A Novel Lipopolysaccharide-response Element Contributes to Induction of Nitric Oxide Synthase*

(Received for publication, January 24, 1997, and in revised form, March 21, 1997)

Qiao-wen Xie Dagger

From the Beatrice and Samuel A. Seaver Laboratory, Department of Medicine, Cornell University Medical College, New York, New York 10021

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The gene encoding the high output isoform of nitric oxide synthase represents a large class of alarm and defense genes transcriptionally induced in response to bacterial lipopolysaccharide (LPS). The promoters of most of these genes contain at least two LPS-response elements, one of which commonly binds transcription factors of the NF-kappa B/Rel family. Here a novel LPS-response element is identified in the inducible nitric oxide synthase promoter, termed LREAA, which contains critical adenosine residues lying 19-20 base pairs downstream of the proximal NF-kappa B binding element (NFkappa Bd). Both NFkappa Bd and LREAA are required for LPS-induced promoter activity. A protein partially recognized by antibody against transcription factor Oct-1 binds to the LREAA element constitutively in untreated macrophages while contributing to a DNA-protein complex that includes NF-kappa B p50 in macrophages treated with LPS. NF-kappa B p50 and the LREAA-binding proteins may together recruit an LPS-triggered transactivator of transcription.


INTRODUCTION

Endotoxic lipopolysaccharide (LPS),1 the major constituent of the cell walls of Gram-negative bacteria, is one of the most potent agonists in biology. Mammalian, avian, and insect phagocytes (1-4) respond to LPS by altering the expression of numerous genes. This response can help protect the host from infection but can also cause systemic inflammation (1, 2). A recent review catalogued 21 mammalian genes induced by LPS, and in 19 of these LPS-response elements have been characterized (5). Transcription factors binding to these promoter elements have been identified with the following frequency: NF-kappa B/Rel, 14 genes; NF-IL6, 9 genes; AP-1, 2 genes; and IL1beta -UNF1, NF-1beta , NF-beta A, c-Jun/ATF2, IRSE-binding protein, NF-M, ATF family members, and c-Jun, 1 gene each.

LPS-inducible genes include NOS2 (iNOS), encoding the high output isoform of nitric oxide synthase (6, 7), which contributes to both antimicrobial defense (8) and LPS-induced hypotension (9). Transcriptional induction of iNOS by LPS (6, 10, 11) requires binding of transcription factor NF-kappa B/Rel to a promoter element, NFkappa Bd (12), at position -85 to -76 (13-16). Another NF-kappa B binding site NFkappa Bu (12) at position -971 to -962 (13, 14) also plays a role (16).

Although NF-kappa B/Rel is essential for the LPS induction of iNOS, it is not sufficient (12, 17). Additional transcription factors are required (12), which is consistent with the precedent that at least two-thirds of LPS-induced genes studied have more than one LPS-response element (5). In the present work, a second LPS-response element (LREAA) is identified in the iNOS promoter that has not been previously recognized in any gene.


EXPERIMENTAL PROCEDURES

Cell Culture

The macrophage cell line RAW 264.7 (American Type Culture Collection) was cultured in complete medium (RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 200 µg/ml penicillin and streptomycin) as described (13).

Reagents

Poly(dI-dC)·poly(dI-dC) was from Pharmacia Biotech Inc. and isotopes were from Amersham Corp. Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) supplied affinity-purified goat or rabbit IgGs against synthetic peptides derived from NF-kappa B/Rel proteins p50, p52, p65 (RelA), c-Rel (C), and RelB, and from transcription factors Oct-1, Oct-2, Pit-1, Stat1(p91), c-Jun/AP-1, c-Fos, Ets-1/Ets-2, and C/EBPalpha . Rabbit IgG against purified recombinant murine IRF-1 was prepared by Teruaki Nomura and Heinz Ruffner and kindly provided by Dr. Luis Reis (University of Zurich, Zurich, Switzerland). LPS from Escherichia coli 0111:B4 was from Sigma.

Plasmids

Plasmids p7iNOS-CAT and p8.11iNOS-CAT contain the iNOS promoter and a reporter gene for chloramphenicol acetyltransferase (CAT). p7iNOS-CAT (18) contains a full-length promoter, including a basal promoter and an upstream enhancer; p8.11iNOS-CAT (12) contains only the basal promoter. Polymerase chain reaction-engineered mutations of element NFkappa Bd (from GGG to CTC) and of element LREAA (from AA to CG) were individually introduced into p8.11iNOS-CAT to form p8.11kappa Bm and p8.11LREm, respectively. Similarly, mutant constructs p7kappa Bm and p7LREm were formed from p7iNOS-CAT. DNA sequence analysis confirmed the mutation of these elements without unwanted mutations elsewhere.

Transient Transfection and CAT Assays

RAW 264.7 cells were transfected by a modification of the DEAE-dextran procedure (19). The DNA used for transfection was prepared by the EndoFree plasmid kit (QIAGEN Inc., Chatsworth, CA). Media and DNA at the concentrations used contained <50 pg/ml LPS (QCL-1000 kit, BioWhittaker, Inc., Walkersville, MD). Cells were washed twice with serum-free RPMI and suspended at 5-9 × 106/ml in RPMI prewarmed to 37 °C containing DEAE-dextran (250 µg/ml) and 50 mM Tris (pH 7.4). 10 µg of DNA was added to 2 ml of cell suspension at 37 °C for 45 min with occasional shaking. Cells were shocked with 10% Me2SO for 1 min at room temperature, washed, and distributed to two 100-mm plates with 10 ml of complete medium. After 24 h at 37 °C, 5% CO2, LPS (100 ng/ml) was added to some of the plates. About 16 h later, the cells were washed with ice-cold phosphate-buffered saline, resuspended in 0.25 mM Tris (pH 8.0), and frozen and thawed 3 times. Lysates were centrifuged (11,700 × g, 10 min, 4 °C), and the supernatant was heated at 65 °C for 10 min to inactivate CAT inhibitors and then centrifuged as above. The supernatant (1 or 10 µg) was assayed for CAT by a TLC method (20), and protein content was determined (21).

Oligonucleotides and Probes

Single-stranded oligonucleotides (Oligos Etc., Inc., Guilford, CT) were annealed with the complementary strand by polymerase chain reaction to form double-stranded oligomers with 5' overhang. To prepare probes (see Table I), double-stranded oligomers were filled in by the Klenow fragment of DNA polymerase I with [alpha -32P]dCTP and the three other nonradiolabeled dNTPs. To prepare competitors, all four dNTPs were nonradiolabeled.

Table I. Oligonucleotides used

Only one strand is shown. Uppercase sequences are from the promoter. The NFkappa Bd site and AA of LREAA are underlined with asterisks indicating mutations.

Name Sequence (5' right-arrow 3')

      -85  NFkappa Bd                   LREAA -50                -31
B     caTGGGGACTCTCCCTTTGGGAACAGTTATGCAAAATAGCTCTGCAGAGCCTGGAGGGG
D     caTGGGGACTCTCCCTTTGGGAACAGTTATGCAAAATAGC
Dkappa Bm     caTGCTCACTCTCCCTTTGGGAACAGTTATGCAAAATAGCTCTGCAG
        ***
DLREm     caTGGGGACTCTCCCTTTGGGAACAGTTATGCACGATAGCTCTGCAG
                                     **
Dkappa BmLREm     caTGCTCACTCTCCCTTTGGGAACAGTTATGCACGATAGCTCTGCAG
                   ***                          **
A gaagctTGGGGACTCTCCCTTTGGGAAC
Akappa Bm       TGCTCACTCTCCCTTTGGGAAC
        ***
C               caagCTTTGGGAACAGTTATGCAAAATAGC
CLREm               caagCTTTGGGAACAGTTATGCACGATAGC
                                     **

Electrophoretic Mobility Shift Assay

Binding was tested in 15 µl of solution by incubating 5 µg of nuclear extract (22) with reaction buffer (20 mM HEPES, pH 7.9, 1 mM EDTA, 60 mM KCl, 12% glycerol, 1 mM dithiothreitol, 2 µg of poly(dI-dC)·poly(dI-dC)) in the presence or absence of competitor or antibody for 20 min, followed by a 20-min incubation at room temperature with probe (>= 20,000 cpm). Products were electrophoresed at 30 mA for at least 3 h on 4.8% polyacrylamide gels in high ionic strength buffer (50 mM Tris, 380 mM glycine, 2 mM EDTA, pH ~8.5) (23), and dried gels were analyzed by autoradiography.

UV Cross-linking Analysis

50 µg of nuclear extract and 106 cpm of bromodeoxyuridine-containing probe Dkappa Bm were reacted in a total volume of 60 µl. The electrophoretic mobility shift assay gel was exposed to x-ray film for 10 min to localize the complex of interest. Excised gel was UV-irradiated (366 nm) 5 cm from an inverted transilluminator at 4 °C for 60 min, boiled with sample buffer for 5 min, and analyzed by 10% SDS-polyacrylamide gel electrophoresis and autoradiography.


RESULTS

An LPS-response Element (LREAA) Distinct from NFkappa Bd Confers Inducibility of the iNOS Promoter by LPS

Construct p8.11iNOS-CAT contains a fragment of the 5'-flanking region of the mouse iNOS gene (-85 to +161) that includes the NFkappa Bd element (-85 to -76) and confers LPS-inducible promoter activity on transfected RAW 264.7 mouse macrophages (12). LPS responsiveness was lost in p8.13iNOS-CAT, from which the the NFkappa Bd element was eliminated (12). In this work, two mutated constructs derived from p8.11iNOS-CAT were prepared by (Fig. 1A) and tested for their promoter activity as induced by LPS (Fig. 1B). In p8.11kappa Bm, three nucleotides of the NFkappa Bd element were mutated, from GGGACTCTCC to CTCACTCTCC. This reduced LPS-induced promoter activity to 3.2 ± 1.3% of wild type (mean ± S.D., three experiments), consistent with an earlier finding that nuclear protein failed to bind to the NFkappa Bd element when it carried the same mutation (12). Surprisingly, mutation of two nucleotides (from AA to CG) located 19-20 base pairs downstream from the 3' end of the NFkappa Bd element, as seen in p8.11LREm, also nearly abolished LPS-induced promoter activity leaving only 2.0 ± 0.3% as much activity as in wild type (mean ± S.D., three experiments). This new LPS-response element will be called LREAA. When the same mutations of elements NFkappa Bd and LREAA were individually introduced into the full-length promoter construct p7iNOS-CAT (Fig. 1A) to form p7kBm and p7LREm, LPS-induced transcription was also substantially reduced (Fig. 1C). Therefore, both elements play an important role in the full-length promoter as well as in small fragments.


Fig. 1. Reporter gene constructs linking CAT with variant portions of the iNOS promoter and their promoter activity affected by NFkappa Bd or LREAA elements. A, construct p7iNOS-CAT contains full-length iNOS promoter (-1468 to +161) with the TATA box (13, 14) and element NFkappa Bu (12) and interferon regulatory factor binding element (IRF-E) (34) indicated by arrows. Construct p8.11iNOS-CAT and its mutants p8.11kappa Bm and p8.11LREm contain a fragment of promoter (-85 to +161). The DNA sequence of the region from -85 to -50 is shown with elements NFkappa Bd and LREAA (underlined) and the mutated nucleotides (asterisks). B and C, mutations of NFkappa Bd or LREAA elements reduced the LPS-induced promoter activity in p8.11iNOS-CAT and in p7iNOS-CAT. Wild type and mutant constructs were transfected into RAW 264.7 cells with or without addition of LPS. The CAT activity of each was compared with that of respective wild type in the presence of LPS, which is set as 100%. Numerical results are means ± S.D. for three or more experiments with each construct and for one the TLC results are illustrated. CAT activity driven by the wild type in response to LPS was 27% acetylation/10 µg of protein/2 h for p8.11iNOS-CAT and 24% acetylation/1 µg of protein/2 h for p7iNOS-CAT.
[View Larger Version of this Image (28K GIF file)]

Contribution of Both LREAA and NFkappa Bd to the Binding of Nuclear Proteins on the iNOS Promoter Correlates with Induction of the Gene by LPS

Oligonucleotides derived from the iNOS promoter with or without mutation (Table I) were used as probes or competitors in electrophoretic mobility shift assays to analyze the DNA binding activity of nuclear extracts from cells cultured with or without LPS. As described earlier (12), a cycloheximide-sensitive DNA-protein complex termed "X" was formed upon LPS induction when probe B was used (Fig. 2A, lane 2). Two other faster migrating complexes, designated "Z" and "Y" in Fig. 2A (not labeled on Fig. 2A in Ref. 12), were present whether or not the cells were exposed to LPS. Without LPS, complex Z was strong and complex Y faint; LPS increased the amount of complex Y without appreciably changing the amount of complex Z (see Fig. 2 in Ref. 12).


Fig. 2. NFkappa Bd- and LREAA-dependent binding of nuclear proteins assessed by electrophoretic mobility shift. A, formation of complexes X, Y, and Z with probes B or D; B, competition assay with wild type probe D by oligonucleotides indicated at the top; C, effects on complex formation of mutated probes D, Dkappa Bm, DLREm, or Dkappa BmLREm; D, competition assay with mutated probe Dkappa Bm; E, competition assay with mutated probe DLREm; F, complex formation with probe C or mutated probe CLREm. In this and all subsequent figures, the lower portion of the gel (not depicted) contained no signal except from free probe.
[View Larger Version of this Image (58K GIF file)]

Probe D is 19 nucleotides shorter than probe B at the 3' end but still contains elements NFkappa Bd and LREAA. Probe D had the same binding activity as probe B, forming complexes X, Y, and Z with nuclear extracts from LPS-induced cells (Fig. 2A, lane 4). Thus, the nucleotides between -85 and -50 of the iNOS promoter are sufficient to sustain the formation of all three complexes.

Competition assays established that LREAA together with NFkappa Bd contributed to the formation of these complexes. Complexes X and Y but not Z were competed by excess nonlabeled oligomer A containing element NFkappa Bd but not LREAA (Fig. 2B, compare lanes 2 and 4). Such competition did not occur with oligomer AkBm, whose NFkappa Bd element was mutated (Fig. 2B, compare lanes 2 and 6). On the other hand, oligomer C, which contained the LREAA element, blocked the formation of complex Z (Fig. 2B, compare lanes 1 and 7 without LPS and lanes 2 and 8 with LPS) and most of complex X (compare lanes 2 and 8). No competition was seen using oligomer CLREm with a mutated LREAA element (lanes 9 and 10).

Results of transcription (Fig. 1B) and competition assays (Fig. 2B) were mirrored by tests of direct binding to mutated probes (Fig. 2, C-E). Taking the formation of complexes X, Y, and Z upon LPS induction as wild type DNA binding activity (Fig. 2C, lane 2), the incomplete formation of these complexes resulted from mutation of either NFkappa Bd (lane 3) or LREAA (lane 4) in the context of probe D. Probe DkBm containing a point mutation of element NFkappa Bd did not sustain formation of complex X or Y but still formed complex Z (Fig. 2C, lane 3; note its slightly slower migration). On the other hand, complex Z and most of complex X were absent using probe DLREm containing the mutated element LREAA; in the meantime, complex Y became stronger and was accompanied by a complex migrating more slowly than Y (lane 4). Probe DkBmLREm, in which both elements are mutated, formed none of the complexes (lane 5). Probe C, containing LREAA but not NFkappa Bd, formed complex Z alone (Fig. 2F, lanes 1 and 2), whereas no complex formed on the mutated probe CLREm (lanes 7 and 8). Thus, LREAA and NFkappa Bd were the only two elements required for the binding of nuclear factors on the iNOS promoter between nucleotides -85 and -50.

The complex Z that formed with probe C or the mutated probe DkBm shared the same characteristics as the complex Z that formed with wild type probe D. Its formation was independent of exposure to LPS (Fig. 2, D and F, lanes 1 and 2) but dependent on the presence of element LREAA. Formation of complex Z on probe D was competed by excess unlabeled oligomer C containing the element LREAA (Fig. 2, D and F, lanes 3 and 4) but not by the oligomer CLREm in which LREAA is mutated (Fig. 2, D and F, lanes 5 and 6).

On the other hand, the complex Y that formed on probe DLREm was more abundant than that formed on wild type probe D (Fig. 2C, compare lanes 2 and 4). Formation of complex Y required only the element NFkappa Bd, because complex Y that was formed with probe DLREm disappeared in a competition assay with NFkappa Bd containing oligomer A (Fig. 2E, lane 4) but not with oligomer AkBm containing the mutated NFkappa Bd element (not shown).

With probe DLREm, a small amount of complex X was still seen after LPS induction (Fig. 2E, lane 2). This residual complex X was competed by oligomer C (Fig. 2E, lane 6).

p50 and Oct-1-like Proteins Contribute to the DNA-Protein Complexes

Supershift assays gave information about some of the proteins comprising complexes X, Y, and Z. No reaction was detected with antibodies against NF-kappa B p52, p65, c-Rel, or RelB, or against IRF-1, STAT1 (p91), c-Jun, c-Fos, C/EBP, Ets-1/Ets-2, NF-AT, Oct-2, or Pit 1, except for a partial supershift of complex X with large amounts of anti-c-Rel (not shown). In contrast, anti-NF-kappa B p50 (a reagent that immunoblotted and supershifted authentic p50 overexpressed upon cell transfection; not shown) completely supershifted complex X (Fig. 3A, lane 2) while leaving complex Z untouched (Fig. 3, A and B, lanes 1 and 2). Moreover, anti-p50 completely supershifted complex Y (marked by an asterisk in lane 2 of Fig. 3A), and such supershift was even clearer when complex Y was formed with probe DLREm (Fig. 3C, lane 3). Thus, p50 is a component of complexes X and Y but is not present (or is not accessible to the antibody) in complex Z. 


Fig. 3. Supershift analyses without (none) or with antibodies (Ab) alpha p50 and alpha Oct-1 for the complexes formed with probe D (A), probe DkBm (B), and probe DLREm (C). The asterisk in lane 2 of panel A marks the supershifted complex Y.
[View Larger Version of this Image (31K GIF file)]

Anti-Oct-1 (a reagent that supershifted authentic Oct-1 overexpressed upon cell transfection; not shown) partly supershifted complexes X and Z (Fig. 3, A and B, lanes 3 and 4). In contrast, anti-Oct-1 was nonreactive with complex Y as formed with probe DLREm (containing NFkappa Bd but not LREAA) (Fig. 3C, lane 4). As noted above, probe DkBm only supported the formation of complex Z and did so whether or not the cells had been exposed to LPS (Fig. 3B, lanes 5 and 6).

Complex Z was subjected to UV cross-linking followed by SDS-polyacrylamide gel electrophoresis, revealing that the region containing element LREAA bound at least three nuclear proteins with apparent molecular masses of ~160, ~100, and ~60 kDa (Fig. 4).


Fig. 4. UV cross-linking analysis of complex Z formed on probe Dkappa Bm. Arrows indicate three oligonucleotide-protein complexes with different molecular weights. Numbers are the molecular mass of standards.
[View Larger Version of this Image (25K GIF file)]


DISCUSSION

The most frequently implicated LPS-response element in mammalian promoters is the 10-base pair kappa B element (GGGRNNYYCC) that binds transcription factors of the NF-kappa B/Rel family (5, 24). NF-kappa B frequently associates with other transcription factors to impart specific regulation (25). The present work identifies an LPS-response element termed LREAA including the dinucleotide AA downstream of NFkappa Bd in the mouse iNOS promoter. An NF-kappa B-like binding site followed closely by an LPS-response element containing an AA dinucleotide was reported in the regulatory region of the major histocompatibility complex class II Aalpha k gene (26) and later in the mouse granulocyte colony-stimulating factor promoter (27), but in neither gene was the AA dinucleotide noted.

The discovery of LREAA focuses attention on the identity of the transcription factors that bind to it and the nature of their interaction with NF-kappa B. The requisite dinucleotide AA of LREAA is embedded in an octamer-like sequence, ATGCAAAA. This sequence departs from the canonical octamer (ATGCAAAT) at the eighth position. Conversion of the eighth nucleotide from T to A may reduce or abolish the binding of the ubiquitous transcription factor Oct-1, as evidenced in the human Ig heavy chain gene enhancer, where the Oct-4 element (ATGCAAAA) bound octamer-binding proteins with only very low affinity (28). Similarly, Oct-1 may bind little to the iNOS LREAA. Other transcription factors known to bind the octamer motif include Pit-1 and the B cell-specific Oct-2 (29). However, with oligonucleotide probes derived from iNOS promoter and nuclear extracts from RAW cells, antibodies against Pit-1 and Oct-2 had no effect, while the antibody against Oct-1 caused a partial supershift in the two complexes (Z and X) whose formation depended on LREAA. Thus, an Oct-1 like protein (OLP) is a candidate for one of the transcription factors interacting with LREAA. UV cross-linking analysis indicated that the LREAA-dependent complex Z included at least three nuclear proteins with molecular masses of ~160, ~100, and ~60 kDa. None of these correspond to the molecular mass of Oct-1 (~70 kDa). It is not known which, if any, of these three proteins binds anti-Oct-1 antibody to cause a partial supershift of complex Z. The ~100-kDa species corresponds in size to a component previously detected in complex X (12). The ~100 kDa protein that binds the LREAA may be the same as the ~100 kDa protein that binds NFkappa Bd.

Sequence context strongly influences the composition of promoter binding complexes. The practice of using minimal probes to sustain complex formation militates against detecting factors that impart specificity to the induction of genes regulated by widely shared transcription systems such as NF-kappa B. The present study used relatively long probes. Results with such probes were consistent with findings from reporter constructs including those representing point mutants in the full-length promoter. In earlier work (12) probe A, containing only the NFkappa Bd element, supported the LPS-activated binding of p50/p65 and p50/c-Rel. However, this did not fully explain LPS-induced promoter activity of iNOS. First, activation of NF-kappa B/Rel is not sensitive to cycloheximide (12), but synthesis of iNOS mRNA in the cells under study was cycloheximide-sensitive (30). Second, activation of p50/p65 and p50/c-Rel peaked at 0.5 h after LPS induction and then decreased (12, 16), but synthesis of iNOS mRNA continued for more than 24 h (11, 18). Finally, NF-kappa B/Rel was activated by LPS in an LPS-hyporesponsive macrophage cell line from C3H/HeJ mice, but iNOS was not induced. Thus, NF-kappa B/Rel was not sufficient for LPS induction of iNOS (17). In contrast, probes B and D (Ref. 12 and present study) included not only the NFkappa Bd element but also downstream sequences that appear to be relatively specific for the iNOS gene. The complexes formed with probes B and D after LPS induction were different from those formed with probe A. In particular, complex X required both NFkappa Bd and LREAA, contained additional protein(s) besides those of the NF-kappa B/Rel family, lacked p65 and c-Rel, and was sensitive to cycloheximide (12).

Sequence analyses suggested that elements for binding of NF-IL6 are present in the iNOS promoter at positions -74 to -66 and -150 to -142 (14); the latter was protected by in vivo footprinting during LPS induction (15). However, mice rendered genetically deficient in NF-IL6 produced iNOS normally in response to LPS and interferon-gamma (31). Footprinting also showed protection of nucleotide at -58 (within the octamer-like sequence ATGCAAAA) after LPS induction (15). The present report demonstrates that the dinucleotide AA at -56 and -55 is critical to the formation of complex Z, which is independent of LPS induction, and that the mutation of AA to CG eliminates both protein binding and promoter activity.

Based on reporter constructs, binding assays, competition experiments, and antibody supershifts, it is hypothesized that both constitutive complex Z and inducible complex X required LREAA and contained OLP, whereas inducible complexes X and Y required NFkappa Bd and contained NF-kappa B p50. Since no direct interaction between NF-kappa B and an Oct-1-containing complex has been reported and since p50 lacks a transactivation domain (25), it is postulated that LPS causes a distinct protein to bridge p50 and OLP, contributes to the formation of complex X, and either furnishes or recruits the transactivation domain that stimulates transcription of iNOS. The bridging protein may be the ~100 kDa species that seems common to complex X and complex Z, or the transactivating protein may bind the ~100 kDa species when it is bound to NFkappa Bd (an LPS-induced event) as well as to LREAA (a constitutive event).

Together, NF-kappa B p50 and the activated OLP-containing complex are proposed to recruit a transactivator to complex X, much as Bcl-3 supplies transactivating capacity by binding p50 dimers on DNA (32, 33). Cloning of the proteins in complexes X and Z may provide fresh approaches to the pharmacologic control of iNOS expression and other responses to LPS.


FOOTNOTES

*   This work was supported by National Institutes of Health Grant AI34543.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Box 57, 1300 York Ave., New York, NY 10021. Tel.: 212-746-2985; Fax: 212-746-8536; E-mail: qwxie{at}med.cornell.edu.
1   The abbreviations used are: LPS, bacterial lipopolysaccharide; CAT, chloramphenicol acetyltransferase; LRE, LPS-response element; iNOS, inducible nitric oxide synthase; IL, interleukin.

ACKNOWLEDGEMENTS

Carl Nathan provided critical discussions and editorial assistance, Mary Leung gave excellent technical help, and Lei Chen critiqued the manuscript.


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