Bacterial Lipopolysaccharide Induces mRNA Expression of an IκB MAIL through ... The MAIL induction was attenuated when the cells were treated with.
FULL PAPER Biochemistry
Bacterial Lipopolysaccharide Induces mRNA Expression of an IκB MAIL through Toll-Like Receptor 4 Hiroshi KITAMURA1,2), Katsushi KANEHIRA1), Takahiko SHIINA3), Masami MORIMATSU3), Bae Dong JUNG1), Sachiko AKASHI4) and Masayuki SAITO1) 1)
Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060–0818, Immunogenetics Team, RIKEN Research Center for Allergy and Immunology, RIKEN Yokohama Institute, Yokahama 230–0045, 3) Department of Veterinary Physiology, Faculty of Agriculture, Iwate University, Morioka, Iwate 020–8550 and 4) Division of Infectious Genetics, Department of Microbiology and Immunology, Tokyo University, Tokyo 108–8639, Japan 2)
(Received 15 November 2001/Accepted 6 February 2002) ABSTRACT.
Molecule possessing ankyrin-repeats induced by lipopolysaccharide (MAIL) is a nuclear IκB protein recently identified as a molecule appearing in immunocompetent organs after administration of bacterial lipopolysaccharide (LPS). Participation of Toll-like receptor (TLR) 4, which is a major form of LPS receptors, in the LPS-induced MAIL expression was investigated. When a human myelomonocytic cell line U937 was treated with phorbol 12-myristate 13-acetate for 3 days, the LPS-induced MAIL expression was much potentiated in parallel with an increase in TLR4 expression. The MAIL induction was attenuated when the cells were treated with a neutralizing antibody against TLR4. The in vivo induction of MAIL in the spleen was smaller in mice having a missense mutation of the Tlr4 gene than in normal control mice. These results collectively indicate that TLR4 contributes, at least in part, MAIL induction after LPS stimulation. KEY WORDS: C3H/HeJ mouse, inflammation, lipopolysaccharide, MAIL, TLR4. J. Vet. Med. Sci. 64(5): 419–422, 2002
Infection with Gram-negative bacteria elicits numerous inflammatory responses, and occasionally leads to septic shock characterized by refractory hypotension and to eventually multi-organ failure and death. Virulence and pathogenic potentials among this group of bacteria are attributed mainly to the presence of lipopolysaccharide (LPS), a component of the outer bacterial membrane [13]. Invasive LPS initially generates a complex with an LPS-binding protein in the plasma, and subsequently binds to glycosylphosphatidyl-inositol-linked membrane receptor CD14 [1, 16]. Since CD14 lacks a cytoplasmic region, another transmembrane co-receptor has been postulated to transduce LPS-signals into cytoplasm. Recent genetic and physical mapping studies of the mouse have demonstrated that missense mutations in Toll-like receptor (TLR) 4 cause hyporesponsiveness to LPS [11, 12]. Moreover, B-lymphocytes and macrophages from TLR4-deficient mice do not respond to LPS [4]. These findings indicate that TLR4 is a critical transmembrane receptor of the LPS-induced inflammatory responses. Recently, we identified a novel nuclear IκB protein, MAIL, as a Molecule possessing Ankyrin-repeats Induced by Lipopolysaccharide in mice [7]. Ectopic expression of MAIL augments interleukin (IL)-6 production in cultured fibroblasts, and inhibits NF-κB activation in macrophages and embryonic kidney cells, suggesting its potential role in the regulation of inflammatory responses [7, 17]. MAIL mRNA expression is undetectably low in normal mice, but as manifested by its name, it increases dramatically and rapidly after LPS-injection in several organs, especially in the spleen and lymph node. These organs are densely populated by B-lymphocytes and macrophages, whose functions are altered by LPS through activation of TLR4 [4]. In fact,
MAIL was dominantly induced in these cells after LPS injection (unpublished observations). Thus, it seems likely that MAIL is induced through TLR4 activation. To test this idea, in the present study, we investigate possible involvement of TLR4 in LPS-induced MAIL expression using an antibody against TLR4 and a mouse strain having a mutated Tlr4 gene. MATERIALS AND METHODS Reagents and cells: LPS (E. coli 055:B5) was purchased from Difco (Detroit, MI, U.S.A.). Phorbol 12-myristate 13acetate (PMA) was from Sigma (St. Louis, MO, U.S.A.). A monoclonal antibody against human TLR4 (HTA125) was described previously [15]. Mouse gamma globulin was from ICN (Costa Mesa, CA, U.S.A.). Human histiocytic leukemia U937 cells were obtained from the American Type Culture Collection (Manassas, VA, U.S.A.), and maintained in RPMI 1640 supplemented with 10% fetal calf serum (FCS). In some experiments, the cells were differentiated by treatment with 32 nM PMA for 3 days. After washing with phosphate-buffered saline (PBS) and a subsequent 24 hr-incubation with RPMI 1640 containing FCS alone, the cells were stimulated with LPS (100 ng/ml) for 1 hr in the presence or absence of 10 µg/ml of HTA125 or control gamma globulin. Animals and treatments: Male C3H/HeN and C3H/HeJ mice (7–8 weeks old) were purchased from SLC (Shizuoka, Japan). They were housed in plastic cages at 24 ± 1°C with a 12 hr light-dark cycle (lights on at 7:00 hr–19:00 hr) and given free access to laboratory chow and water. Mice were injected intraperitoneally with either LPS (3 mg/kg) or ster-
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ile PBS, and were sacrificed by cervical dislocation at 1 hr after the injection. Tissues were freshly removed for total RNA extraction. The experimental procedure and care of animals were in accordance with the guidelines of the Animal Care and Use Committee of Hokkaido University. Northern blot analysis: Thirty µg of total RNA extracted with TRIzol solution (Gibco BRL, Gaithersburg, MD, U.S.A.) was separated on 1% agarose/formaldehyde gel, and transferred to and fixed on a nylon membrane (Amersham Pharmacia Biotech, Pixcataway, NJ, U.S.A.). cDNA probes for mouse MAIL (+1,329 to +1,709 bp; accession number, AB020974), human TLR4 (+163 to +842 bp; accession number, U88880), and human CD14 (+159 to +926 bp; accession number, M86511) were labeled with α32P-dCTP using a multiprime DNA labeling kit (Amersham Pharmacia Biotech). The membranes were hybridized to the labeled probes at 42°C for 20 hr in a buffer containing 50% formamide, 5 × SSPE, 0.1% sodium dodecyl sulfate, 5 x Denhardt’s solution, and 400 µg/ml salmon sperm DNA. Subsequently, the membranes were washed, and exposed to X-ray films (RX-U; Fuji Film, Tokyo, Japan) for 1 or 2 weeks. The mouse cDNA MAIL probe was also used for RNA samples from a human cell line, U937. The radioactivity was quantified using a BAS-1000 bioimage analyzer (Fuji Film). The membranes were also hybridized with mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH, +566 to +1,017 bp; accession number, M32599) cDNA as a reference. Statistics: All values were expressed as means ± S.E.M. Statistical comparison was done by analysis of variance, followed by Fisher’s protected least significant difference test.
Fig. 1. mRNA expression of MAIL, TLR4, CD14, and GAPDH in U937 cells treated with PMA and LPS. U937 cells were treated with either PMA (32 nM: +) or medium alone (–) for 3 days, and stimulated for 1 hr with either LPS (100 ng/ml: +) or PBS (–). Total RNA (30 µg) was subjected to Northern blot analysis. Results are representative of three independent experiments.
RESULTS A human myelomonocytic cell line, U937, is known to exhibit mature macrophage-like properties when treated with PMA [2]. Using this cell line, we examined the effects of PMA on the LPS-induced MAIL mRNA expression and also on the mRNA levels of TLR4 and CD14 (Fig. 1). Untreated U937 cells showed low and/or undetectable mRNA levels of TLR4 and CD14, but the cells pretreated with PMA expressed higher mRNA levels of these molecules. After LPS stimulation, the PMA-treated cells revealed much exaggerated MAIL induction compared with untreated cells. To assess the possible role of TLR4 in the LPS-induced MAIL response, we examined the effect of a neutralizing antibody against human TLR4 (HTA125) in PMA-treated U937 cells (Fig. 2). Stimulation with 100 ng/ml of LPS produced a 5-fold increase of MAIL mRNA levels. Nonimmunized gamma globulin did not show any effect. However, HTA125 significantly suppressed the LPS-induced MAIL expression. These results suggested that the LPSinduced MAIL expression was mediated, at least in part, through activation of a TLR4-dependent pathway. To confirm this idea, we next examined the MAIL induction in vivo in a mouse strain having mutated TLR4 (Fig. 3).
Fig. 2. Effects of a neutralizing antibody against TLR4 on MAIL mRNA expression in U937 cells. U937 cells were treated with PMA as in Fig. 1, and stimulated with LPS (100 ng/ml). The anti-TLR4 antibody (HTA125, 10 µg/ml) and control non-immunized gamma globulin (NIGG, 10 µg/ml), or vehicle (PBS) was added. Thirty µg of total RNA was subjected to Northern blot analysis. The MAIL mRNA levels were normalized by those of GAPDH, and expressed as relative to the value of cells treated with PBS alone. Values are means ± S.E.M. for four wells. * P
