The Role of NF-kB as a Survival Factor in Environmental Chemical-Induced Pre-B Cell Apoptosis

Polycyclic aromatic hydrocarbons (PAH) are ubiquitous environmental chemicals that suppress the immune system at multiple levels, including at the level of B cell development in the bone marrow microenvironment. Specifically, PAH induce preB cell apoptosis in primary bone marrow cultures and in cocultures of an early preB cell line (BU-11) and a bone marrow stromal cell line (BMS2). Previous studies focused on the molecular mechanisms through which PAH induce stromal cells to deliver an apoptosis signal to adjacent preB cells. Apoptosis signaling within the preB cell itself was not investigated. Here, the role of NF-kB, a lymphocyte survival factor, in PAH-induced preB cell apoptosis was assessed. Analysis of DNA-binding proteins extracted from the nuclei of untreated BU-11 cells indicated DNA-binding complexes comprising NF-kB subunits p50, c-Rel, and/or Rel A. NF-kB down-regulation with previously described inhibitors induced BU-11 cell apoptosis, demonstrating that the default apoptosis pathway blocked by NF-kB is functional at this early stage in B cell development. Similarly, exposure of BU-11/BMS2 cocultures to 7,12-dimethylbenz[a]anthracene (DMBA), a prototypic PAH, down-regulated nuclear Rel A and c-Rel before overt apoptosis. Finally, ectopic expression of Rel A or c-Rel rescued BU-11 cells from DMBA-induced apoptosis. These results extend previous observations by demonstrating that 1) NF-kB is a survival factor at an earlier stage of B cell development than previously appreciated and 2) NF-kB down-regulation is likely to be part of the molecular mechanism resulting in PAH-induced preB cell apoptosis. These results suggest nonclonally restricted, PAH-mediated suppression of B lymphopoiesis. Polycyclic aromatic hydrocarbons (PAH), relatively common environmental contaminants, are immunotoxic (Day et al., 1990; Davis et al., 1991; Ladics et al., 1992; Temple et al., 1993; Szczeklik et al., 1994; Davilla et al., 1996). In animal models and/or human lymphocyte cultures, PAH decrease resistance to infectious agents and transplantable tumors, impair B and T lymphocyte proliferation, inhibit B cell antibody responses, suppress cytokine production, and decrease bone marrow cellularity. However, the molecular mechanisms through which these outcomes are effected have not been adequately described. To dissect the intracellular signals activated by PAH and resulting in adverse effects on the immune system, we have evaluated the effects of low PAH doses on early preB cell growth with in vitro models of B cell development in the bone marrow microenvironment (Hinoshita et al., 1992; Yamaguchi et al., 1997a,b; Mann et al., 1999; Near et al., 1999). In primary bone marrow cultures and in a coculture system consisting of an early (CD43) preB cell line (BU-11) and a cloned bone marrow stromal cell line on which preB cells depend for growth (BMS2), it was shown that low doses ($10 M) of prototypic PAH (benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene) rapidly induce preB cell apoptosis. In evaluating the role of stromal cells in PAH-induced preB cell apoptosis, it was shown that 1) stromal cells are required for preB cell apoptosis (Yamaguchi et al., 1997a,b), 2) PAH-treated stromal cells or liver parenchymal cells deliver a death signal to adjacent preB cells (Mann et al., 1999; Near et al., 1999), 3) induction of this death signal is, at low PAH doses, dependent on activation of the aryl hydrocarbon receptor/transcription factor within stromal cells (Mann et al., 1999; Near et al., 1999), and 4) PAH metabolism within stromal cells likely plays a role in the generation of the apoptosis signal (Mann et al., 1999). Recent results in a related system in which apoptosis of a stromal cell-independent preB cell line (70Z/3) was induced Supported by National Institutes of Health Grant RO1-ES06086, Superfund Basic Research Grant #1P42ES 07381, an EPA STAR fellowship to K.K.M., and an NRSA fellowship to J.J.S. D.H.S. and S.Q. contributed equally to this project. ABBREVIATIONS: PAH, polycyclic aromatic hydrocarbon(s); DMBA, 7,12-dimethylbenz[a]anthracene; EMSA, electromobility gel shift assay(s); NF-kB, nuclear factor kB; IkB, inhibitor kB; MG-132, Z-Leu-Leu-Leu-CHO; PDTC, pyrrolidinedithiocarbamate; PI, propidium iodide; sIg, surface Ig; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone; PBS, phosphate-buffered saline; BrdU, bromodeoxyuridine; PAGE, polyacrylamide gel electrophoresis; IL, interleukin. 0026-895X/01/5902-302–309$3.00 MOLECULAR PHARMACOLOGY Vol. 59, No. 2 Copyright © 2001 The American Society for Pharmacology and Experimental Therapeutics 262/879751 Mol Pharmacol 59:302–309, 2001 Printed in U.S.A. 302 at A PE T Jornals on Sptem er 0, 2017 m oharm .aspeurnals.org D ow nladed from with DMBA are consistent with the latter conclusion (Heidel et al., 1998, 1999). These studies were extended herein to evaluate the molecular signals activated within early preB cells that result in their demise following delivery of the stromal cell-derived, PAH-induced apoptosis signal. In particular, the role of NFkB, a survival factor in several mature cell types (Antwerp et al., 1996; Beg and Baltimore, 1996; Wang et al., 1996; Wu et al., 1996a,b; Karin, 1998), was investigated. Our previous studies indicated that cross-linking surface Ig receptors on cells of an immature B cell line, WEHI-231, resulted in NF-kB down-regulation and apoptosis induction (Wu et al., 1996a,b). These results implicate NF-kB modulation in B cell clonal deletion induced by high-affinity Ig receptor interactions with autoantigens. Although clonal deletion begins when immature B cells acquire Ig heavy-chain and surrogate light-chain receptors, it is formally possible that the NF-kBdependent apoptosis pathway is intact at an earlier stage of B cell development. Consequently, PAH-induced preB cell apoptosis could be mediated by a clonally nonrestricted down-regulation of NF-kB. This hypothesis was tested by 1) determining whether an early (i.e., CD43) stromal celldependent preB cell line constitutively expresses nuclear, DNA-binding NF-kB, 2) testing if NF-kB down-regulation with specific inhibitors induces BU-11 cell apoptosis, 3) analyzing NF-kB-DNA binding following exposure of BU-11/ BMS2 cell cultures to DMBA, and 4) attempting to rescue BU-11 cells from DMBA-induced apoptosis by transfection with the NF-kB subunits c-Rel or Rel A (p65). The data are consistent with NF-kB expression at an earlier point in B cell development than previously demonstrated and with a critical role for NF-kB regulation in PAH-induced early preB cell apoptosis. Materials and Methods Cell Culture and Treatments. The stromal cell-independent late preB line 70Z/3 and the sIg WEHI-231 line were maintained in RPMI 1640 media with 10% fetal bovine serum, penicillin/streptomycin, L-glutamine, and 2-mercaptoethanol at 37°C in a humidified 10% CO2 chamber. The stromal cell-dependent C57BL/6-derived BU-11 cell line has been previously characterized (Near et al., 1999; Mann et al., 1999; Yamaguchi et al., 1997a,b). BU-11 cells express both CD43 and B220 and do not contain rearranged Ig heavy chain genes. Therefore they represent B cells at the transition point between the proand early preB cell stages (Hardy et al., 1991). For convenience, it is referred to as an early preB cell line. BMS2 is a culture dish-adherent, cloned bone marrow stromal cell line which supports preB cell growth (Pietrangeli et al., 1988). Cultures of BU-11 cells maintained on BMS2 cell monolayers were grown in RPMI 1640 media with 5% fetal bovine serum, penicillin/streptomycin, L-glutamine, and 2-mercaptoethanol. Cocultures were treated with vehicle (acetone, final concentration of 0.1%), DMBA (Sigma/ BRL Chemical Co., St. Louis, MO), 125 to 12.5 mM N-tosyl-L-phenylalanine chloromethyl ketone (TPCK; Sigma/BRL), 50 to 0.5 mM pyrrolidinedithiocarbamate (PDTC; Sigma), 10 to 2.5 mM lactacystin (Alexis Biochemicals, San Diego, CA), or 0.1 to 0.4 mM Z-Leu-LeuLeu-CHO (MG-132; Biomol Inc., Plymouth Meeting, PA). BU-11 cells were harvested 12 to 24 h later, and the percentage of cells undergoing apoptosis was quantitated by propidium iodide staining and