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Breast Cancer Research

, 8:R5

First Online: 16 December 2005Received: 03 September 2005Revised: 24 October 2005Accepted: 01 November 2005

Abstract

IntroductionDendritic cells DCs are key antigen-presenting cells that play an essential role in initiating and directing cellular and humoral immunity, including anti-tumor responses. Due to their critical role in cancer, induction of DC apoptosis may be one of the central mechanisms used by tumors to evade immune recognition.

MethodsSpontaneous apoptosis of blood DCs lineage negative HLA-DR positive cells was assessed in peripheral blood mononuclear cells PBMCs using Annexin-V and TUNEL assays immediately after blood collection. The role of tumor products was assessed by culturing cells with supernatants derived from breast cancer cell lines TDSN or PBMCs PBMC-SN, as a control. The capacity of DC stimulation to prevent apoptosis was assessed by incubating DC with inflammatory cytokines, poly I:C, IL-12 or CD40 ligand CD40L prior to culture with TDSN. Apoptosis was determined by flow cytometry and microscopy, and Bcl-2 expression determined by intracellular staining.

ResultsIn this study we document the presence of a significantly higher proportion of apoptotic Annexin-V and TUNEL blood DCs in patients with early stage breast cancer stage I to II; n = 13 compared to healthy volunteers n = 15. We examined the role of tumor products in this phenomenon and show that supernatants derived from breast cancer lines induce apoptosis of blood DCs in PBMC cultures. Aiming to identify factors that protect blood DC from apoptosis, we compared a range of clinically available maturation stimuli, including inflammatory cytokines tumor necrosis factor-α, IL-1β, IL-6 and prostaglandin PGE2 as a cytokine cocktail, synthetic double-stranded RNA poly I:C and soluble CD40 ligand. Although inflammatory cytokines and poly I:C induced robust phenotypic maturation, they failed to protect blood DCs from apoptosis. In contrast, CD40 stimulation induced strong antigen uptake, secretion of IL-12 and protected blood DCs from apoptosis through sustained expression of Bcl-2. Exogenous IL-12 provided similar Bcl-2 mediated protection, suggesting that CD40L effect is mediated, at least in part, through IL-12 secretion.

ConclusionCumulatively, our results demonstrate spontaneous apoptosis of blood DCs in patients with breast cancer and confirm that ex vivo conditioning of blood DCs can protect them from tumor-induced apoptosis.

AbbreviationsAPCallophycocyanin

CCcytokine cocktail

DCdendritic cell

FACSfluorescence activated cell sorting

FITCfluorescein isothiocyanate

FSCForward side characteristic

ILinterleukin

Linlineage markers

LinHLA-DRlineage negative HLA-DR positive

PBMCperipheral blood mononuclear cell

PBMC-SNPBMC-conditioned supernatant

PBSphosphate-buffered saline

PEphycoerythrin

PGprostaglandin

SSCside scatter characteristic

TDSNtumor-derived supernatant

TNFtumor necrosis factor

TLRToll-like receptor

ΔMFIdelta mean fluorescence intensity

7-AAD7-Aminoactinomycin D.

Electronic supplementary materialThe online version of this article doi:10.1186-bcr1361 contains supplementary material, which is available to authorized users.

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Autor: Alberto Pinzon-Charry - Tammy Maxwell - Michael A McGuckin - Chris Schmidt - Colin Furnival - J Alejandro López

Fuente: https://link.springer.com/



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