Induction of monocyte-to-dendritic cell maturation by extracorporeal photochemotherapy: initiation via direct platelet signaling

Abstract

Extracorporeal Photochemotherapy (ECP) is a widely used therapy for cutaneous T cell lymphoma (CTCL). Although the mechanism of clinical action of ECP is not precisely established, previous studies have shown evidence of induction of dendritic cells (DCs). Here we show that, under flow conditions similar to those in post-capillary venules, ECP promotes platelet immobilization and activation, initiating stepwise receptor-ligand interactions with monocytes, which then differentiate into DC. These findings clarify how ECP directly stimulates DC maturation; suggest a new clinically applicable approach to the obtainment of DC; and identify a novel mechanism that may reflect physiological induction of DC.

Keywords: Dendritic cells; Extracorporeal photochemotherapy; Monocytes; P-selectin; Platelets.

Conflict of interest statement

Conflict of Interest Statement: Yale University owns patents deriving from the dendritic cell research of RT and RE. Although no products have been derived from these laboratory studies, it is possible that, along with their parent institution, these two authors could personally benefit from commercialization of these discoveries in the future.

Copyright © 2013 Elsevier Ltd. All rights reserved.

Figures

Figure 1. Effect of platelet density on number of monocyte-platelet interactions and subsequent monocyte phenotype

Monocytes were passed through parallel plates coated with platelets at low, medium, or high density. (A) The number of monocyte-platelet interactions increased substantially for plates coated with higher densities of platelets. (B) After overnight incubation, monocytes which were exposed to high levels of platelets were significantly more likely to develop a phenotype consistent with DC differentiation, as assessed by expression of membrane CD83 and HLA-DR (high versus medium or low density: p

Figure 2. Gene expression following exposure to platelets

Monocytes were exposed to high or low levels of platelets in flow. Following overnight incubation, cells were assessed for differences in gene expression using RT-PCR. Figure shows gene expression changes in monocytes exposed to high levels of platelets relative to those exposed to low levels. Seven genes associated with DC-differentiation and/or function were found to be upregulated, while three were downregulated. Of the genes downregulated, GPNMB and FPRL2 have known functions in decreasing cytokine production and inhibiting DC maturation, respectively. Of the genes upregulated, all have either pro-immune functions or miscellaneous roles in DC biology. See text for specific description of genes. Data shown are the means (± SE) of 2 independent experiments.

Figure 3. Plasma protein influence on platelet adhesion to plastic plates

(A) Platelets were passed through plates coated with fibrinogen (blue), plasma (red), fibronectin (purple), or RMPI (green) at the shear stress level indicated by the x-axis. Platelets in flow adhered optimally to fibronectin. For all proteins, platelet adhesion occurred maximally between 0.5 and 1.0 dyne/cm2. (B) Platelets were either untreated (baseline), or pretreated with either RGD fragments (+RGD) or (gamma fragments (+Gamma) and their subsequent adhesion to fibrinogen (left panel) and fibronectin (right panel) was assessed. Platelet binding to fibrinogen was decreased by gamma fragments (p < 0.05), while binding to fibronectin was decreased by RGD peptides (p < 0.001). lpf, low power field. Data shown are the means (± SD) of at least 2 independent experiments.

Figure 4. Effect of p-selectin exposure on monocyte integrins

Plastic plates were coated with platelets at the relative density indicated by the x-axis. Platelets were then pretreated with anti p-selectin (dashed line) or an isotype control (gray line), or received no pretretment (black line). Monocytes were passed through the plates at 0.5 dyne/cm2 and then immediately assessed by flowcytometry for expression of active β1 integrins. The y-axis indicates the percent of monocyte which bound an antibody directed at an epitope only exposed when the integrin is in the open confirmation. Data shown are the means (+/- SD) of 3 independent experiments.

Figure 5. Effect of P-selectin exposure on monocyte phenotype after overnight incubation

Platelet-coated plates were either untreated (first column), or pretreated with an isoptype control (second column) or anti-P-selectin (third column). Monocytes were passed through the plates at 0.5 dyne/cm2 then incubated overnight. The y-axis indicates the percent of monocytes which developed a phenotype consistent with DC differentiation, i.e., membrane HLA-DR+/CD83+. Data shown are the means (+/- SD) of 3 independent experiments.

Figure 6. Proposed mechanism for induction of monocyte-to-DC differentiation

Based on data presented in this manuscript, the following sequence of events is postulated: (1) plasma fibrinogen coats the plastic surface of the flow chamber; (2) through their αIIbβ3 receptor, unactivated platelets bind to the gamma-component of immobilized fibrinogen; (3) platelets become activated and instantaneously express preformed P-selectin and other surface proteins; (4) passaged monocytes transiently bind P-selectin via PSGL-1, causing partial monocyte activation and integrin receptor conformational changes; (5) partially-activated monocytes, now capable of further interactions, bind additional platelet-expressed ligands, including those containing RGD domains; (6) finally, so influenced, monocytes efficiently enter the DC maturational pathway within 18 hours. Note that, in-vivo, step (1) above may be replaced physiologically by inflammatory signals from tissue acting on local endothelium, causing it to recruit and activate platelets in a similar manner (see text).