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. 2014 Mar;175(3):449-57.
doi: 10.1111/cei.12231.

Integrin-driven monocyte to dendritic cell conversion in modified extracorporeal photochemotherapy

A L Gonzalez  1 , C L Berger, J Remington, M Girardi, R E Tigelaar, R L Edelson
Affiliations

Integrin-driven monocyte to dendritic cell conversion in modified extracorporeal photochemotherapy

A L Gonzalez et al. Clin Exp Immunol. 2014 Mar.

Abstract

Due to clinical efficacy and safety profile, extracorporeal photochemotherapy (ECP) is a commonly used cell treatment for patients with cutaneous T cell lymphoma (CTCL) and graft-versus-host disease (GVHD). The capacity of ECP to induce dendritic antigen-presenting cell (DC)-mediated selective immunization or immunosuppression suggests a novel mechanism involving pivotal cell signalling processes that have yet to be clearly identified as related to this procedure. In this study we employ two model systems of ECP to dissect the role of integrin signalling and adsorbed plasma proteins in monocyte-to-DC differentiation. We demonstrate that monocytes that were passed through protein-modified ECP plates adhered transiently to plasma proteins, including fibronectin, adsorbed to the plastic ECP plate and activated signalling pathways that initiate monocyte-to-DC conversion. Plasma protein adsorption facilitated 54·2 ± 4·7% differentiation, while fibronectin supported 29·8 ± 7·2% differentiation, as detected by DC phenotypic expression of membrane CD80 and CD86, as well as CD36, human leucocyte antigen D-related (HLA-DR) and cytoplasmic CD83. Further, we demonstrate the ability of fibronectin and other plasma proteins to act through cell adhesion via the ubiquitous arginine-glycine-aspartic (RGD) motif to drive monocyte-to-DC differentiation, with high-density RGD substrates supporting 54·1 ± 5·8% differentiation via αVβ3 and α5β1integrin signalling. Our results demonstrate that plasma protein binding integrins and plasma proteins operate through specific binding domains to induce monocyte-to-DC differentiation in ECP, providing a mechanism that can be harnessed to enhance ECP efficacy.

Keywords: DC; integrins; monocyte; transimmunization.

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Figures

Figure 1
Figure 1
Extracorporeal photochemotherapy (ECP) model systems and potential signalling mechanism. (a) Benchtop mock ECP system using in hospital ECP plastic exposure plate (Therakos®), ultraviolet (UVA) source and peristaltic pump. (b) Integrin-specific induction of ECP-type monocyte to dendritic cell (DC) differentiation using polyethylene glycol based hydrogels with and without bioactive arginine–glycine–aspartic (RGD) sequence. (c) Hypothesis of ECP-induced signal transduction pathway, initiated through ECP plate adsorbed plasma protein binding to monocyte-presented integrins. Integrins associate with intracellular proteins to form focal adhesions, capable of signalling through mitogen-activated protein kinase/extracellular-regulated kinase (MEK/ERK) pathways to induce survival and differentiation.
Figure 2
Figure 2
Plastic adherent plasma protein and fibronectin stimulated monocyte-to-dendritic cell (DC) conversion and characterization. Extracorporeal photochemotherapy (ECP)-treated cells were cultured overnight and analysed by flow cytometry gated on the monocyte/DC population. DC were identified by co-expression of membrane CD80 and CD86 and membrane staining for CD36 or human leucocyte antigen D-related (HLA-DR) combined with cytoplasmic reactivity with CD83. (a) Two-colour histograms from freshly isolated monocytes (pre-ECP) and overnight cultured ECP treated using plasma-coated [post-ECP (plasma)] or fibronectin-coated [post-ECP (fibronectin)] exposure plates. (b) Average fraction of DC obtained using plasma-coated [post ECP (plasma), mean ± standard error (s.e.)] or fibronectin-coated [post-ECP (fibronectin), mean ± s.e.] exposure plates compared to freshly isolated monocytes (pre-ECP, mean ± s.e.). *P < 0·05; **P < 0·01 as confirmed by analysis of variance (anova) and paired Student's t-test. (c) Monocyte-to-DC conversion was supported further by an increase in the mean fluorescence intensity (MFI) of the co-stimulatory molecule CD86 and a decrease in the MFI of CD36 post-ECP. Both trends are characteristic of DC maturation, signifying an increase in antigen-presenting capacity and decrease in the ability to phagocytose apoptotic cells. One-colour histograms were normalized to 100% of peak value. (d) Phase-contrast microscopy demonstrates appropriate dendrite formation on ECP differentiated DC on plasma- and fibronectin-coated plates.
Figure 3
Figure 3
Monocyte-to-dendritic cell (DC) conversion is stimulated by arginine–glycine–aspartic (RGD)-conjugated polyethylene glycol (PEG) hydrogels. (a) Modulation of integrin subunit expression after overnight incubation of extracorporeal photochemotherapy (ECP)-treated samples (post-ECP, n = 3) in comparison to peripheral blood monocytes (pre-ECP, n = 4). **P < 0·01 as confirmed by Student's t-test. (b) Purified monocytes were cultured overnight on PEG hydrogels conjugated with RGD (PEG–RGD) or unmodified PEG hydrogels (PEG). Dendritic cells were identified by co-expression of membrane CD80 and CD86 and membrane staining for CD36 or human leucocyte antigen D-related (HLA-DR) combined with cytoplasmic reactivity with CD83. Representative two-colour histograms from freshly isolated monocytes cultivated on PEG or PEG–RGD hydrogels. (c) Average fraction of DC obtained using PEG–RGD or PEG hydrogels. *P < 0·05; **P < 0·01; ***P < 0·0001 as confirmed by analysis of variance (anova) and paired Student's t-test. (d) Phase-contrast microscopy demonstrates poor dendrite formation on ECP differentiated DC on PEG with improved morphology on PEG–RGD.
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