Tumor-endothelium cross talk blocks recruitment of neutrophils to endothelial cells: a novel mechanism of endothelial cell anergy

Abstract

Tumor cells have evolved effective strategies to escape the host immune response. The objective of this study was to determine whether tumor cells can condition endothelial cells in a specific manner to prevent subsequent adhesion of polymorphonuclear neutrophils (PMNs) and/or peripheral blood lymphocytes (PBLs). Human umbilical vein endothelial cells (HUVECs) and UKF-NB-4 neuroblastoma tumor cells were established in coculture on opposite sides of porous transwell filters. After 24 hours with and without HUVEC conditioning, PMNs or PBLs were added to the HUVEC monolayer. Adhesion to conditioned HUVEC versus adhesion to nonconditioned HUVEC was compared. Effects on endothelial CD44v4, CD44v5, CD44v7, intercellular adhesion molecule 1 (ICAM-1), E-selectin, and vascular cell adhesion molecule 1 (VCAM-1) adhesion receptor expression were analyzed by flow cytometry, intracellular signaling proteins of the mitogen-activated protein kinase pathway and protein kinase C (PKC) subtypes quantified by Western blot analysis. Endothelial conditioning led to a distinct reduction in PMN but not in PBL adhesion to HUVEC. CD44 was significantly reduced, whereas ICAM-1, E-selectin, and VCAM-1 were not altered during HUVEC conditioning. Antibody blockade against CD44v4, CD44v5, and CD44v7 inhibited PMN but not PBL binding. The observed effects were caused by direct tumor cell-HUVEC contact because addition of isolated tumor cell membrane fragments but not of soluble cell culture supernatant to HUVEC induced the CD44 receptor loss. PKCalpha activity was strongly enhanced in conditioned HUVEC. Blocking PKC prevented the reduction in PMN binding, indicating that this protein is involved in PMN adhesion regulation. A novel tumor escape strategy is presented here. Cell contact-dependent adhesion of tumor cells to the vascular wall promotes down-regulation of endothelial CD44 receptor expression, impairing an effective neutrophil attack.

Figure 1

Fluorometric analysis of CD56 surface expression on HUVECs (A) versus UKF-NB-4 cells (B). Both cell types were stained separately with PE-conjugated monoclonal antibody anti-CD56, which detects the NCAM 120-, 140-, and 180-kDa isoforms. CD56 expression is depicted as dot blot (sideward scatter vs CD56) and FL-2H (log) channel histogram analysis. (C) A 24-hour contamination rate of CD56^- HUVEC by traversed CD56⁺ UKF-NB-4 cells in the transwell coculture assay. Cell populations growing on the upper surface of 8-µm pore-sized membranes were stained with an anti-CD56 monoclonal antibody and dot blot analysis carried out by a FACscan. One representative of three tests is shown.

Figure 2

PMNs' versus PBLs' adhesion to HUVECs. Isolated PMNs or PBLs were added to preconditioned HUVECs as described in the Materials and Methods section. Anti-CD45 PerCP monoclonal antibody was then used to distinguish between HUVECs (R1) and PMNs (R2; A) or PBLs (R2; B) by flow cytometry (SSC gating) and to display the percentage of PMNs or PBLs, which bound to HUVECs (%R2). Controls, indicating PBL/PMN binding to unstimulated or TNFα-stimulated HUVECs, were each set to 100%. “HUVEC/NB” indicates percent PBL/PMN binding to preconditioned HUVEC; “HUVEC^act/NB” indicates percent PBL/PMN binding to preconditioned, TNFα-activated HUVECs (C). One representative of n = 6 experiments. *Significantly different from controls. ^#Significantly different from HUVEC-NB.

Figure 3

Analysis of endothelial adhesion receptor expression. HUVECs were cocultivated with UKF-NB-4. CD56⁺ populations were gated out by flow cytometry (R1 vs R2) as described in the Materials and Methods section (A). E-selectin (CD62E), ICAM-1 (CD54), and P-selectin (CD106; B), and CD44v4, v5, and v7 (C) of CD56-negative HUVECs were then evaluated by FACS analysis (MFU indicates mean fluorescence units). “HUVEC” depicts isolated, nonconditioned endothelial cells (controls), MFU of which were set to 100%. “HUVEC/NB” indicates endothelial cells preconditioned by UKF-NB-4 tumor cells. One representative of n = 6 experiments. *Significantly different from control HUVECs.

Figure 4

Evaluation of CD44 expression level on HUVECs. HUVECs and UKF-NB-4 cells were grown on opposite sides of 3-µm cell inserts and endothelial CD44 expression analyzed thereafter (A), or HUVECs were subjected to isolated UKF-NB-4 membrane fragments and CD44 expression was evaluated subsequently (B). CD44 analysis was carried out by a FACscan using the appropriate monoclonal antibodies as listed in the Materials and Methods section (MFU indicates mean fluorescence units). Nonconditioned HUVECs served as controls; MFU values were set to 100%. One representative of six experiments is shown. *Significant difference from control HUVECs.

Figure 5

CD44 blockade prevents PMNs' adhesion to HUVECs. PMNs or PBLs were added to HUVEC monolayers (nonactivated vs TNFα activated) and then added at a density of 1.5 x 10⁵ cells per milliliter for 60 minutes. HUVECs have been preincubated for 60 minutes with anti-CD44v4, anti-CD44v5, or anti-CD44v7 monoclonal antibodies. Controls remained untreated. Nonadherent PMNs or PBLs were washed off in each sample; the remaining cells were fixed and counted in five different fields (5 x 0.25 mm²) using a phase-contrast microscope. Mean values were calculated from five counts. Mean adhesion capacity is depicted as counted cells per squared millimeter and related to control values that were set at 100%. One representative of six experiments is shown. *Significant difference from controls.

Figure 6

Cell signaling in conditioned versus nonconditioned HUVECs. UKF-NB-4 cells were added to subconfluent HUVEC monolayer for 16 hours. Cells were then detached from the culture flask by Accutase treatment and HUVECs were isolated by magnetic separation (negative selection). The selected population (R1) obtained by this procedure was greater than 95% as controlled by flow cytometry (A). ERK and PKC signaling was explored in control HUVECs and HUVECs preconditioned with UKF-NB-4 cells (HUVEC-NB) by Western blot analysis (B). β-Actin served as the internal control. One representative of three experiments is shown. (C) Relevance of PKC blocking for PMNs' adhesion. A total of 0.5 x 10⁶ PMNs per milliliter were added to preconditioned HUVECs (HUVEC-NB), to preconditioned HUVECs incubated with the PKC blocking antibody BIM I, or to control HUVECs for 60 minutes as described in the Materials and Methods section. Bound PMN were identified by flow cytometry using the leukocyte-specific anti-CD45 monoclonal antibody. SSC gating was set up to display percentage of PMNs that bound to HUVECs (controls = 100%). *Significant difference from controls. ^#Significant difference from HUVEC-NB.