2008 PBL Cat_mech_r.qxd:PBL Catalog.mech 11/29/07 1:50 PM Page 5
Chapter 1: Interferons, Cytokines & Growth Factors Application Note
Stat2 dependent regulation of MHC class II expression
Wenli Zhao*
1
, Edward N. Cha*
1
, Carolyn Lee
1
, and Christian Schindler
1,2,†
Departments of
1
Microbiology &
2
Medicine, Columbia University, New York, NY, 10032
Introduction
IFN-γ (type II IFN), whose expression is tightly regulated, is a potent inducer of conditioned media for 5 -10 days on 10 cm petri dishes (5). HEK-293T cells
Major Histocompatibility Complex type II (MHC II), especially in macrophages (1, and L929 cells (L-cells), from ATCC (Manassas, Virginia, USA), were cultured
2). In contrast, more pervasively expressed type I IFNs (IFN-Is; e.g., IFN-α/IFN-β) in DMEM (GIBCO; Grand Island, NY), supplemented with 10% fetal bovine
fail to induce MHC II expression, even though they activate an overlapping serum. Splenocytes were harvested, filtered through a nylon mesh, counted,
intracellular signaling pathway. Specifically, both classes of IFNs transduce and then cultured in RPMI 1640 / 10% FCS. IFN stimulation entailed the
signals through Stat1 (Signal transducer and activator of transcription 1), the addition of “universal” human IFN-αA/D (1000 U/ml; PBL product #11200-1),
critical regulator of MHC II expression (3, 4). However, IFN-Is also uniquely or murine IFN-γ(66 U/ml; PBL), as indicated.
activate a second intracellular signaling molecule, Stat2, which directs the • Flow cytometry: After IFN treatment (24-72 hrs), peritoneal and BM
formation of a distinct transcription factor, ISGF-3 (i.e., Stat1 + Stat2 + IRF-9). macrophages were fixed and and stained with CD11b (M1/70, eBioscience,
The surprising observation that IFN-α acquired the ability to effectively San Diego, CA), CD11c (HL3, BD Pharmingen, San Diego, CA), I-A
b
(BD
induce MHC II expression in Stat2-/- macrophages provided an opportunity to Pharmingen), streptavidin-PerCP (BD Pharmingen), or an isotype matched
explore how the expression of this important regulator of immune response is control. Recovered (i.e., by gentle scraping), stained samples were analyzed
controlled (4, 5). These studies determined that deletion of Stat2 is associated on a FACS-Calibur with Cellquest software (BD Biosciences, San Jose, CA), as
with a loss in the IFN-αdependent induction of an important negative regulator, previously reported (5).
Socs-1 (Suppressor of cytokine signaling-1; (4, 6)). In the absence of Socs-1, • Biochemical studies: Whole cell and nuclear extracts were prepared from IFN
IFN-α stimulated Stat1 activation is significantly prolonged, exceeding the treated cells and evaluated by immunoblotting or electrophoretic mobility
threshold required to induce MHC-II expression. Thus, IFN-I dependent Socs-1 shift assay (EMSA), as previously reported (5, 8). Antibodies employed in
expression plays an important role in distinguishing the biological response super-shifts, immunoblotting studies and ChIP assays included: Stat1 (9),
between type I and II IFNs in macrophages (4). Stat2 (5), phosphotyrosine-Stat1 (#9171S, Cell Signaling Technology Inc.,
Beverly, MA), phosphotyrosine-Stat2 (#07-224, Upstate Group,
Materials and Methods Charlottesville, VA) and β-actin (sc-1616, Santa Cruz Biotechnology, Santa
• Mice: Stat2-/-, Stat1-/-, and wild type (5, 7), all on a pure 129/Sv background, Cruz, CA), as previously reported (10). EMSAs were carried out with either an
were housed under specific pathogen-free conditions. Stat1/Stat2 double IRF-1 GAS or OAS ISRE probe (see Table 1; (5)).
knockout mice, obtained by crossing the appropriate strains, were • RNA: Total RNA was prepared from BMMs by Trizol (Invitrogen, Carlsbad, CA)
monitored by PCR-based genotyping and confirmed by immunoblotting, as extraction. For Q-PCR, five ∝gs of total RNA was treated with RQ1 DNAse
previously reported (5). (Promega, Madison WI) and then reverse transcribed with SuperScript™ II
• Cell culture: Peritoneal macrophages (nonelicited) were harvested by lavage (Invitrogen, Carlsbad, CA). The cDNA was quantitatively amplified in an ABI
and cultured in RPMI 1640 (GIBCO; Grand Island, NY) supplemented with 10% Prism 7700 in a SYBR green master mix (Applied Biosystems, Foster City,
FCS (Hyclone, Logan, UT). Bone marrow macrophages (BMM), harvested CA), as previously described ((11); see Table 1). Gene expression was
from femurs, were grown in RPMI 1640 supplemented with 20% L-cell normalized to a β-actin control.
Q-PCR ß-actin Forward - 5' GCT CCT CCT GAG CGC AAG T3' / Reverse - 5' TCG TCA TAC TCC TGC TTG CTG AT 3'
Socs-1 Forward - 5' ACT CAC TTC CGC ACC TTC C 3' / Reverse - 5' AAG CAG TTC CGT TGG CGA C 3'
Socs-3 Forward - 5' GCC GTT GAC AGT CTT CCG A 3' / Reverse - 5' TCT TTG CCA CCC ACG GAA CC 3'
EMSA dsOAS ISRE† 5' agct TCTGAG GAAAC GAAAC CAACAG 3'
dsIRF-1 GAS† 5' gatc GATTT CCCCG AAAT 3'
Table 1. Sequence of oligonucleotides
†Palindromic oligonucleotides are self-annealed to generate double stranded probe.
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