Down-Modulation of L-Selectin by Lipopolysaccharide Is Not Required for Lipopolysaccharide-Induced Expression of CD14 in Mouse Bone Marrow Granulocytes

Abstract

We established in previous studies that a constitutive lipopolysaccharide (LPS) receptor of low affinity is present on mouse bone marrow granulocytes (BMG). This yet-unidentified receptor is involved in the LPS-induced expression of a second LPS receptor, CD14. Because it has been claimed that L-selectin (CD62L) is a low-affinity LPS receptor in mature granulocytes (polymorphonuclear leukocytes), it may be asked whether this molecule could be the constitutive LPS receptor in BMG. We show in this study that l-selectin is constitutively present on BMG and is down-regulated after exposure of the cells to LPS. A phorbol ester induced a down-regulation of CD62L and blocked the LPS-induced expression of CD14. However, a metalloproteinase inhibitor (BB-3103) blocked the former but not the latter effect of PMA. We also observed an absence of cross-reactivity between LPS and a CD62L ligand (fucoidan) in binding studies with radiolabeled derivatives of the two agents. Furthermore, BMG from l-selectin-deficient mice expressed normal levels of CD14 in response to LPS. Taken together, these results demonstrate that in BMG, l-selectin is not the constitutive LPS receptor required for the LPS-induced expression of CD14.

Host responses to pathogens require the recruitment of circulating leukocytes and their extravasation into tissues. This process is regulated by specific leukocyte-endothelial cell interactions mediated by several families of adhesion receptors. The initial interaction with endothelium that allows leukocytes to “roll” along the venular wall is mediated by selectin, a class of adhesion receptors that bind carbohydrate structures. Subsequently, other classes of adhesion receptors, including integrins and immunoglobulin (Ig) superfamily members, mediate “firm attachment” of the leukocytes to the endothelium.

The selectin family consists of three closely-related members: L-selectin (CD62L), constitutively expressed on all classes of leukocytes; E-selectin (CD62E), expressed on endothelium following activation with inflammatory cytokines; and P-selectin (CD62P), rapidly mobilized to the surface of activated platelets (4, 6, 12). The extracellular region of the three selectins includes a C-type lectin domain, an epidermal growth factor (EGF)-like domain, and several repeat units homologous to complement-binding sequences.

The function of selectins under certain pathological conditions has been investigated by several authors (5, 28). L-selectin-deficient mice were shown to be dramatically resistant to the lethal effects of high doses of lipopolysaccharide (LPS) in a model of septic shock (30). Other studies indicated that L-selectin can act as a low-affinity LPS receptor (16) and that the interaction of LPS with L-selectin in neutrophils can be blocked by fucoidan and lactoferrin and mediates cell activation and superoxide production (3, 17). Further studies have shown that LPS binds to P-selectin as well as to L-selectin (18). A pathophysiological role for selectins in LPS-induced sepsis is supported by the observation that sulfatides, which inhibit both L- and P-selectins, markedly decreased LPS-induced mortality in mice (11).

The observation that L-selectin can act as a low-affinity LPS receptor in neutrophils is reminiscent of our previous results showing that a constitutive LPS receptor of low affinity is present on mouse bone marrow granulocytes (BMG) and is involved in LPS-induced expression of the differentiation antigen CD14 (9). Because bone marrow is the site of differentiation and maturation of neutrophilic granulocytes (10) and because inflammatory stimuli increase the rate of polymorphonuclear leukocyte (PMN) production from the precursors, shorten their maturation time, and cause both mature and immature PMN to enter the circulation (20), in the present study, we examined whether L-selectin is involved in responses of BMG to LPS and whether down-regulation of L-selectin by different agents can influence these responses.

MATERIALS AND METHODS

Animals and cells.

LPS-responsive C3H/HeOU and LPS-hyporesponsive C3H/HeJ mice were bred and maintained in the animal facility of the Pasteur Institute (Paris, France). L-selectin-deficient (L^−/−) C57BL/6J × 129S3/SvImJ F₂ hybrid mice (strain B6129SF2/J) were obtained from Jackson Laboratory (Bar Harbor, Maine). Eight- to 10-week-old mice were used in all experiments. Bone marrow cells were collected by flushing femurs of mice and were used without further purification.

Media and reagents.

Fetal calf serum (FCS) was obtained from ATGC Biotechnologie (Noisy le Grand, France). Culture medium (CM) was made up of RPMI-1640 (GIBCO, Grand Island, N.Y.) containing 2 mM l-glutamine, 100 IU of penicillin per ml, 100 μg of streptomycin per ml, and 2-mercaptoethanol (5 × 10⁻⁵ M) and supplemented with 10% heat-inactivated (56°C, 30 min) FCS. Phorbol 12-myristate 13-acetate (PMA), fucoidan, dibutyl phthalate, and dinonyl phthalate were purchased from Sigma Chemical Co. (St. Louis, Mo.). The metalloproteinase inhibitor BB-3103 was obtained from British Biotech Pharmaceutical (Oxford, United Kingdom). The protein kinase C (PKC) inhibitor GF-109203X was obtained from Calbiochem (La Jolla, Calif.). The rat anti-mouse CD14 monoclonal antibody (Rm-C5–3), the rat anti-mouse CD16/CD32 monoclonal antibody (clone 2.4G2), and the biotin-labeled antimouse CD18 monoclonal antibody were obtained from PharMingen (San Diego, Calif.). The biotin-labeled anti-CD11b and anti-CD62L (clone MEL 14) antibodies were obtained from Caltag (Burlingame, Calif.). In fluorescence-activated cell sorting (FACS) experiments, fluorescein isothiocyanate (FITC)- or biotin-labeled goat anti-rat Ig antibodies (Southern Biotechnology Associates, Birmingham, Ala.) and FITC-labeled goat anti-hamster Ig antibody (Caltag) were used as secondary antibodies, and biotin-labeled antibodies were stained with FITC-labeled streptavidin (Amersham-Pharmacia Biotech, Buckinghamshire, United Kingdom). In Western blot experiments, the biotin-labeled antibody was stained with a streptavidin-peroxidase conjugate (Southern Biotechnology Associates). Autoradiography Hyperfilm MP and all electrophoresis reagents, including molecular mass standards (rainbow markers), were obtained from Amersham (Buckinghamshire, United Kingdom).

LPS and fucoidan and their radiolabeled derivatives.

LPS from Salmonella enterica serovar Choleraesuis (serotype 6₂,7,14) was prepared as described previously (9). LPS and fucoidan were activated with cyanogen bromide, coupled to tyramine, and iodinated with ¹²⁵I by the chloramine-T method, as described previously (9). Radiolabeled LPS (2.1 × 10⁶ cpm/μg) and fucoidan (6.1 × 10⁵ cpm/μg) were suspended in water and stored at −30°C.

Cell binding assay.

The binding of ¹²⁵I -LPS and ¹²⁵I-fucoidan to bone marrow cells was carried out at 0°C for 30 min in a standard binding medium (SBM) consisting of RPMI-1640 containing 100 IU of penicillin per ml, 100 μg of streptomycin per ml, 20 mM HEPES, 2 mM l-glutamine, 50 μM 2-mercaptoethanol, and 1 mM sodium pyruvate. Bone marrow cells (5 × 10⁶ cells in 1-ml polystyrene tubes) were incubated in SBM (total volume of 400 μl), with ¹²⁵I-fucoidan (1.25 μg/ml) or ¹²⁵I -LPS (5 μg/ml) presonicated in the presence or absence of various concentrations of an unlabeled competitor. Unbound ligand was removed by a modification (7) of the method of Tsudo et al. (31). The cells were resuspended and layered on cold mixtures (0°C, 200 μl) of 30% dinonyl phthalate–70% dibutyl phthalate (density of 1.025) in 1.5-ml conical microcentrifuge tubes. After centrifugation for 3 min at 10,000 × g and removal of the supernatant, the tips of the tubes containing the cell pellets were cut off, and the radioactivity was measured with a gamma counter (Kontron MR 480C). Assays were done in triplicate. The nonspecific bindings of radiolabeled fucoidan and LPS represent the bindings in the presence of 50- and 10-fold excesses of the homologous unlabeled ligands, respectively. Specific bindings represent the differences between total and nonspecific bindings.

FACS analysis.

Bone marrow cells (5 × 10⁵ cells in 400 μl of CM without FCS) were incubated at 37°C with (10 ng/ml) or without LPS. When used, inhibitors were added into cell cultures 30 to 60 min before LPS. For detection of membrane antigens, the cells were incubated first (30 min, 4°C) with the primary antibody and stained by reincubation (30 min, 4°C) with a labeled secondary antibody. Stained cells were layered on a 50% FCS solution and centrifuged, and the cell pellet was resuspended in 0.5 ml of staining buffer (phosphate-buffered saline [PBS], 5% FCS, 0.02% sodium azide) containing propidium iodide (0.2 μg/ml) to stain dead cells. Fluorescent cells were detected by analysis (5,000 cells per sample) on a FACS flow cytometer (FACScan; Becton-Dickinson Electronic Laboratories, Mountain View, Calif.) with Cell Quest software. Dead cells, which incorporated propidium iodide, were gated out of analysis. Cells with a fluorescence intensity higher than the maximal level of autofluorescence were scored as fluorescent cells.

SDS-PAGE analysis of membrane CD14.

Membrane proteins were extracted from the cell pellet with 1% CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate} in 300 mM NaCl–50 mM Tris (pH 7.5), supplemented with a cocktail of protease inhibitors (aprotinin, 10 μg/ml; phenylmethylsulfonyl fluoride, 1 mM; pepstatin and leupeptin, 2 μg/ml [each]; iodoacetamide, 2 mM.). Solubilized proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 10% polyacrylamide slab gels according to the method of Laemmli. Molecular mass markers from 14.3 to 220 kDa were run in parallel. Gels were fixed in transfer buffer (20 mM Tris, 150 mM glycine, 20% methanol), and proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, Mass.) with a semidry blotting system at 30 V for 90 min. Membranes were blocked (18 h at 20°C) with 2% bovine serum albumin (BSA) in PBS and incubated (1 h, 20°C) with the rat anti-mouse antibody rmC5–3 (1:1,000 in PBS containing 2% BSA). The blots were washed with 0.1% Tween 20 in PBS and then incubated for 1 h at 20°C with a biotin-labeled goat anti-rat antibody (1:2,500 in the same buffer). After extensive washing and incubation with peroxidase-labeled streptavidin (1:20,000 in 2% nonfat milk in PBS), sites with peroxidase activity were detected by chemiluminescence with the Super Signal system (Pierce, Rockford, Ill.) according to the guidelines of the manufacturer.

RESULTS

LPS down-regulates CD62L in BMG of endotoxin-responsive mice.

Unlike CD14, which is not constitutively expressed on BMG, several other cluster differentiation antigens are constitutively present on the surface of these cells. This is particularly the case of CD11b, CD11c, CD16, CD18, and CD62L. Using specific antibodies directed against these antigens, we examined by FACS whether exposure of BMG to LPS can modulate the expression of one or more of these antigens. After incubation for 20 h with 10 ng of LPS per ml, the cells were incubated (30 min, 4°C) with the specific antibodies, washed, stained by incubation (30 min, 4°C) with the appropriate secondary antibody, and analyzed by FACS. The results in Table 1 show that among the five antigens examined, CD62L (L-selectin) was the only one that was clearly down-regulated in response to the LPS treatment. (In a paired Student t test of LPS-treated versus untreated cells, we found P = 8 × 10⁻⁵ for the percentage of fluorescent cells and P = 3 × 10⁻⁶ for the mean fluorescence, indicating a highly significant statistical difference in CD62L expression.) To assess whether this effect is actually linked to the LPS responsiveness of the cells, we compared the down-modulation of CD62L in BMG from LPS-responsive C3H/HeOU and LPS-hyporesponsive C3H/HeJ mice. We found (Fig. 1) that incubation for 20 h with 10 ng of LPS per ml induced a 34.3% down-modulation of CD62L in C3H/HeOu cells and a 3.9% down-modulation of the antigen in C3H/HeJ cells. Analysis of the kinetics of this response in C3H/HeOU cells indicated (Fig. 2) that this effect appears rapidly after exposure of BMG to LPS. Fifty percent of the maximal decrease of cell surface CD62L was reached within 10 min, and the optimal effect required only 1 h.

TABLE 1.

Influence of LPS on levels of CD antigens in bone marrow cells

Antigen	Level in cellsa:
	Untreated with LPS		Pretreated with LPS
	Fluorescent cells (%)	Mean fluorescence (a.f.u.)	Fluorescent cells (%)	Mean fluorescence (a.f.u.)
CD11b	17	13	71	40
CD11c	15	13	64	30
CD16	99	317	99	292
CD18	90	86	85	137
CD62L	86	92	69	60

^aBone marrow cells (0.5 × 10⁶ cells), pretreated or not with 10 ng of LPS per ml (20 h, 37°C), were incubated (4°C, 30 min) with biotin-labeled antibodies directed against different CD antigens. The binding of the antibodies was detected by further incubation (4°C, 30 min) with FITC-labeled streptavidin or FITC-labeled secondary antibodies, followed by analysis of cell fluorescence by FACS. Granulocytic cells were gated on the basis of their forward scatter-side scatter characteristics, and only viable (propidium iodide negative) cells belonging to this population were analyzed. Values represent the results obtained in one representative experiment. 

FIG. 1.

Comparative levels of CD62L in untreated and LPS-treated C3H/HeOU and C3H/HeJ cells. Bone marrow cells (0.5 × 10⁶ cells) from C3H/HeOU mice (A and C) and C3H/HeJ mice (B and D) were incubated (20 h, 37°C) without (A and B) or with 10 ng of LPS per ml (C and D). The cells were then incubated (4°C, 30 min) with the biotin-labeled anti-CD62L antibody MEL-14. The binding of the antibody was detected by further incubation (4°C, 30 min) with FITC-labeled streptavidin, followed by analysis of cell fluorescence by FACS. Granulocytic cells were gated on the basis of their forward scatter-side scatter characteristics, and only viable (propidium iodide negative) cells belonging to this population were analyzed. The mean fluorescence of the population analyzed is indicated in each panel.

FIG. 2.

Kinetics of the LPS-induced down-regulation of CD62L. Bone marrow cells (0.5 × 10⁶ cells) of C3H/HeOU mice were incubated at 37°C for different times with 1 μg of LPS per ml. The level of CD62L on the viable granulocyte population was determined as described in the legend to Fig. 1. Values represent the mean fluorescence of the population analyzed.

PMA down-regulates CD62L in BMG.

To analyze the influence of L-selectin down-regulation on LPS responses, we looked for another agent able to reduce the expression of L-selectin. It has been reported that PMA down-regulates CD62L in PMN (12). We thus examined whether this agent can induce the same effect in BMG. The results obtained by FACS analysis of BMG pretreated with PMA indicated that down-modulation of CD62L requires concentrations of PMA of 1 ng/ml or higher (Fig. 3A). At these concentrations of PMA, BMG remained fully viable (95% viability with up to 100 ng of PMA per ml). The level of constitutive CD62L (80 arbitrary fluorescence units [a.f.u.]) decreases to 9 a.f.u. after treatment for 30 min at 37°C with 100 ng of PMA per ml (Fig. 3B). This is very close to the autofluorescence level of the cells (5 a.f.u.) and represents a 96.5% down-modulation of cell surface L-selectin.

FIG. 3.

PMA-induced down-modulation of CD62L. Bone marrow cells (0.5 × 10⁶ cells) of C3H/HeOU mice were incubated for 30 min at 37°C with different concentrations of PMA. The level of CD62L on the viable granulocyte population was then determined as described in the legend to Fig. 1. Ninety-five percent of the cells were viable after treatment with 100 ng of PMA per ml. Panel A illustrates the percentage of fluorescent (CD62L⁺) cells and the mean fluorescence of the granulocyte population. Panel B illustrates the fluorescence histograms of BMG untreated (bold line) or treated with 100 ng of PMA per ml (thin line), obtained by FACS.

Influence of a PKC inhibitor on PMA- and LPS-induced shedding of L-selectin.

Because both LPS and PMA induce the down-regulation of CD62L, and because many of the PMA effects are mediated by PKC, we examined whether a PKC inhibitor can block the two effects. We used the specific PKC inhibitor GF-109203X. The results in Fig. 4 show that GF-109203X partially inhibits the down-regulation of CD62L inhibited by PMA, but has no significant influence on the LPS-induced effect. This indicates that LPS and PMA induce L-selectin down-regulation by different mechanisms.

FIG. 4.

Influence of a PKC inhibitor on LPS- and PMA-induced shedding of CD62L. Bone marrow cells (0.5 × 10⁶ cells) of C3H/HeOU mice were preincubated (30 min at 37°C) with or without 4 μM GF109203X. LPS (1 μg/ml) or PMA (10 ng/ml) was then added, and the cells were reincubated for 30 min at 37°C. The level of CD62L on the viable granulocyte population was then determined as described in the legend to Fig. 1.

Influence of a metalloproteinase inhibitor on PMA-induced shedding of L-selectin and on the response of BMG to LPS.

We reported previously (21) that as a consequence of their stimulation with LPS, BMG express membrane CD14, detectable by Western blotting. We have also shown recently that PMA (100 ng/ml) blocks this LPS-induced expression of CD14 (22). Because PMA induces a down-modulation of L-selectin and blocks one of the responses of BMG to LPS, it was tempting to speculate that L-selectin could be required for this LPS response. One possible approach to examine this hypothesis was to block the PMA-induced down-regulation of L-selectin and determine if this would restore the LPS response. Because one of the main mechanisms of receptor down-regulation is a shedding of the receptor and because metalloproteinases are often involved in shedding, we used the metalloproteinase inhibitor BB-3103 to examine the role of L-selectin in the response to LPS. The results in Fig. 5A show that the complete loss of membrane CD62L induced by PMA can be partially reversed by increasing doses of BB-3103. The FACS histogram obtained with 0.1 μg of PMA per ml and 25 μg of BB-3103 per ml (Fig. 5B) also shows the inhibition by BB-3103 of the PMA-induced shedding of CD62L. We then examined whether BB-3103 can also reverse in BMG the PMA-induced inhibition of the LPS effect (i.e., the LPS-induced expression of membrane CD14). We found (Fig. 5C) that even in the presence of 25 μM BB-3103, the LPS-induced expression of membrane CD14 is still markedly inhibited by PMA. Because the dose of BB-3103 (25 μM) that inhibits 97% of PMA-induced shedding of CD62L (Fig. 5A) does not inhibit the PMA-induced down-regulation of the LPS effect (Fig. 5C), we can conclude either that CD62L (L-selectin) is not involved in the LPS effect examined (expression of CD14) or that PMA acts downstream of the LPS receptor.

FIG. 5.

Influence of a BB-3103 on PMA-induced effects. Two effects of PMA were analyzed: the shedding of CD62L (A and B) and the inhibition of LPS-induced expression of CD14 (C). After preincubation (30 min at 37°C) with various concentrations of the metalloproteinase inhibitor BB-3103, bone marrow cells (0.5 × 10⁶ cells) of C3H/HeOU mice were incubated (30 min, 37°C) with (100 ng/ml) or without PMA. The level of CD62L on the viable granulocyte population was then determined as described in the legend to Fig. 1 (A). Panel B illustrates the FACS fluorescence histograms of BMG untreated (thin line), directly treated with 100 ng of PMA per ml (dotted line), or treated with 25 μg of BB-3103 per ml before exposure to 100 ng of PMA per ml (bold line). After treatments with BB-3103 and/or PMA, the cells were exposed for 3 h to 10 ng of LPS per ml, and the induction of CD14 expression was analyzed by Western blotting (C). m.w., molecular mass.

Binding of an L-selectin ligand is not inhibited by LPS.

Fucoidan is a homopolymer of sulfated l-fucose known to inhibit the interactions of L-selectin with its natural endothelial cell ligands. Because it has been shown that both fucoidan and LPS bind to L-selectin in neutrophils, we examined whether the binding of fucoidan to bone marrow cells can be inhibited by LPS. We used a preparation of fucoidan radiolabeled with ¹²⁵I. The results in Fig. 6 show that the binding of ¹²⁵I-labeled fucoidan (1.25 μg/ml) to the cells is efficiently inhibited by unlabeled fucoidan (62.5 μg/ml), but is not inhibited by the same concentration of LPS. Furthermore, using ¹²⁵I-LPS (5 μg/ml), we found that the specific binding of this radiolabeled LPS (difference between bindings in the absence or presence of a 10-fold excess of unlabeled LPS) to constitutive LPS-binding sites of bone marrow cells (25,900 ± 2,700 cpm) was not significantly different from that obtained in the presence of 20 μg of unlabeled fucoidan per ml (22,700 ± 2,700 cpm). Therefore, LPS did not inhibit the binding of fucoidan, and fucoidan did not inhibit the binding of LPS.

FIG. 6.

Inhibition of the binding of ¹²⁵I-fucoidan by unlabeled fucoidan and LPS. Bone marrow cells (5 × 10⁶ cells) were incubated for 30 min at 0°C with ¹²⁵I -fucoidan (1.25 μg/ml) in SBM (0.4 ml) in the presence or absence of various amounts (1.25 to 62.5 μg/ml) of fucoidan (●) or LPS (▴). Bound ligand was measured after centrifugation of the cells through phthalate. Nonspecific binding (10,300 ± 1,500 cpm) was subtracted from total binding values. Results represent the specific binding (mean ± standard deviation) of triplicate determinations.

Responsiveness of L-selectin-deficient BMG to LPS.

To obtain a clear-cut answer to our question on the possible role of L-selectin in responses of BMG to LPS, we used mice lacking L-selectin because of a targeted mutation on the Sell gene (Sell^tm1Hyn) of their chromosome 1. It has been reported that the numbers of leukocytes rolling per min in tumor necrosis factor alpha (TNF-α)-stimulated venules were markedly reduced in these L-selectin-deficient (L^−/−) animals (25). We prepared bone marrow cells of these L^−/− mice. As expected, these cells did not express detectable levels of CD62L on their surface (Fig. 7). We then examined their ability to express CD14 after a 3-h exposure to 10 ng of LPS per ml. The expression of CD14 was analyzed by Western blotting. The results in Fig. 7 show that this response of L^−/− bone marrow cells to LPS was not different from that of L^+/+ cells. This observation clearly demonstrates that L-selectin is not the constitutive LPS receptor required for LPS-induced expression of CD14.

FIG. 7.

Responsiveness of bone marrow cells from L-selectin-deficient mice to LPS. Bone marrow cells (0.5 × 10⁶ cells) of normal (A) and L-selectin-deficient (B) mice were incubated (4°C, 30 min) with (bold line) or without (thin line) the biotin-labeled anti-CD62L antibody MEL-14. The level of CD62L on the viable granulocyte population was then determined by FACS. BMG from normal (C3H/HeOU) and L-selectin-deficient (L ^−/−) mice were also exposed for 3 h at 37°C to 10 ng of LPS per ml, and the induction of CD14 expression was analyzed by Western blotting (C). M.W., molecular mass.

DISCUSSION

CD62L (L-selectin) is a cell surface adhesion molecule involved in the recruitment and homing of leukocytes. We observed that this well-known constituent of mature lymphocytes and granulocytic neutrophils is also present on the immature (CD14⁻) granulocytes of bone marrow, and we demonstrated that LPS, one of the most biologically active agents of gram-negative bacteria, induces down-regulation of this antigen. As for many other biological effects of LPS, we did not observe this down-regulation of CD62L in BMG from animals with a defect on their TLR4 gene (C3H/HeJ mice) (Fig. 1).

We show, however, that many other CD antigens (CD11b, CD11c, CD16, and CD18) are not down-regulated in response to LPS (Table 1). Moreover, previous studies in our laboratory indicated that CD14 is not constitutively present on BMG and that its level increases progressively upon exposure of the cells to LPS. This second effect of LPS on BMG has been thoroughly studied in our laboratory, and we established that an LPS receptor of low affinity (K_d = 480 nM), yet unidentified, triggers this CD14 expression. This was deduced from the observation that inhibition of LPS binding by different treatments (incubation of the cells with serum or with the synthetic lipid PPDm2) correlated with inhibition of induction of CD14 by LPS (9).

In spite of some nonconcordant reports, the mechanism of action of enterobacterial LPS on monocytes or macrophages is currently often accepted as a paradigm of the action of any LPS in any cell type. According to this paradigm, an interaction of LPS with Tlr4 triggers the signaling cascade. Concerning a possible relationship between the low-affinity receptor of BMG and Tlr4, data from our laboratory (still unpublished) suggest that the molecular mass of the low-affinity receptor in BMG is much smaller than that of Tlr4. In this respect, it is noteworthy that it has never been clearly established that LPS has direct contact or any affinity with Tlr4, although some experiments may indirectly suggest this. For example, it has been reported that in macrophages, antibodies against Tlr4 can partially block signal transduction initiated by LPS (1, 26) and that the discrimination between LPS and LPS partial structures is dependent on the species origin of Tlr4 (15, 24). However, these data are not direct evidence that Tlr4 binds LPS. Furthermore, because Tlr4 also transduces the signal delivered by lipoteichoic acid (29), which is structurally very different from LPS, it seems unreasonable to postulate that Tlr4 directly binds to these two ligands. Rather than a direct contact between LPS and Tlr4, an alternative possibility is that Tlr4-mediated signal requires the interaction of LPS with other LPS-binding molecules, depending on the cell type concerned (CD14 and Mac-1 in macrophages, L-selectin in mature circulating neutrophils, and the yet-unidentified low-affinity receptor in BMG). The observation of Perera and colleagues (23) indeed supports this concept of various coreceptors required for Tlr4 signaling.

Since CD14 is considered to be a differentiation antigen and since down-regulation of L-selectin is regarded as a hallmark of granulocyte activation (13), the observation that LPS triggers both CD14 expression and L-selectin down-regulation in BMG indicates that LPS induces both activation and differentiation of these cells. Because studies of Malhotra et al. (16) suggested that L-selectin can act as a low-affinity LPS receptor and is involved in LPS-induced activation of mature granulocytes (17), it was tempting to speculate that the yet-unidentified LPS receptor of low affinity required for CD14 expression could be L-selectin. In this hypothesis, LPS would first interact with L-selectin, induce its down-regulation, and trigger further biochemical events leading to CD14 expression. In line with this sequence of events, we found (Fig. 2) that the LPS-induced decrease in CD62L is evident after only 10 min, whereas expression of CD14 requires several hours (8). The ability of L-selectin to trigger cell signaling events is also well documented: this adhesion molecule has been shown to transduce signals leading to cytokine production (14), enhanced oxidative burst (32), protein phosphorylation (33), and integrin function (27).

To determine if L-selectin can function as an LPS receptor, a first approach can be to ask whether after complete down-regulation of this molecule, the cells can or cannot respond to LPS. We found that treatment with PMA induced a marked down-regulation of L-selectin and completely blocked the LPS-induced expression of CD14 detectable by Western blotting. However, this result did not prove that L-selectin is involved in the LPS-induced expression of CD14, because in the presence of the metalloproteinase inhibitor BB-3103, PMA appeared unable to down-regulate L-selectin, but was still able to block the response to LPS (Fig. 5).

Another way to determine if L-selectin can be considered as an LPS receptor is to examine whether LPS actually binds to L-selectin. Fucoidan is generally regarded as a good competitor of L-selectin ligands. For example, it has been shown that the specific binding of human factor H to L-selectin is inhibited by fucoidan (19). Our results with radiolabeled fucoidan and radiolabeled LPS in competition binding assays indicated a complete absence of cross-reactivity between fucoidan and LPS on bone marrow cells. This can be taken as an indication that LPS does not bind to L-selectin on BMG. However, the possibility that LPS and fucoidan bind to L-selectin at different sites of the molecule cannot be ruled out by these experiments.

Direct and indisputable evidence that L-selectin is not a functional LPS receptor in BMG was definitely obtained by the use of L-selectin-deficient mice. We found that BMG from these mice produced normal levels of CD14 in response to LPS, although L-selectin was completely absent from their cell surface, as assessed by FACS analysis (Fig. 7).

Therefore, our data suggest that in bone marrow cells, the two effects of LPS examined (early down-regulation of CD62L and later expression of CD14) are not the result of an L-selectin-dependent signaling pathway. These results do not seem to accord with those of Tedder et al. showing that L-selectin-deficient mice are resistant to endotoxic shock (30) and those of Malhotra et al. suggesting that L-selectin can act as an LPS receptor involved in the activation of neutrophils (16, 17). These discrepancies could be due to the state of maturation of the cells examined. We used bone marrow granulocytes, which are relatively immature cells, whereas the neutrophils used by other authors represent a more mature population of circulating granulocytes. It is therefore possible that in mature granulocytes, L-selectin could take part in a receptor complex, in association with CD14, TLR4, and other surface molecules, whereas in the immature granulocytic population of the bone marrow, L-selectin would not yet be associated with this complex. CD14 could be a good candidate for anchoring L-selectin in the receptor complex, since CD14 is not present in BMG, is expressed in circulating granulocytes, and is associated with the TLR4 constituent of the LPS-receptor complex. Further investigations are required to test this hypothesis.