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F4/80 is a monoclonal antibody that recognizes a murine macrophage-restricted cell surface glycoprotein and has been extensively used to characterize macrophage populations in a wide range of immunological studies. Apart from the tightly regulated pattern of expression of the F4/80 antigen, little is known about its possible role in macrophage differentiation and function. We have sought to characterize the molecule at the molecular level, through the isolation of cDNA clones, and now describe the sequence of the F4/80 protein. The primary amino acid sequence demonstrates homology to two protein superfamilies. The NH2-terminal region consists of seven epidermal growth factor-like domains, separated by approximately 300 amino acids from a COOH-terminal region that shows homology to members of the seven transmembrane-spanning family of hormone receptors. The potential role of these distinct domains is discussed with respect to the possible function of the F4/80 molecule.

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At present, approximately 150 different members of the adhesion-G protein-coupled receptor (GPCR) family have been identified in metazoans. Surprisingly, very little is known about their function, although they all possess large extracellular domains coupled to a seven-transmembrane domain, suggesting a potential role in cell adhesion and signaling. Here, we demonstrate how the human-restricted adhesion-GPCR, EMR2 (epidermal growth factor-like module-containing mucin-like hormone receptor), regulates neutrophil responses by potentiating the effects of a number of proinflammatory mediators and show that the transmembrane region is critical for adhesion-GPCR function. Using an anti-EMR2 antibody, ligation of EMR2 increases neutrophil adhesion and migration, and augments superoxide production and proteolytic enzyme degranulation. On neutrophil activation, EMR2 is rapidly translocated to membrane ruffles and the leading edge of the cell. Further supporting the role in neutrophil activation, EMR2 expression on circulating neutrophils is significantly increased in patients with systemic inflammation. These data illustrate a definitive function for a human adhesion-GPCR within the innate immune system and suggest an important role in potentiating the inflammatory response. Ligation of the adhesion-GPCR EMR2 regulates human neutrophil function.

C-type lectins are the most diverse and prevalent lectin family in immunity. Particular interest has recently been attracted by the C-type lectin-like receptors on NK cells, which appear to regulate the activation/inhibitory balance of these cells, controlling cytotoxicity and cytokine production. We previously identified a human C-type lectin-like receptor, closely related to both the beta-glucan receptor and the lectin-like receptor for oxidized-LDL, named MICL (myeloid inhibitory C-type lectin-like receptor), which we had shown using chimeric analysis to function as an inhibitory receptor. Using a novel MICL-specific monoclonal antibody, we show here that human MICL is expressed primarily on myeloid cells, including granulocytes, monocytes, macrophages, and dendritic cells. Although MICL was highly N-glycosylated in primary cells, the level of glycosylation was found to vary between cell types. MICL surface expression was down-regulated during inflammatory/activation conditions in vitro, as well as during an in vivo model of acute inflammation, which we characterize here. This suggests that human MICL may be involved in the control of myeloid cell activation during inflammation. IntroductionIn order to execute their immune (and non-immune) functions, leukocytes must interact with a broad range of endogenous and exogenous ligands and must be able to respond to these interactions appropriately. This requires a wide and flexible, yet specific and regulated, repertoire of cell surface molecules. One large family of such molecules, which are particularly important to immunity, are the C-type lectin and lectin-like receptors. C-type lectin-like receptors were first identified as dimeric molecules on NK cells. On these cells, much attention has been drawn toward their ability to regulate the balance between cellular activation and inhibition, such as cytotoxicity and cytokine production. This is achieved through signalling via intracellular ITIM present in the cytoplasmic tails of these receptors or via ITAM, of which the majority are located in the cytoplasmic tails of associated molecules, such as DAP12. The study of these activationand inhibitory NK cell receptorshas generated a number of interesting hypotheses, including immune privilege [1], the recognition of 'missing self', 'non-self' and 'induced self' [2][3][4][5], as well elucidating the underlying mechanisms of some of the immune responses to viruses [6] and malignancy [7]. Others and we have demonstrated that C-type lectinlike receptors are not restricted to NK cells, but are expressed by many other cell types including myeloid cells [8][9][10]. This introduces the novel concept that the myeloid paralogs of NK cell receptors might govern myeloid cell activation/inhibition in the same manner as those on NK cells. The genes of most C-type lectin-like molecules are located within the 'natural killer complex' (NKC), but many of those expressed by myeloid and other cells are found in a distinct cluster of genes within the NKC [10]. This cluster includes Decti...

A down-modulation of both the 55-kDa (TNF-R55) and the 75-kDa (TNF-R75) TNF receptors is observed in neutrophils exposed to a variety of stimuli. Proteolytic cleavage of the extracellular region of both receptors (shedding) and, with TNF, internalization of TNF-R55 and shedding of TNF-R75 are the proposed mechanisms. We have characterized the TNF-induced shedding of TNF receptors in neutrophils and determined the nature of the involved proteinase. Neutrophils exposed to TNF release both TNF receptors. A release of TNF receptors comparable to that observed with TNF was induced with TNF-R55-specific reagents (mAbs and a mutant of TNF) but not with the corresponding TNF-R75-specific reagents. A hydroxamic acid compound (KB8301) almost completely inhibited shedding of TNF-R55 and to a lesser degree shedding of TNF-R75. KB8301 also inhibited FMLP-induced shedding to a similar extent. Shedding was also inhibited by 1,10-phenanthroline, but this effect was considered nonspecific as the compound, at variance with KB8301, almost completely inhibited TNF and FMLP-induced PMN activation. Diisopropylfluorophosphate partially inhibited shedding of TNF-R75, suggesting the contribution of a serine proteinase to the release of this receptor. Shedding activity was not affected by matrix metalloproteinases inhibitors nor was it released in the supernatants of FMLP-stimulated neutrophils. These results suggest that TNF induces release of its receptors, that such a release is mediated via TNF-R55, and that a membrane-bound and non-matrix metalloproteinase is involved in the process. The possibility that ADAM-17, which we show to be expressed in neutrophils, might be the involved proteinase is discussed.

Abstract. Chloride ion efflux is an early event occurring after exposure of neutrophilic polymorphonuclear leukocytes (PMN) in suspension to several agonists, including cytokines such as tumor necrosis factor-a (TNF) and granulocyte/macrophage-colony stimulating factor (Shimizu, Y., R.H. Daniels, M,A. Elmore, M.J. Finnen, M.E. Hill, and J.M. Lackie. 1993. Biochem. Pharmacol. 9:1743-1751. We have studied TNFinduced C1-movements in PMN residing on fibronectin (FN) (FN-PMN) and their relationships to adherence, spreading, and activation of the respiratory burst. Occupancy of the TNF-R55 and engagement of [32 integrins cosignaled for an early, marked, and prolonged C1-efflux that was accompanied by a fall in intracellular chloride levels (Cl-i). A possible causal relationship between CI-efflux, adherence, and respiratory burst was first suggested by kinetic studies, showing that TNF-induced CI-efflux preceded both the adhesive and metabolic response, and was then confirmed by inhibition of all three responses by pretreating PMN with inhibitors of CI-efflux, such as ethacrynic acid. Moreover, CI-efflux induced by means other than TNF treatment, i.e., by using Cl--free media, was followed by increased adherence, spreading, and metabolic activation, thus mimicking TNF effects. These studies provide the first evidence that a drastic decrease of CI-i in FN-PMN may represent an essential step in the cascade of events leading to activation of proadhesive molecules, reorganization of the cytoskeleton network, and assembly of the O2--forming NADPH oxidase. N 'EtrrRoamLtC polymorphonuclear leukocytes (PMN) 1 respond to both particulate and soluble stimuli with a vigorous respiratory burst. This leads to the release of toxic oxygen molecules that contribute to both the PMN microbicidal activity and the tissue inflammatory damage. Among the physiologically relevant soluble stimuli, cytokines such as tumor necrosis factor-a (TNF), granulocyte/macrophage-colony stimulating factor, and granulocyte-colony stimulating factor are peculiar, since they activate the respiratory burst only in PMN residing on biologic surfaces, e.g., on proteins of the extraAddress all correspondence to R. Menegazzi, Istituto di Patologia Generale, Universit~t di Trieste, via A. Fleming, 22, 34127 Trieste, Italy. Tel.: (39) 40 572012. Fax: (39) 40 567862.1. Abbreviations used in this paper: 9-AC, anthracene-9-carboxylic acid; CHC, c~-cyano-4-hydroxy-cinnamic acid; C1-1, intracellular chloride content; DIDS, 4,4'diisothiocyanatostilbene-2,2'-disulfonic acid; EA, ethacrynic acid; FBG, fibrinogen; FMLP, N-formyl-methyonil-leucyl-phenylalanine; FN, fibronectin; FN-PMN, PMN residing on FN-coated surfaces; G-buffer, glucuronate-containing buffer; MA, o-[(3-hydroxymercuri-2-methoxypropyl)carbamoyl] phenoxyacetic acid; Oz-, superoxide anion; PMN, neutrophilic polymorphonuclear leukocytes; s-PMN, PMN in suspension; poly (HEMA), poly(2-hydroxyethyl methacrylate); TNF, tumor necrosis factor-a; TNF-R, TNF receptor. cellular matrix immobilized on a solid support, but...