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(Journal of Leukocyte Biology. 2000;68:267-276.)
© 2000 by Society for Leukocyte Biology

Lipopolysaccharide-triggered desensitization of TNF-{alpha} mRNA expression involves lack of phosphorylation of I{kappa}B{alpha} in a murine macrophage-like cell line, P388D1

Mitsuhiro Fujihara*, Shinobu Wakamoto*, Takatoshi Ito*, Masashi Muroi{dagger}, Tsuneo Suzuki{ddagger}, Hisami Ikeda* and Kenji Ikebuchi*

* Japanese Red Cross, Hokkaido Red Cross Blood Center, Sapporo;
{dagger} Division of Microbiology, National Institutes of Health Sciences, Tokyo, Japan; and
{ddagger} Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City

Correspondence: Dr. Mitsuhiro Fujihara, Japanese Red Cross, Hokkaido Red Cross Blood Center, Yamanote 2-2, Nishi-ku, Sapporo 063-0002 Japan. E-mail: fujihara{at}hokkaido.bc.jrc.or.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Activation of nuclear factor {kappa}B (NF-{kappa}B) is thought to be required for cytokine production by lipopolysaccharide (LPS)-responsive cells. Here, we investigated the contribution of NF-{kappa}B in preventing LPS-induced transcription of the tumor necrosis factor {alpha} (TNF-{alpha}) gene in a murine macrophage cell line, P388D1, when tolerance was induced in the cells with a short exposure to a higher dose of LPS. Electrophoretic mobility shift assays with the {kappa}B elements of the murine TNF-{alpha} promoter and enhancer revealed that nuclear mobilization of heterodimers of p65/p50, c-rel/p50 and p65/c-rel, and homodimers of p65 was markedly reduced in LPS-tolerant cells, whereas that of p50 homodimers was only slightly increased. Western blot analysis showed that the phosphorylation of Ser32 on I{kappa}B{alpha} and its transient degradation did not occur in LPS-tolerant cells. These results thus suggest that desensitization of TNF-{alpha} gene expression in this LPS-tolerant state is closely associated with down-regulation of transactivating NF-{kappa}B and may involve a defect in the LPS-induced I{kappa}B{alpha} kinase pathway.

Key Words: NF-{kappa}B • signal transduction • Toll-like receptor 4


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Desensitization or tolerance is a well-known process that is characterized by the loss of responses due to repeated stimulation. Lipopolysaccharide (LPS), a cell wall constituent of gram-negative bacteria, is a potent activator of macrophage functions [1 , 2 ]. LPS plays major roles in septic shock, and also has the ability to induce desensitization to its own effects in a variety of primary monocyte/macrophage cells and cell lines [3 4 5 6 7 8 9 10 11 12 13 14 ]. The macrophages in an LPS-tolerant state typically respond to a secondary LPS stimulation to a much lesser extent (such as LPS-induced proinflammatory cytokine production) than the initial stimulation. LPS tolerance is now regarded as an adaptation or reprogramming of cellular responses such as cytokine autocrine/paracrine regulatory control [15 ], and appears to require the synthesis of one or more proteins that interfere with LPS-signaling processes [8 , 9 , 11 12 13 14 ].

LPS-induced signal transmission is thought to entail binding to specific cellular receptors, which trigger intracellular signaling cascades leading to activation of the transcription factor nuclear factor {kappa}B (NF-{kappa}B) and mitogen-activating protein kinases among others [16 ]. NF-{kappa}B is essential for tumor necrosis factor {alpha} (TNF-{alpha}) gene expression in LPS-stimulated murine primary macrophages [17 18 19 ]. NF-{kappa}B consists of multiple proteins belonging to the Rel family that include p105/p50 (NF-{kappa}B1), p65 (RelA), p100/p52 (Lyt10, NF-{kappa}B2), c-rel, and RelB [20 ]. The p50/p65 heterodimer has been most thoroughly studied and is known to have transactivating function, whereas p50 homodimer appears to function most commonly as a transcriptional suppressor [21 22 23 ]. In most cell types, NF-{kappa}B dimers are kept in the cytoplasm through interaction with the I{kappa}B inhibitory proteins [24 , 25 ]. Among I{kappa}Bs, the most important appear to be I{kappa}B{alpha}, I{kappa}Bß, and the newly discovered I{kappa}B{varepsilon}. I{kappa}B proteins show distinct specificity for various NF-{kappa}B protein dimer combinations. For instance, I{kappa}B{alpha} binds to both p50 homodimers and p50/p65 heterodimers but inhibits only p50/p65 heterodimers [26 , 27 ]. In response to cell stimulation with proinflammatory cytokines (IL-1, TNF), LPS, and phorbol myristate acetate (PMA), at least two of the I{kappa}Bs (I{kappa}B{alpha} and I{kappa}Bß) undergo rapid phosphorylation at two sites within their amino-terminal regulatory domain. The phosphorylation of the I{kappa}Bs results in their polyubiquitination, which in turn leads to their 26S proteasome-mediated degradation, allowing NF-{kappa}B to translocate into the nucleus and activate target genes [28 , 29 ]. This phosphorylation occurs on Ser-32 and Ser-36 of I{kappa}B{alpha} and Ser-19 and Ser-23 of I{kappa}Bß [30 , 31 ]. Thus, the signal-induced phosphorylation of I{kappa}B is the key event that triggers the cascade of events leading to activation of NF-{kappa}B. Hence, I{kappa}B kinase (IKK)-{alpha}/IKK1 and IKK-ß/IKK2 have been molecularly cloned [32 ].

CD14, the 53- to 55-kDa glycoprotein expressed on phagocytic leukocytes, has been identified as the principle LPS-binding molecule [33 ]. The serum LPS-binding protein (LBP) significantly increases LPS binding of CD14, and facilitates transfer of LPS monomers into high-density lipoproteins [34 , 35 ]. Thus, CD14 and LBP are regarded as one of the components of an LPS receptor complex [16 , 33 ]. However, because CD14 is a glycosylphosphatidylinositol-anchored protein without transmembrane domain, its interaction with other signal-transducing molecules has been assumed. Recently, several members of a mammalian Toll-like receptor (TLR) family have been identified [36 ]. Members of the TLR family share homology in their intracellular portions with the signaling domains of the IL-1 receptor family, and Drosophila Toll and several mammalian TLRs have been shown to activate NF-{kappa}B [37 , 38 ]. Overexpression of TLR2 or TLR4 cDNA was originally shown to confer responsiveness to LPS stimulation in human embryonic kidney HEK293 cells through NF-{kappa}B signaling cascade, suggesting the implication of TLR2 in LPS signal transduction [39 , 40 ]. Identification studies of the causative gene of the LPS hyporesponsiveness of C3H/HeJ and C57BL/10ScCr mice demonstrated that defective LPS signaling corresponds to mutations of the TLR4 gene [41 , 42 ]. Vogel et al. demonstrated that mutation of the TLR4 gene in C3H/HeJ exerts a dominant-negative effect on LPS sensitivity in vivo [43 ]. In addition, TLR-4 knockout mice are shown to be hyporesponsive to LPS [44 ]. Furthermore, Chow reported that human TLR4 activates NF-{kappa}B-mediated expression by stimulation with LPS/CD14 in HEK293 cells [45 ]. It has also been reported that TLR4 can confer CD14- dependent LPS responsiveness on HEK293 cells, depending on the concomitant expression of an additional protein, MD-2 [46 ]. More recently published data suggested that TLR4 is a strong candidate as an LPS signal transducer in normal phagocytes and that differential roles of TLR2 and TLR4 in recognition of negative and gram-positive bacterial cell wall components [47 48 49 50 51 ]. With regard to LPS tolerance, TLR4 mRNA was shown to be strongly and transiently suppressed by LPS treatment in murine macrophage cell line RAW264.7 cells [41 ]. This result suggested that down-regulation of TLR4 mRNA contributes to endotoxin tolerance.

The involvement of NF-{kappa}B has been studied to understand the mechanisms of desensitization of TNF-{alpha} gene expression in response to LPS [3 , 4 , 9 , 11 12 13 14 ]. Several studies showed that an up-regulation of NF-{kappa}B p50 homodimers and the binding of p50 homodimers to the {kappa}B#3 element of mouse TNF-{alpha} promoter is attributed to down-regulation of TNF-{alpha} gene expression in an LPS-tolerant state, because p50 can bind to DNA but lacks a transactivation domain [8 , 12 13 14 ]. This notion is supported by the result that the expression of TNF-{alpha} mRNA was not diminished by prolonged exposure to LPS in macrophages from p50-/- mice [52 ]. Furthermore, it has been shown that TNF-{alpha} transcription in mouse macrophages is attenuated by an autocrine factor that preferentially induces p50 homodimers, thereby causing their binding to the distal {kappa}B sites of mouse TNF-{alpha} promoter, including {kappa}B#3 [53 ].

Although LPS tolerance, in general, requires a prolonged primary LPS challenge, a short exposure to LPS can induce a tolerant state in vitro [10 , 11 , 54 , 55 ]. We also recently demonstrated that the protooncogene junB, a member of the Jun family, is down-regulated in the LPS-tolerant state that is induced by a short exposure of primary LPS, in the mouse macrophage cell line, P388D1, with the decreased expression of TNF-{alpha} gene [56 ]. In this study, therefore, we addressed the question of whether an up-regulation of p50 is instrumental in the down-regulation of TNF-{alpha} expression, in an LPS tolerant state caused by a short exposure to primary LPS. In addition, the phosphorylation as well as degradation of I{kappa}Bs was examined in the LPS-tolerant state with regard to the diminished expression of TNF-{alpha} mRNA. We also investigated whether the suppression of TLR4 mRNA expression occurs in response to LPS in our system. The results show that: (1) the nuclear translocation of heterodimers of transactivating NF-{kappa}B (p50/p65, p50/c-rel, and p65/c-rel heterodimers and p65 homodimers) was markedly reduced in the LPS-tolerant state, whereas the nuclear translocation of p50 homodimers was slightly increased; (2) the phosphorylation of I{kappa}B{alpha}, and subsequent loss of its degradation was not observed in the LPS-tolerant state; and (3) the down-regulation of TLR4 mRNA expression was not observed in either the control or the LPS-tolerant state in our system.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture and induction of endotoxin tolerance
The mouse macrophage-like cell line, P388D1 cells, which were isolated from a methylcholanthren-induced lymphoid neoplasm of a DBA/2 mouse and has been shown to possess characteristics typical of macrophages, were grown in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum, supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin. Endotoxin tolerance was induced by incubating cells with 1 µg/mL LPS (Escherichia coli 0111:B4; Sigma Chemical, St. Louis, MO) for 1 h. The cells (1 x 107/experiment) were washed twice with phosphate-buffered saline (PBS), followed for 2 h in LPS-free complete medium. Then the cells were restimulated with LPS as described in the figure legends. For all assays, control cells were treated similarly, but were not given the primary dose. In some experiments, cells (3 x 106/10cm-dish) were cultured for 2 days in the presence or absence of 100 ng/mL LPS. The cells were then washed and stimulated at 1 µg/mL LPS for the indicated periods [12 , 13 ].

Northern blot analysis
Northern analysis of TNF-{alpha} and ß-actin mRNAs was carried out as described previously [58 ]. P388D1 cells (1 x 107/experiment) treated as indicated in the text were washed with cold PBS, quickly frozen, and stored at -80°C until use. Total cellular RNA was extracted from the cells through the use of the guanidinium thiocyanate procedure. The isolated RNA (10 µg) was electrophoresed in 1% agarose gels containing 2.2 M formaldehyde and then transferred to Nytran nylon membranes (Schleicher and Schuell, Dassel, Germany) by capillary action using 10x SSC. The membranes were prehybridized for at least 2 h at 42°C in a solution containing 50% (v/v) formamide, 5x SSPE, 5x Denhardt’s solution, 0.5% sodium dodecyl sulfate (SDS), 0.01 M EDTA, and 100 µg/mL salmon sperm DNA. Membranes were then incubated for 20 h at 42°C in the same solution with 32P-labeled cDNA probes specific for TNF-{alpha} previously labeled by the random hexamer priming method using [{alpha}-32P]dCTP. Membranes were then washed twice at 25°C in 0.1% SDS and 2x SSPE for 30 min, twice at 42°C in 0.1% SDS and 0.1x SSPE, and autoradiographed on a Kodak XAR-5 film at -80°C with intensifying screens. Membranes were then stripped and reprobed with 32P-labeled ß-actin cDNA. The mouse TLR4 cDNA fragments were obtained by reverse-transcription polymerase chain reaction (RT-PCR) from total RNA extracted from mouse macrophage cell line, RAW264.7 cells. The primers used were identical to those reported [41 ]. The resultant 2.6-kb TLR4 fragment was cloned into pCRII (Invitrogen, Carlsbad, CA). The identity of this fragment as a TLR4 cDNA fragment was confirmed by recognition sites of several restriction enzymes as well as partial DNA sequencing. The linearized plasmids containing the properly oriented insert were transcribed using an in vitro transcription kit (Ambion, Austin, TX) with [{alpha}-32P]UTP, and used for hybridization. The membranes were prehybridized for at least 6 h at 55°C in a solution containing 50% (v/v) formamide, 5x SSPE, 5x Denhardt’s solution, 0.5% SDS, and 100 µg/mL salmon sperm DNA. Membranes were then incubated for 20 h at 55°C in the same solution with 32P-labeled TLR4 riboprobe. Membranes were then washed three times at 65°C in 0.5% SDS and 1x SSPE for 15 min, and autoradiographed on a Kodak MS film at -80°C with intensifying screens. After analysis with TLR4 probe, the membrane was rehybridized with 32P-labeled ß-actin riboprobe. In some experiments, blots were hybridized using ULTRAhyb solution (Ambion) with 32P-labeled DNA probe obtained by RT-PCR. The primers used were 5’-TGACACCCTCCATAGACTTC-3’ and 5’-GTTCTCCTCAGGTCCAAGTTGCCGTTTC-3’ [42 ]. As a negative control, the RNA from a mouse T cell hybridoma, HTB 176.10 was used.

Preparation of nuclear and cytosolic extracts
Nuclear extracts and cytosolic fractions were prepared by the method of Sadowski and Gilman [57 ], as follows. After the stimulation period, the cells (1 x 107/experiment) were rinsed twice with ice-cold PBS, once with PBS containing 1 mM Na3VO4 and 5 mM NaF, and once with hypotonic buffer. Ice-cold hypotonic buffer with 0.4% Nonidet P-40 (NP-40; 0.5 mL) was added directly to the cells to lyse them. Lysates were scraped into microfuge tubes and mixed, and the nuclei were removed by centrifugation at 15,000 rpm for 30 s. Supernatants were supplemented with NaCl to 120 mM, clarified (15,000 rpm for 20 min), and glycerol was added to 10% as the cytosol fraction. The nuclei were then washed twice with 500 µL of hypotonic buffer containing 0.4% NP-40 and centrifuged at 12,000 rpm for 5 min. The nuclear pellets were resuspended in 75 µL of high-salt buffer on ice for 30 min with occasional gentle mixing. The nuclear suspension was centrifuged for 15 min at 15,000 rpm, and then 5 min at 15,000 rpm to remove the insoluble fraction. Hypotonic buffer consisted of: 20 mM HEPES (pH7.9), 20 mM NaF, 1 mM Na3VO4, 1 mM Na4P2O7, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 1 µg/mL of pepstatin, and 10 µg/mL each of leupeptin and aprotinin. This buffer containing 420 mM NaCl and 20% glycerol (high-salt buffer) was used for nuclear extraction. Cytosolic and nuclear extracts were then frozen and stored at -80°C. The protein concentration of each extract was determined by the Bradford assay (Bio Rad Laboratories, Hercules, CA).

Electrophoretic mobility shift assay (EMSA)
This was carried out as described previously [58 ]. The following NF-{kappa}B-specific oligonucleotides containing one NF-{kappa}B site were used: {kappa}B#3 motif (the -510) of the murine TNF-{alpha} promoter [17 ] (5’-TTCAGGGGGCTTTCCCTACA-3’) and {kappa}B#4 motif (+2830) of the downstream of the murine TNF-{alpha} gene [18 ] (5’-CATGGGGGCATGGGAATTTCCCACTCTGCATG-3’). The oligonucleotide and its complementary strand were annealed, and labeled by a T4 kinase-mediated phosphorylation reaction in the presence of [{gamma}-32P]ATP. Nuclear protein extracts (5–6 µg) with 2.5 µg of poly (dI-dC) · poly (dI-dC) (Amersham Pharmacia Biotech, Uppsala, Sweden) and end-labeled DNA (~40,000 cpm) were mixed in 25 µL of EMSA buffer (10 mM Tris · HCl, pH 7.5, containing 40 mM NaCl, 1 mM EDTA, 1 mM ß-mercaptoethanol, 4% glycerol, 0.1% NP-40, 1 mM DTT, and 1 µg/µL BSA) and incubated for 30 min at 25°C after mixing. After this initial binding reaction, 20 µL of the mixture was electrophoresed at 15 V/cm for 1.5 h at room temperature through a native 4.5% polyacrylamide gel in 0.25x TBE. Gels were processed for autoradiography. To quantitate the amount of NF-{kappa}B proteins specifically bound to the probe, the radioactive bands identified by autoradiography were cut out and counted in a ß counter. For the supershift assay, nuclear proteins (2 µg/25 µL) extracted from P388D1 cells treated with LPS (1 µg/mL) for 30 min were first incubated for 60 min at 25°C with 1 µL of polyclonal antibodies directed against p50 (#sc-1192x), p65 (#sc-372x), c-rel (#sc-70x), or STAT-3 (#sc-482x) from Santa Cruz Biotechnology (Santa Cruz, CA). Antiserum to carboxy terminus of mouse cRel (#1266) that was kindly provided by Dr. N. Rice (NCI-FCRDC, Frederick, MD) was also used. These mixtures were then subjected to EMSA as described above.

Western blot analysis
Nuclear extracts (10 µg) and cytosolic extracts (30 µg) were subjected to an electrophoresis in 10% SDS-PAGE. The proteins separated by SDS-PAGE were transferred to a nitrocellulose membrane (Schleicher and Schuell) using electrophoretic transfer cell. Subsequently, the nitrocellulose membrane was blocked with TBST (20 mM Tris, 137 mM NaCl, 0.1% Tween 20, pH 7.6) containing 5% w/v nonfat dry milk for 3 h at room temperature. The membrane was then incubated with TBST containing the relevant Abs in the presence of 5% BSA at 4°C overnight with gentle agitation. The primary Abs used were the rabbit polyclonal antibodies of anti-I{kappa}B{alpha} (New England Biolabs, Beverly, MA), anti-I{kappa}Bß (N-20), anti-p50 (H-119), anti-p65 (C-20), and anti-c-rel (N-466) from Santa Cruz Biotechnology at a 1/1000 dilution. After being rinsed with three changes of TBST, the blot was incubated with horseradish peroxidase-conjugated secondary antibody at 1/4000 dilution in TBS with 5% nonfat dry milk for 1 h at room temperature. The blots probed with relevant antibodies were then washed three times with TBST, and developed with the use of the Phototope chemiluminescence detection system (New England Biolabs). To detect the posphorylation of Ser32 of I{kappa}B{alpha}, Abs that specifically recognize Ser32 on I{kappa}B{alpha} were purchased from New England Biolabs and used in immunoblot analyses. Detection of Ab binding was conducted as described in the manufacturer’s instructions.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TNF-{alpha} mRNA expression by the treatment with LPS in P388D1 cells
The kinetics and dose-response characteristics for TNF-{alpha} mRNA expression in mouse macrophage cell line, P388D1 cells in response to LPS treatment were first examined by Northern analysis. P388D1 cells (1 x 107) were incubated with LPS (1 µg/mL) for varying periods of time (0–6 h). Total RNAs were then extracted from the cells and were subjected to Northern analysis. The expression of TNF-{alpha} mRNA was substantially increased at 30 min of exposure to LPS, further increased at 1 h, and then greatly reduced after a 3-h exposure to LPS (Fig. 1A ).



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  		Figure 1. (A) Induction of TNF-{alpha} mRNA expression in P388D1 cells exposed to LPS. P388D1 cells (1 x 107 cells) were treated with LPS (1 µg/mL) for the indicated periods. (B) Effect of pretreatment with LPS on TNF-{alpha} mRNA expression in response to secondary exposure to LPS. P388D1 cells (1 x 107 cells) were treated for 1 h with varying concentrations (10 pg/mL to 1 µg/mL) of LPS. The cells were washed twice in PBS, incubated for 2 h in LPS-free medium, and restimulated with 1 µg/mL LPS for 1 h. Total RNAs extracted from the cells were subjected to Northern analysis. After analysis with TNF-{alpha} probe, the membrane was stripped and rehybridized with ß-actin cDNA. Additional experiments gave similar results.

 
Prior exposure to LPS reduced TNF-{alpha} expression in response to secondary LPS treatment
Next, the question of how repeated exposure of P388D1 cells to LPS affects TNF-{alpha} expression was investigated. To this end, P388D1 cells were exposed for 1 h at 37°C to varying doses of LPS (10 pg/mL to 1 µg/mL). Cells were then washed, incubated for an additional 2 h in LPS-free medium, and restimulated with 1 µg/mL of LPS for 1 h. Northern analysis of total RNAs extracted from the cells (Fig. 1B) showed that the secondary responses of cells became more refractory, when cells were first exposed to >10 ng/mL LPS. The initial exposure of cells to <1 ng/mL LPS did not substantially alter the TNF-{alpha} mRNA expression on secondary exposure of the cells.

NF-{kappa}B translocation by the treatment with LPS in P388D1 cells
NF-{kappa}B proteins have been shown to regulate the expression of TNF-{alpha} mRNA in murine macrophages [17 18 19 ]. Indeed, pyrrolidine dithiocarbamate, a relatively specific inhibitor of the activation of NF-{kappa}B in macrophages [59 ], remarkably inhibited the LPS-triggered TNF-{alpha} expression in P388D1 cells (data not shown). To investigate the mode of activation of NF-{kappa}B in response to LPS treatment, P388D1 cells were incubated with LPS (1 µg/mL) for up to 3 h. Nuclear extracts were prepared and incubated with end-labeled oligonucleotide probe containing {kappa}B#3 motif (the -510) of the murine TNF-{alpha} promoter [12 ]. The reaction mixtures were then subjected to EMSA. Figure 2A showed that the {kappa}B#3 site bound two major complexes constitutively, the fast-moving C1 complex and slower-moving C2 complex. The C1 complex gradually increased in nuclear extract and was about 1.6-fold the constitutive level (based on the radioactive counts of the C1 complex band cut out) at 3 h. The level of C2 complex substantially increased as early as 10 min after exposure to LPS, reached a maximum at 30 min (about 4.8-fold the constitutive level), and then declined. To identify the components of the C1 and C2 complexes, supershift analyses were performed. Both C1 and C2 complexes were supershifted markedly when the nuclear extract was preincubated with anti-p50 antibody before binding to the probe. A significant reduction of C2 complex with supershift was observed with anti-p65 or anti-c-rel antibody, but the level of C1 complex was not decreased by these antibodies. The antiserum to carboxy-terminal of mouse c-rel (#1266) showed the same result (data not shown). Unrelevant antibody against STAT-3 affected neither C1 nor C2 complexes. Thus, C1 complex appeared to consist predominantly of homodimer of p50, whereas C2 complex appeared to contain heterodimers of p50/p65 and p50/c-rel (Fig. 2C) .



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  		Figure 2. LPS-induced activation of NF-{kappa}B. P388D1 cells were treated with LPS (1 µg/mL) for indicated periods. Nuclear extracts (prepared as outlined in Materials and Methods) were subjected to EMSA with radiolabeled oligonucleotides corresponding to one of the distal NF-{kappa}B sites ({kappa}B#3) of the TNF-{alpha} promoter (A) or the downstream {kappa}B element ({kappa}B#4) of the TNF-{alpha} gene (B). Protein-DNA complexes were electrophoresed on 4.5% polyacrylamide gels (left panel). The autoradiograph shows a representative of two separate experiments that gave similar results. NS, nonspecific band (right panel). The degree of increase in each complex over the respective constitutive level was determined by counting the radioactivity of each cut-out band, then subtracting the nonspecific radioactivity. Value was expressed as the mean of two experiments. (C) Supershift analysis of nuclear proteins from P388D1 cells. Nuclear extracts from P388D1 cells treated with LPS for 30 min were mixed with no antibody (lane 1) or with antibodies against p65, p50, c-rel, or STAT-3. These mixtures were then added to the radiolabeled oligonucleotide probes corresponding to the {kappa}B#3.

 
Because {kappa}B#3 motif was shown to be minimally important for LPS-induced transcription of the extended TNF-{alpha} promoter [19 ], we tested an mTNF-{kappa}B#4 element that was known to have enhancer activity and to bind two conventional complexes I and II as well as a slower-migrating complex III in LPS-activated mouse bone marrow-derived macrophages or mouse macrophage cell line, ANA-1 cells [18 ]. Combination of high-resolution EMSA with monospecific antibodies and ultraviolet-cross-linking indicated that the p50 homodimer as a protein constituent of complex I, p50/c-rel and p50/p65 heterodimers as the fraction of complex II, and the p65/c-rel heterodimer and p65 homodimer as a constituents of complex III [18 ]. In the nuclear extract from LPS-activated P388D1 cells the mTNF-{kappa}B#4 probe bound three complexes designated as C1, C2, and C3, similar to the results obtained in the LPS-activated murine macrophages or ANA-1 cells (Fig. 2B) . The C1 complex (p50 homodimer) gradually increased in nuclear extract and was about 1.6-fold the constitutive level at 3 h. The level of C2 complex (p50/p65 and p50/c-rel) substantially increased as early as 10 min after exposure to LPS, reached a maximum at 30–60 min (about eightfold the constitutive level), and then declined. The level of C3 complex (presumably p65 homodimer and p65/c-rel) also increased, reached a maximum at 30–60 min (about 4.3-fold the constitutive level), and then declined. Thus, EMSA using two {kappa}B elements revealed that transient and significant increases in levels of heterodimers including p50/p65, p50/c-rel, and p65/c-rel, and homodimers of p65 occurred with LPS stimulation, whereas levels of p50 homodimers were gradually and slightly elevated during the observed periods.

I{kappa}B{alpha} degradation by treatment with LPS in P388D1 cells
Activation of NF-{kappa}B by TNF-{alpha} or IL-1 is achieved through phosphorylation-dependent degradation of the I{kappa}Bs, which results in the release and subsequent nuclear translocation of NF-{kappa}B [28 , 29 ]. We performed immunoblotting experiments with I{kappa}B{alpha} and I{kappa}Bß antibodies to examine whether LPS causes degradation of these I{kappa}B proteins in this cell line. Almost complete degradation of I{kappa}B{alpha} had occurred by 30 min after LPS treatment, with reexpression observed by 60 min. The phosphorylation of Ser32 of I{kappa}B{alpha} occurred as early as 10 min after LPS treatment, and persisted thereafter (Fig. 3A ). By contrast, no degradation of I{kappa}Bß was observed over 3 h after LPS treatment (Fig. 3B) . Thus, the degradation of I{kappa}B{alpha}, due to the phosphorylation, is mainly responsible for the translocation of NF-{kappa}B in this cell line in response to LPS.



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  		Figure 3. LPS treatment of P388D1 cells caused degradation of I{kappa}B{alpha}, but not I{kappa}Bß. (A) Cells were treated with LPS (1 µg/mL) for indicated periods. Cytosolic extracts were subjected to 10% SDS-PAGE, and blotted on nitrocellulose membrane. The total I{kappa}B{alpha} level and the phosphorylation of Ser32 on I{kappa}B{alpha} were detected with rabbit polyclonal anti-I{kappa}B{alpha} and phosphospecific I{kappa}B{alpha} (Ser32) Ab. NS, nonspecific band. (B) The I{kappa}Bß level was detected with rabbit polyclonal anti-I{kappa}Bß Ab.

 
Prior exposure to LPS reduced NF-{kappa}B activation in response to secondary LPS treatment
We then examined whether the reduced level of steady-state TNF-{alpha} mRNA observed in endotoxin-tolerant cells could be due to the decrease in NF-{kappa}B activation, as follows. P388D1 cells were incubated for 1 h with or without 1 µg/mL LPS. Cells were then washed, incubated for 2 h in LPS-free medium, and incubated with LPS for 0, 15, 30, and 90 min. Nuclear extracts were subjected to EMSA using the oligonucleotide probe containing {kappa}B#3 (Fig. 4A ). Treatment with LPS in the control cells increased the C2 complex binding with a peak at 30 min (Fig. 4 , lanes 1–4), whereas the increase of C2 complex activation in LPS-pretreated cells by secondary stimulation with LPS was very small (Fig. 4 , lanes 5–8). The fold increase of C2 complex binding of control cells was significantly higher than that of LPS-treatment cells at 30 min after LPS treatment (3.58 ± 0.45 vs. 1.91 ± 0.52, P < 0.05 by unpaired Student’s t test). The C1 complex slightly increased in both control and LPS-tolerant cells, but there was no significant difference between control and LPS-tolerant cells (1.13 ± 0.13 vs. 1.81 ± 0.44).



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  		Figure 4. Effect of pretreatment with LPS on translocation of NF-{kappa}B activation in response to subsequent exposure to LPS. P388D1 cells (1 x 107/experiment) were incubated in the absence (lanes 1–4) or presence (lanes 5–8) of 1 µg/mL LPS for 1 h, washed twice in PBS, and incubated again for 2 h in LPS-free medium. Cells were then stimulated for 0 (lanes 1, 5), 15 (lanes 2, 6), 30 (lanes 3, 7), or 90 min (lanes 4, 8) with 1 µg/mL LPS. Nuclear extracts were subjected to EMSA using radiolabeled oligonucleotides corresponding to the {kappa}B#3 of the TNF-{alpha} promoter (A) or the downstream {kappa}B element ({kappa}B#4) of the TNF-{alpha} gene (B). The autoradiograph (left panel) shown is a representative result of three different experiments (right panel). Degree of increase in each complex over the constitutive level, respectively, was determined as shown in Figure 2 , and was expressed as the mean ± SE of three experiments.

 
We also performed EMSA with the probe corresponding to {kappa}B#4 site (Fig. 4B) . Treatment with LPS in the control cells resulted in increase in the C2 and C3 complex binding with a peak at 30 min (lanes 1–4), whereas the increases of C2 and C3 complex activation by restimulation with LPS in LPS-pretreated cells were very small (lanes 5–8). The degree of increases of C2 and C3 complex binding in control cells were significantly higher than those in LPS-treatment cells at 30 min after LPS treatment ( 5.23 ± 0.28 vs. 1.51 ± 0.20 in C2, 4.20 ± 0.25 vs. 1.16 ± 0.23 in C3, P < 0.05 by unpaired Student’s t test). Again, there was no significant difference between control and LPS-tolerant cells in the C1 complex binding. These results suggest that decreased activation of C2 complex (p50/p65 and p50/c-rel) as well as C3 complex (presumably p65/c-rel and p65 homodimer) in LPS-tolerant cells contributed to decreased levels of LPS-triggered TNF-{alpha} mRNA expression.

Translocation of p65 and c-rel was greatly reduced in the LPS-tolerant state
To analyze the down-regulation of NF-{kappa}B activation in the LPS-tolerant state, we performed immunoblotting analysis of NF-{kappa}B proteins, p65, p50, and c-rel, in the nuclear fraction of the control and LPS-tolerant cells (Fig. 5 ). In the control cells, the level of p65 increased at 15 and 30 min and then decreased at 90 min, whereas the level of p65 did not increase in the LPS-tolerant cells. The level of p50 also increased at 30 min and declined at 90 min in the control cells, whereas the level of p50 in LPS-tolerant cells remained higher than the constitutive level of the control cells. The level of c-rel clearly increased at 30 min after LPS treatment in the control cells, but not in the LPS-tolerant cells. These results support the EMSA patterns that the binding of heterodimers of p50/p65, p50/c-rel, and p65/c-rel and homodimers of p65 to the {kappa}B#4 in LPS-tolerant cells were significantly less than that in the control cells. In addition, the slightly higher binding of p50 homodimers to the {kappa}B#3, in the LPS-tolerant cells may be related to the persistent presence of p50 in the nucleus in the tolerant cells.



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  		Figure 5. Effect of pretreatment with LPS on translocation of p65, p50, and c-rel in response to subsequent exposure to LPS. Cells were treated as described in the legend of Figure 4 . Nuclear extracts were subjected to 10% SDS-PAGE, and blotted on nitrocellulose membrane. The blots were probed with rabbit polyclonal anti-p65, anti-p50, or anti-c-rel antibody. The position of each band is indicated by an arrow. A representative blot of three independent experiments is shown.

 
Lack of phosphorylation and degradation of I{kappa}B{alpha} in the LPS-tolerant state
To examine whether the degradation as well as phosphorylation of I{kappa}B{alpha} occurred in the LPS-tolerant state, we performed immunoblotting analysis of the I{kappa}B{alpha} in the cytosolic fraction of control and LPS-tolerant cells (Fig. 6 ). In the control cells, the level of I{kappa}B{alpha} decreased at 15 and 30 min and then increased at 90 min, suggesting that LPS stimulation induced the transient degradation and resynthesis of I{kappa}B{alpha}. In contrast, the level of I{kappa}B{alpha} did not change significantly in the LPS-tolerant cells. Furthermore, the increased phosphorylation of Ser32 of I{kappa}B{alpha} was observed in the control cells, but not in the tolerant cells. The level of I{kappa}Bß did not change in either control or LPS-tolerant cells. These results suggest that the absence of phosphorylation of I{kappa}B{alpha} contributes to the down-regulated TNF-{alpha} mRNA expression in LPS-tolerant cells.



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  		Figure 6. Effect of pretreatment with LPS on phosphorylation of Ser32 on I{kappa}B{alpha} and its degradation in response to subsequent exposure to LPS. Cells were treated as described in the legend of Figure 4 . Cytosolic extracts were subjected to 10% SDS-PAGE, and blotted on nitrocellulose membrane. (A) The phosphorylation of Ser32 on I{kappa}B{alpha} was determined by phospho I{kappa}B{alpha}-specific Ab, and the total level of I{kappa}B{alpha} was probed with polyclonal rabbit I{kappa}B{alpha} antibody. (B) The total level of I{kappa}Bß was probed with polyclonal rabbit I{kappa}Bß antibody. The position of each band is indicated by an arrow. A representative blot of three independent experiments is shown.

 
Effect of LPS treatment on TLR4 mRNA expression in control and LPS-tolerant cells
It was recently shown that TLR4 primarily acts in the recognition of LPS and transduces the LPS signal in mice. In addition, LPS treatment was shown to cause strong and transient suppression of TLR4 mRNA in murine RAW264.7 cells, suggesting that the down-regulation of TLR4 mRNA contributes to endotoxin tolerance [41 ]. Therefore, we investigated the expression pattern of TLR4 mRNA in both the control and the LPS-tolerant state in response to LPS. P388D1 cells were incubated for 1 h with or without 1 µg/mL LPS. Cells were then washed, incubated for 2 h in LPS-free medium, and incubated for 0, 0.5, 1, 2, and 3 h with 1 µg/mL LPS. Figure 7 shows that the TLR4 mRNA expression was suppressed in neither the control nor LPS-tolerant state. This result suggests that the down-regulation of TNF-{alpha} mRNA expression detected in our system was not due to the change of TLR4 mRNA level.



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  		Figure 7. Effect of pretreatment with LPS on the TLR4 mRNA expression in response to subsequent exposure to LPS. P388D1 cells (1 x 107 cells) were incubated in the absence (lanes 1–5) or presence (lanes 6–10) of 1 µg/mL LPS for 1 h, washed twice in PBS, and incubated again for 2 h in LPS-free medium. Cells were then stimulated for 0 (lanes 1, 6), 0.5 (lanes 2, 7), 1 (lanes 3, 8), 2 (lanes 4, 9), or 3 h (lanes 5, 10) with 1 µg/mL LPS. Total RNAs extracted from the cells were subjected to Northern analysis. TLR4 mRNA was detected on Northern blots with riboprobe as described in Materials and Methods. After analysis with TLR4 probe, the membrane was rehybridized with ß-actin riboprobe. Additional experiments gave similar results. Right panel: untreated P388D1 (lane 1), a mouse T cell hybridoma HTB 176.10 as a negative control (lane 2).

 
Preincubation with a lower LPS dose for a longer period resulted in a substantial increase in p50 homodimer in response to secondary LPS treatment
Previously, Ziegler-Heitbroch et al. demonstrated that LPS tolerance involves a preferential increase in p50 homodimers using the same cell line and {kappa}B#3 oligonucleotides as those we used in this study. The differences in outcome may be due to differences in the experimental conditions to induce LPS tolerance. The earlier study used a much longer preincubation and a lower dose of LPS to induce LPS tolerance. To test whether these differences may account for the disagreement, P388D1 cells were preincubated for 2 days with 100 ng/mL LPS, washed, and then restimulated with 1 µg/mL LPS for up to 1 h. The result of EMSA using the {kappa}B#3 oligonucleotides revealed that p50 homodimers (C1 complex) were significantly increased in LPS-tolerant cells and were further up-regulated by LPS restimulation (compare Fig. 4A ), confirming the earlier study by Ziegler-Heitbroch et al. [12 ] (Fig. 8A ).



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  		Figure 8. (A) Effect of longer pretreatment with a lower LPS dose on translocation of NF-{kappa}B activation in response to subsequent exposure to LPS. P388D1 cells (3 x 106/experiment) were incubated in the absence (lanes 1–4) or presence (lanes 5–8) of 100 ng/mL LPS for 2 days, and washed once with LPS-free medium. Cells were then stimulated for 0 (lanes 1, 5), 15 (lanes 2, 6), 30 (lanes 3, 7), or 60 min (lanes 4, 8) with 1 µg/mL LPS. Nuclear extracts were subjected to EMSA using radiolabeled oligonucleotides corresponding to the {kappa}B#3 of the TNF-{alpha} promoter (left panel). The autoradiograph shows a representative of two separate experiments that gave similar results (right panel). The degree of increase in each complex over the constitutive level, respectively, was determined as shown in Figure 2 , and was expressed as the mean of two experiments. (B) The effect of longer pretreatment with a lower LPS dose on phosphorylation of Ser32 on I{kappa}B{alpha} and its degradation in response to subsequent exposure to LPS. Cells were treated as described above. Cytosolic extracts were subjected to 10% SDS-PAGE, and blotted on nitrocellulose membrane. The phosphorylation of Ser32 on I{kappa}B{alpha} was determined by phospho I{kappa}B{alpha}-specific Ab, and the total level of I{kappa}B{alpha} was probed with polyclonal rabbit I{kappa}B{alpha} antibody. Another experiment showed similar results.

 
We also performed immunoblotting analysis to examine whether degradation as well as phosphorylation of I{kappa}B{alpha} occurred in LPS-tolerant cells (Fig. 8B) . Immunoblotting analysis of the total I{kappa}B{alpha} level and the phosphorylation of I{kappa}B{alpha} showed that LPS restimulation failed to induce the transient degradation and resynthesis of I{kappa}B{alpha} in LPS-tolerant cells. In addition, LPS-triggered phosphorylation of I{kappa}B{alpha} was not observed in LPS-tolerant cells. These results suggest that the proximal level of LPS-signal transduction pathway was impaired in LPS-tolerant cells, regardless of experimental conditions to induce LPS tolerance.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The notion that NF-{kappa}B pathways play an important role in LPS-induced desensitization of TNF-{alpha} gene expression is supported by several investigators. Although multiple mechanisms have been proposed, the up-regulation of p50 homodimers is currently recognized as instrumental to LPS-tolerance in TNF-{alpha} gene expression. Ziegler-Heitbrock et al. [8 , 12 ] provided evidence that human Mono Mac 6 cells translocate predominantly p50/p65 or p50/c-rel heterodimers after primary LPS challenge, whereas LPS-tolerant cells had a persistent nuclear localization of p50 homodimers that was up-regulated in response to secondary LPS stimulation. They also examined whether similar processes occur in murine monocyte/macrophage cells, and detected a pronounced up-regulation of p50 homodimers in LPS-tolerant P388D1 cells. Because the p50 homodimers are not effective in transcriptional activation, and the strong up-regulation of p50 homodimers is thought to be attributed to down-regulation of TNF-{alpha} gene expression in the LPS-tolerant state, they assumed that the increase of p50 homodimer DNA binding activity is due to the synthesis of p50 protein from its precursor, p105, and demonstrated that p105 mRNA levels were up-regulated upon stimulation of Mono Mac 6 cells with LPS, whereas p65 mRNA levels were unaffected [8 ]. This is in agreement with reports that NF-{kappa}B itself participates in the regulation of p105 expression through NF-{kappa}B motifs in the p105 promoter [60 , 61 ], whereas the p65 promoter does not contain such a motif [62 ]. Furthermore, it has been speculated that up-regulation of p50 in the cytoplasm favors formation of p50 homodimers in cytoplasm, and resultant p50 homodimers may directly translocate to the nucleus [54 ], as at least I{kappa}B{alpha} does not inhibit p50 homodimers [26 , 27 ].

Bohuslav et al. provided evidence of an essential role for p50 in LPS tolerance [52 ]. They demonstrated that murine peritoneal exudate macrophages obtained from p50 knock-out mice did not become LPS tolerant, as measured by TNF-{alpha} gene expression, after an 18-h pretreatment with LPS, whereas macrophages from wild-type mice did. Also, they reported the persistent nuclear localization of p50 homodimers, which bound probe DNA containing the {kappa}B#3 site from the mouse TNF-{alpha} promoter in cells after induction of LPS tolerance.

In this study, we addressed the question of whether up-regulation of p50 homodimers is also essential, when the LPS-tolerant state was induced by a shorter exposure (1 h) to a higher dose of primary LPS (1 µg/mL) in P388D1 cells: these were 2- to 3-day and 100 ng/mL in the study by Ziegler-Heitbrock et al. We used the same oligonucleotide ({kappa}B#3 motif of the mouse TNF-{alpha} promoter) together with the {kappa}B#4 of the downstream of TNF-{alpha} gene as a comparison. The results of the gel shift assay revealed a remarkable decrease in mobilization of heterodimers consisting of homodimers of p65 and heterodimers of p50/p65, p50/c-rel, and p65/c-rel in the LPS-tolerant state. Although constitutive p50 homodimers and the gradual increase in response to LPS were detected, only a slight up-regulation of p50 homodimers in the LPS-tolerant state was observed. Western analysis of nuclear proteins in the LPS-tolerant state showed a markedly decreased nuclear translocation of p65 and c-rel. In contrast, the levels of nuclear p50 remained high in the tolerant state. This up-regulation of nuclear p50 levels presumably contributed to the slight increase of p50 homodimers in the LPS-tolerant state, although the up-regulation of p50 homodimers was much less extensive than those reported by Ziegler-Heitbrock et al. Our results thus suggest that desensitization of TNF-{alpha} mRNA expression in our system is mainly associated with down-regulation of homodimers of p65 and heterodimers of p50/p65, p50/c-rel, and p65/c-rel, whereas the slight increase of p50 homodimers may involve the decreased cytokine expression to some extent. The slight increase of p50 homodimers in our system seems to be due to the short period of primary LPS exposure (1 h), because the up-regulation of p50 homodimers after primary LPS exposure requires transcriptional activation of the p50-precursor p105, and subsequent protein synthesis. Analysis of {kappa}B#3 site-bound nuclear complexes of mouse macrophages by ultraviolet cross-linking revealed a substantial increase in the binding of p50 in cells that were activated with LPS for increasing periods of time and a concomitant decrease in p65 [54 ]. The predominance of p50 over p65, which allows for p50/p65 to be gradually displaced with p50 homodimers was detected after 4 h of exposure to LPS [52 ]. In fact, as shown in Figure 8A , the significant increase in p50 homodimers occurred in LPS-tolerant cells and further up-regulation was observed in response to LPS, when the cells were preincubated with 100 ng/mL LPS for 2 days (compare Figure 4A ).

I{kappa}B{alpha} and I{kappa}Bß are mediators of either transient or persistent NF-{kappa}B activation in response to stimulators like TNF-{alpha} and LPS, respectively [63 ]. We estimated the amount of I{kappa}B{alpha} and I{kappa}Bß proteins at different time points after LPS treatment with Western blots. The results, outlined in Figure 3 , clearly indicate that LPS treatment produced a transient decrease of I{kappa}B{alpha}, in parallel to the transient phase in the increase of homodimers of p65 and heterodimers consisting of p50/p65, p50/c-rel, and p65/c-rel. In contrast, the I{kappa}Bß protein level in the cytoplasm was not altered after LPS treatment in this cell line. Thus, I{kappa}B{alpha} appears to be mainly responsible for the regulation of NF-{kappa}B activation in P388D1 cells. Furthermore, LPS treatment did not induce the phosphorylation of Ser32 of I{kappa}B{alpha} in the LPS-tolerant state, nor the degradation of I{kappa}B{alpha}. These results suggest that the down-regulation of homodimers of p65 and heterodimers of p50/p65, p50/c-rel, and p65/c-rel involves a defect in the LPS-induced I{kappa}B{alpha} kinase pathway.

In view of I{kappa}B effect on LPS tolerance, the absence of LPS-inducible I{kappa}B kinase activity was previously shown in human ovarian carcinoma cells pretreated with LPS for 1 h, when the cells were challenged with secondary LPS after incubation for 4 h in the absence of LPS [55 ]. Our findings are in agreement with the results reported in the non-myeloid cells, suggesting that LPS-inducible I{kappa}B kinase plays an important role in LPS tolerance in both non-myeloid and myeloid cells, particularly when the tolerant state was induced by a short exposure to primary LPS. In contrast, our results differ from another published report in which the degradation of I{kappa}B{alpha} occurred by restimulation with LPS in human THP-1 cells treated with LPS for 16 h, suggesting that the pathway leading to I{kappa}B{alpha} phosphorylation and degradation is intact in LPS-tolerant THP-1 cells [9 ]. This disagreement was not simply due to the difference in exposure periods and/or dose of primary LPS, because the inability of the I{kappa}B{alpha} phosphorylation as well as degradation by LPS restimulation was still observed in LPS-tolerant P388D1 cells when LPS tolerance was induced by longer incubation with a lower dose of primary LPS as outlined in Figure 8B . It is interesting that, although I{kappa}B{alpha} degradation occurred in LPS-tolerant THP-1 cells, the I{kappa}B{alpha} level was more rapidly returned to the steady-state level in endotoxin-tolerant cells compared with control cells. From this finding, the authors speculated that the enhanced rate of synthesis of I{kappa}B{alpha} in the LPS-tolerant state is partially responsible for LPS tolerance, judged by the down-regulated IL-1ß expression, thereby causing only transient activation of NF-{kappa}B. The susceptibility of I{kappa}B{alpha} pathway to LPS stimulation in an LPS-tolerant state may depend on the particular cell type.

Recent studies demonstrated that TLR4 may be the primary mediator of LPS signaling [41 42 43 44 45 46 47 48 49 50 51 ]. The engagement of TLR4 with LPS induces NF-{kappa}B. It is interesting that LPS treatment caused a strong and transient suppression of TLR4 mRNA expression in mouse macrophage cell line RAW264.7 cells, suggesting that down-regulation of TLR4 mRNA contributes to endotoxin tolerance [41 ]. However, this is unlikely in our system, because our Northern analysis demonstrated that TLR4 mRNA was not suppressed in response to LPS in either the control or the LPS-tolerant state up to 3-h exposure of LPS (Fig. 7) . The down-regulation of TLR4 mRNA observed in RAW 264.7 cells may not be a generalized phenomenon, because TLR4 mRNA was significantly induced after treatment with LPS in human monocytes [64 ]. Further work is required to identify the crucial step of LPS signaling pathway, resulting in the lack of phosphorylation of I{kappa}B{alpha} in the LPS-tolerant state.

In conclusion, when an LPS-tolerant state is induced by short exposure to primary LPS, LPS-induced desensitization of TNF-{alpha} gene expression is closely associated with down-regulation of transactivating NF-{kappa}B (homodimers of p65 and heterodimers consisting of p50/p65, p50/c-rel, and p65/c-rel) activation, and may be involved in a defective LPS-inducible I{kappa}B{alpha} kinase pathway.


   ACKNOWLEDGEMENTS
 
We express our gratitude to the late Dr. Sadayoshi Sekiguchi, the ex-director of Hokkaido Red Cross Blood Center, for his support of this study.

Received July 6, 1999; revised March 20, 2000; accepted April 12, 2000.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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