates on the phago- cytes. Similarly to opsonophagocytosis, the nonopsonic lectin-mediated binding of the bacteria to the phagocytes may be followed by activation of the latter cells, as well as by uptake and killing of the target cells, a phenomenon called lectinophagocytosis [6]. Many reports have appeared on lectinophagocytosis of type 1 fimbriated E. coli by mouse, human, and rat peritoneal macrophages, as well as by human granulocytes [6, 7]. Lectinophagocytosis of other bacteria that bind to cell surface sugars, e.g., Actinomyces naesuundii [8], has also been investigated, although only to a limited extent. All these studies were done in vitro. Indirect evidence that mannose- specific lectinophagocytosis of E. coli may take place in vivo was obtained in experimental infections with mixed bacterial strains (or isogens), one of which was fimbriated and the other nonfimbriated. Such experiments revealed that when- ever the organisms reached phagocyte-rich sites, the non- fimbriated phenotype survived, whereas at phagocyte-poor sites the fimbriated phenotype survived, irrespective of the bacterial species or experimental animals used or the route or site of the infection [9-14]. It was assumed that the selec- tive survival of the fimbriated phenotype is due to its binding to the mucosal surfaces of epithehial cells, whereas the selec- tive survival of the nonfimbriated phenotype in deep tissues is the result of the elimination of the fimbriated phenotype by the macrophages and granulocytes present. In this report, we demonstrate for the first time that lec- tinophagocytosis can occur in vivo by showing that type 1 fimbriated E. coli activate macrophages in the mouse peritoneum in a mannose-specific manner. Activation was monitored by measuring degranulation, as determined by the release of lysosomal N-acetyl-/3-D-glucosaminidase (/3- GlcNAc-ase), following intraperitoneal injection of the bac- teria. We chose to work with this enzyme because it is abun- dant in murine macrophages, is stable, and can be assayed easily by the fluorogenic substrate 4-methylumbelhiferyl N- acetyl-/3-D-glucosaminide (MeUmb-/3-G1cNAc). Although degranulation by type I fimbriated E. coli has been demon- strated in vitro with human granulocytes [15, 16], it was not shown with mouse peritoneal macrophages, either in vitro or in vivo. It was therefore necessary to examine first whether the bacteria can cause degranulation of the latter cells in vitro. Methyl a-D-mannoside (MeaMan), an inhibitor of the binding of type 1 fimbriated bacteria to eukaryotic cells, was used to demonstrate the specificity of the interaction be- tween the bacteria and the macrophages. Because this sugar was reported to inhibit f3-GlcNAc-ase from a variety of animal sources [17], conditions had to be worked out at which its interference in the enzyme assay is minimal. Abbreviations: BSA, bovine serum albumin; f3-GIcNAc-ase, N-acetyl-(3-D- glucosaminidase; MeaGal, methyl cs-D-galactoside; MeaMan, methyl a-D- mannoside; MeUmb, 4-methylumbelliferone; MeUmb-l3-GlcNAc, 4-methyl- umbelliferyl N-acetyl- -D-glucosaminide; PBS, phosphate-buffered saline (0.15 M NaC1 in 0.05 M phosphate buffer, pH 7.4); PBS-CaMg, PBS con- taining 1 mM Ca2” and 1 mM Mg2”. Wolfgang Bernhard’s present address: Pharmacology and Toxicology Center, Hannover Medical School, Hannover 61, Germany. Reprint requests: Nathan Sharon, Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot 76100, Israel. Received January 24, 1992; accepted April 24, 1992.