Abstract. In a previous study we observed that neutrophils respond with a rapid rise in [Ca2+]; during adherence to cytokine-activated endothelial cells (EC), caused by EC membrane-associated platelet-activating factor (PAF) . In the present study, we investigated whether this form of PAF was important in neutrophil adherence and migration across monolayers of rIL-lß-or ITNFa-prestimulated EC. PAF receptor antagonists prevented neutrophil migration across cytokine-pretreated EC by -60% (P < 0.005) without interfering with the process of adherence . The antagonists WEB 2086 and L-652,731 had no effect on neutrophil migration across resting EC induced by formylmethionyl-leucyl-phenylalanine (FMLP) . A murine T RANSMIGRATION of neutrophils from the peripheral circulation into inflammatory sites requires neutrophil adherence to the endothelial cell lining of the blood vessels. The CD18 complex proteins are: lymphocyte function-associated antigen-1 (LFA-1),' complement receptor 3 (CR3) or Mac-1, and glycoprotein 150,95 (p150,95) . On neutrophils they are involved in both migration and adherence (Anderson et al ., 1985 ;Harlan, 1985 ;Kuijpers and Roos, 1989). In these processes, endothelial intercellular adhesion molecule-1 (ICAM-1) may function as a cellular ligand for LFA-1 and CR3 (Rotplein et al., 1986 ;Diamond et al ., 1990;Springer, 1990) . Its expression can be increased by the inflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor (TNF) Springer, 1990) .Several other adhesion molecules can play an accessory role in the process of neutrophil adherence . The role of a 1. Abbreviations used in this paper: CR3, complement receptor 3 ; EC, endothelial cells ; ELAM-1, endothelial leukocyte adhesion molecule-l ; FMLP, formyl-leucyl-methionyl-phenylalanine ; HSA, human serum albumin ; ICAM-1, intercellular adhesion molecule-1 ; IL-1, interleukin-1 ; LAM-1, leukocyte adhesion molecule-1 ; LFA-1, lymphocyte-function associated antigen-1; MFI, mean fluorescence intensity ; PAF, platelet-activating factor, p150,95, (glyco)protein 150,95 ; rIL-1p, recombinant interleukin-lß ; rIL-8, recombinant interleukin-8 ; rTNFct, recombinant tumor necrosis fkctor-a ; TNF, tumor necrosis factor.

The general goal of this discussion paper is to contribute toward the further harmonization of human health risk assessment. It first discusses the development of a formal, harmonized set of assessment factors. The status quo with regard to assessment factors is reviewed, that is, the type of factors to be identified, the range of values assigned, as well as the presence or absence of a scientific basis for these values. Options are presented for a set of default values and probabilistic distributions for assessment factors based on the state of the art. Methods of combining default values or probabilistic distributions of assessment factors are also described. Second, the effect parameter, the no-observed-adverse-effect level (NOAEL), is discussed. This NOAEL as selected from the toxicological database may be a poor substitute for the unknown, true no-adverse-effect level (NAEL). New developments are presented with respect to the estimation of the NAEL. The already widely discussed Benchmark Dose concept can be extended to obtain an uncertainty distribution of the Critical Effect Dose (CED). This CED distribution can be combined with estimated uncertainty distributions for assessment factors. In this way the full distribution of the Human Limit Value will be derived and not only a point estimate, whereas information on dose-response relations is taken into account. Finally, a strategy is proposed for implementation of the new developments into human health risk assessments.

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Pretreatment of endothelial cells with cytokines enhances the adherence of leukocytes, a process that is mediated by surface proteins expressed on both cell types. A three-dimensional model system for the simultaneous determination of leukocyte adherence and migration was used to study the contribution of CD11/CD18, endothelial leukocyte- adhesion molecule-1 (ELAM-1) and VLA-4 in neutrophil and monocyte adherence to and migration through cytokine-activated endothelial cells. Pretreatment of endothelial cells for 4 hours with recombinant interleukin-1 beta (rIL-1 beta) was found to enhance neutrophil adherence and migration to a much greater extent than monocyte adherence and migration. Neutrophil adherence was almost completely prevented by the combined use of monoclonal antibodies (MoAbs) against ELAM-1 and CD18. Although ELAM-1 has been designated an endothelial cell-specific cytokine-inducible receptor for neutrophils, we observed that ENA2, an anti-ELAM-1 MoAb, significantly reduced monocyte adherence about 30%. MoAbs against VLA-4, the ligand of the cytokine- inducible receptor VCAM-1, did not affect monocyte adherence. However, the combined use of the MoAbs against CD18, ELAM-1, and VLA-4 had a very strong and additive inhibitory effect on rIL-1 beta-induced monocyte adherence. The anti-CD18 MoAb reduced both rIL-1 beta-induced neutrophil and monocyte migration far below the level of the unstimulated controls, whereas neither the anti-ELAM-1 nor the anti-VLA- 4 MoAb significantly affected the process of migration. Our results indicate that neutrophils and monocytes initially adhere to cytokine- activated endothelial cells by CD18-independent and (to a lesser extent) by CD18-dependent mechanisms and subsequently change gears to a completely CD18-dependent migratory mechanism.

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