Cytotoxicity Mediated by the Fas Ligand (FasL)-activated Apoptotic Pathway in Stem Cells

Molly Thomas,Julia Mazar,Joshua Zimmerberg,Ludmila Bezrukov,Alexander Chanturia,Gulcin Pekkurnaz,Pamela G. Robey,Shurong Yin,Sergei A. Kuznetsov

doi:10.1074/jbc.m109.032235

Abstract

Whereas it is now clear that human bone marrow stromal cells (BMSCs) can be immunosuppressive and escape cytotoxic lymphocytes (CTLs) in vitro and in vivo, the mechanisms of this phenomenon remain controversial. Here, we test the hypothesis that BMSCs suppress immune responses by Fas-mediated apoptosis of activated lymphocytes and find both Fas and FasL expression by primary BMSCs. Jurkat cells or activated lymphocytes were each killed by BMSCs after 72 h of co-incubation. In comparison, the cytotoxic effect of BMSCs on non-activated lymphocytes and on caspase-8(-/-) Jurkat cells was extremely low. Fas/Fc fusion protein strongly inhibited BMSC-induced lymphocyte apoptosis. Although we detected a high level of Fas expression in BMSCs, stimulation of Fas with anti-Fas antibody did not result in the expected BMSC apoptosis, regardless of concentration, suggesting a disruption of the Fas activation pathway. Thus BMSCs may have an endogenous mechanism to evade Fas-mediated apoptosis. Cumulatively, these data provide a parallel between adult stem/progenitor cells and cancer cells, consistent with the idea that stem/progenitor cells can use FasL to prevent lymphocyte attack by inducing lymphocyte apoptosis during the regeneration of injured tissues.

Highlights

In the present study, we hypothesize that bone marrow stromal cells (BMSCs)-mediated cytotoxicity of lymphocytes involves the FasL-activated apoptotic machinery
We show that BMSCs counterattack activated lymphocytes using the same mechanism as cancer cells: Fas-mediated apoptosis
Fas and FasL are co-expressed on primary BMSCs

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—Normal human BMSCs were isolated according to a previously published protocol [1] or were purchased (passage 1) from Cambrex Bio Science (Baltimore, MD). Cells were positive for CD105, CD166, CD29, and CD44 and negative for CD14, CD34, and CD45 (Ͼ95% of total cell number). BMSCs were cultured as a monolayer in mesenchymal stem cell growth medium (MSCGM; Cambrex Bio Science) containing 10% fetal bovine serum and antibiotics (Cambrex). When the cells had grown to 70 – 80% confluence, they were detached with trypsin/EDTA (Invitrogen). Wild-type Jurkat cells (TIB-152 ATCC) and caspase-8-negative (I 9.2; ATCC) Jurkat cells with a mutation in the cysteine protease, caspase-8/FLICE, were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, and 100 ␮g/ml streptomycin (Invitrogen). The lymphocytes were resuspended in RPMI 1640 medium supplemented

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RESULTS

DISCUSSION