A novel selective leukocyte depletion human whole blood model reveals the specific roles of monocytes and granulocytes in the cytokine response to Escherichia coli

Abstract

The lepirudin-based human whole blood model is a well-established ex vivo system to characterize inflammatory responses. However, the contribution of individual cell populations to cytokine release has not been investigated. Thus, we modified the model by selectively removing leukocyte sub-populations to elucidate their contribution to the inflammatory response. Lepirudin-anticoagulated whole blood was depleted from monocytes or granulocytes using StraightFrom® Whole Blood Microbeads. Reconstituted blood was incubated with Escherichia coli (108/mL) for 2 hours at 37°C. CD11b, CD62P and CD63 were detected by flow cytometry. Complement (C3bc, sC5b-9) and platelet activation (PF4, NAP-2) were measured by ELISA. Cytokines were quantified by multiplex assay. A significant (p < 0.05) specific depletion of the monocyte (mean 86%; CI:71-92) and granulocyte (mean 97%; CI:96-98) population was obtained. Background activation induced by the depletion protocol was negligible for complement (C3bc and sC5b-9), leukocytes (CD11b) and platelets (NAP-2). Upon Escherichia coli incubation, release of 10 of the 24 cytokines was solely dependent on monocytes (IL-1β, IL-2, IL-4, IL-5, IL-17A, IFN-γ, G-CSF, GM-CSF, MIP-1α, and FGF-basic), whereas eight were dependent on both monocytes and granulocytes (IL-1ra, IL-6, IL-8, IL-9, IL-10, MIP-1β, TNF, and eotaxin). Six cytokines were not monocyte- or granulocyte-dependent, of which PDGF and RANTES were mainly platelet-dependent. We document an effective model for selective depletion of leukocyte sub-populations from whole blood, without causing background activation, allowing in-depth cellular characterization. The results are in accordance with monocytes playing a major role in cytokine release and expands our knowledge of the significant role of granulocytes in the response to E. coli.