The triggering of fibrinogen receptors on mononuclear phagocytes of healthy subjects and of a thrombasthenia Glanzmann patient promotes the generation of reactive oxygen species.

The interaction of human fibrinogen (Fg) with homologous mononuclear phagocytes was investigated. Using a radioligand binding test, no evidence for high-affinity binding of either monomeric Fg, of the fibrin fragment E, or of radiolabelled Gly-Pro-Arg-Pro(-Try) to mononuclear phagocytes and myelomonocytic cell lines could be obtained, although commercial labelled Fg specifically bound to monocytes (MO), macrophages (M phi), U937 and HL60 cells. MO pretreated with either commercial or monomeric Fg and washed showed an oxidative burst upon treatment with anti-Fg antibodies, as evidenced by chemiluminescence (CL) measurement in the presence of luminol. The reaction depended on the Fg concentration used for pre-incubation, was divalent cation-independent and was inversely related to the time for which Fg was allowed to dissociate from the cell surface. Induction of the CL response required specific divalent antibodies, but not an intact Fc portion. MO pre-treated with fibronectin (Fn), washed and treated with anti-Fn antibodies exhibited no CL response. MO from a patient with thrombasthenia Glanzmann showed a similar reaction upon pre-incubation with Fg and stimulation with antibody. CL was also triggered by exposure of MO to surface-adsorbed Fg. The response was smaller than that induced by equivalent amounts of IgG, but was larger than that promoted by Fn. Pre-treatment of MO with ADP, fMLP or LPS did not enhance CL induced by surface-adsorbed Fg. Our results suggest that unstimulated mononuclear phagocytes of healthy subjects and of patients lacking the Fg receptors on platelets have receptors that bind the C-gamma-terminus of fibrin(ogen) with relatively low affinity, and that crosslinking of these receptors by surface-bound ligand promotes an oxidative burst but fails to induce cytokine secretion.

Fibrinogen association with human monocytes: evidence for constitutive expression of fibrinogen receptors and for involvement of Mac-1 (CD18, CR3) in the binding.

Radiolabeled fibrinogen (Fg) specifically binds to mononuclear leukocytes (MNL) and to purified monocytes, but not to nylon-nonadherent lymphocytes. The association is rapid, Ca++-dependent and reversible. MNL containing Fg-binding monocytes had not been exposed to endotoxin (less than 4 pg/mL) during the isolation and the binding test, and Fg binding was not altered by preincubation of MNL with lipopolysaccharide. The binding of Fg was inhibited by anti-Mac-1 antibodies (OKM1). Antibodies to surface-bound Fg were able to induce luminol-dependent chemiluminescence, indicating that Fg binding sites have receptor function. Emission of a signal depended on MNL exposure to Fg, on specific, divalent antibodies, but not on the antibody Fc portion. These data show that human monocytes constitutively express specific Fg receptors and suggest that Mac-1, a member of the integrin superfamily, is involved in Fg recognition.

Low-affinity interaction of fibrinogen carboxy-gamma terminus with human monocytes induces an oxidative burst and modulates effector functions.

The interaction of highly purified, monomeric fibrinogen (Fg) with human monocytes (MO) was investigated. In contrast to commercial Fg, no high-affinity binding of monomeric Fg to MO or mononuclear cells could be demonstrated. MO preincubated with Fg in the presence or absence of Ca++ elicited an oxidative burst when triggered with anti-Fg antibodies. Divalency of the antibody and specificity were required, but an intact Fc portion was not. Surface-adsorbed monomeric Fg also promoted an oxidative burst. Evidence is presented that Fg-MO interaction is mediated by the carboxy-gamma terminus of Fg. MO treated with monomeric Fg or exposed to Fg-coated surfaces show a reduced oxidative burst upon triggering with unrelated stimuli. Thus, MO function may be modulated upon interaction with surface-adsorbed Fg or with fibrin.

Evidence that exposure to fibrinogen or to antibodies directed against Mac-1 (CD11b/CD18; CR3) modulates human monocyte effector functions.

We have recently shown that the treatment of fibrinogen-coated monocytes (MO) with anti-fibrinogen as well as the exposure of MO to surface-bound fibrinogen (Fg) or to albumin haptenized with the Fg C-gamma-terminal pentadecapeptide, induces on oxidative burst. Using chemiluminescence (CL) for indicating an oxidative burst, and the ingestion of IgG-coated erythrocytes as a test of phagocytosis, we have now studied the impact of Fg on MO effector functions. MO that had been either pretreated with Fg and washed, or that were exposed to surface-adsorbed Fg, exhibited impaired Fc receptor-mediated phagocytosis. A similar impairment was observed when MO were pretreated with activating agents such as phorbol myristate acetate, n-formyl-methionyl-leucyl-phenylalanine (fMLP) or the calcium ionophore A23187. Moreover, following exposure to Fg or IgG, MO exhibited a reduced oxidative burst upon stimulation with a variety of agents. Similarly, MO pretreated or coincubated with anti-Mac-1 exhibited a reduced oxidative burst upon stimulation. Our results raise the possibility that inflammatory mononuclear phagocytes experience a functional modulation upon encountering fibrin by interacting with specific receptors for fibrin(ogen). This type of modulation is analogous to effects induced by the triggering of Fc gamma receptors. MO showed a decreased oxidative burst when either pretreated or coincubated with anti-Mac-1 antibodies, whereas antibodies directed against other MO surface constituents had no, or a weak effect only. This is compatible with the suggestion that Mac-1 acts as a fibrin (ogen) receptor.

Human monocytes CD36 and CD16 are signaling molecules. Evidence from studies using antibody-induced chemiluminescence as a tool to probe signal transduction.

A panel of monoclonal antibodies (mAb) directed against human monocyte surface antigens was tested for their capacity to mediate signal transduction by measuring luminol-enhanced chemiluminescence (CL). The response patterns fell into three categories. The mAb Mo4, OKM3, OKM6 and antibodies specific for Fc receptor (FcR) type I and II did not mediate signal transduction directly or were weak triggers, but upon second-order cross-linking by goat anti-mouse immunoglobulin (Ig) F(ab')2 or rabbit anti-mouse Ig, a strong CL response was induced. The mAb recognizing different epitopes on CD11b (complement receptor type III alpha chain) were unique in their ability to induce a CL response either by direct stimulation or by second-order cross-linking. The mAb 3G8 recognizing FcR type III (FcRIII; CD16) on a monocyte subpopulation and CD36-specific monoclonals directly elicited a CL response. A response of similar magnitude was obtained with 3G8 F(ab')2 or with intact 3G8. Furthermore, elutriation-centrifugation-purified monocytes were stimulated by 3G8 to a similar extent as unseparated mononuclear cells, whereas lymphocytes did not respond. This suggests that a FcRIII-expressing monocyte subpopulation may mediate effector functions, including the generation of reactive oxygen species, via FcRIII triggering. Our finding that anti-CD36 F(ab')2 directly induces an oxidative burst is the first evidence that CD36 itself is a trigger molecule. CD36, which is thought to interact with erythrocytes infected with Plasmodium falciparum and with thrombospondin, may constitute a signal-transducing element and thus may have functions extending beyond mediation of adherence. The present CL system constitutes a simple assay for detecting mAb directed against monocyte surface signalling elements. Probing mAb for signal transduction requires suspended cells and antibodies in the fluid phase in order to avoid inadvertent FcR triggering, which may occur when cells are stimulated by surface-adherent whole antibodies.