Role of Galectin-3 as an Adhesion Molecule for Neutrophil Extravasation During Streptococcal Pneumonia

Cells

Human endothelial cell lines ECV304, HL-60, and hybridoma M3/38.1.2.8 (Mac-2, anti-galectin-3 Ab) were obtained from American Tissue Culture Collection (Manassas, VA). HUVECs were obtained from Cell Applications (San Diego, CA).

Anti-galectin-3 Ab

A rat mAb against galectin-3 was purified from the cultured medium (serum-free hybridoma medium (Life Technologies, Burlington, Ontario, Canada)) of the hybridoma M3/38.1.2.8 by a protein G-Sepharose column (Amersham Pharmacia Biotech, Piscataway, NJ).

Mouse streptococcal pneumonia model

A murine model of streptococcal pneumonia was used as described previously (4, 34). Briefly, lightly anesthetized female CD1 mice (18–20 g) received an inoculum of 10⁷ CFU of S. pneumoniae serotype 3 in 50 μl of PBS applied at the tip of the nose and involuntarily inhaled. Infected animals were sacrificed by decerebration (under anesthesia) at different time points after infection over a 3-day period. In some experiments, lightly anesthetized female CD1 mice received an inoculum of 10⁷ CFU of E. coli instead of S. pneumoniae to induce the extravasation of neutrophils to the alveoli.

In some experiments, the extravasation of neutrophils in S. pneumoniae-infected alveoli of female mice of strains C57BL/6 and BALB/c (instead of CD1) was also examined 6 h postinfection. The other S. pneumoniae strain, Harvard strain (kindly provided by Dr. C. M. Doerschuk, Case Western Reserve University, Cleveland, OH, 10⁸ CFU/mice (7)), which was used in the papers suggesting β₂ integrin-independent neutrophil extravasation, was also used in some experiments to induce neutrophil extravasation (6 h postinfection) to the infected alveoli of CD1, C57BL/6, and BALB/c mice.

Bronchoalveolar lavage (BAL) fluid was collected by gently washing the alveolar space twice with 1 ml of PBS through the trachea. The BAL fluid was centrifuged at 550 × g for 10 min to separate the cells from the supernatants. The cell-free BAL supernatants (BAL sup) were used for the estimation of galectin-3 and TNF-α. The cells were resuspended in PBS for multiple analyses, including a total cell count by hemacytometer and differentiation of cell populations by Diff-Quick (Baxter, Quebec, Canada) staining of cytospin preparations.

Analysis of galectin-3 in cell-free supernatants of BAL fluids or exudated leukocytes of BAL fluids

Cell-free supernatants of BAL fluids (50 μl) were fractionated by SDS-PAGE and transferred to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH) using a Tris-glycine buffer system. After incubation with 10% skimmed milk in TBST (20 mM Tris-HCl (pH 7.5),150 mM NaCl, 0.2% Tween 20), membranes were incubated with anti-galectin-3 Ab followed by anti-rat IgG-peroxidase (Amersham Pharmacia Biotech). Ab complexes bound onto membranes were detected by exposing to Blue XB-1 film (NEN Life Science, Boston, MA) after incubating with chemiluminescence substrate for peroxidase (NEN Life Science).

Exudated leukocytes (cell fractions) of BAL fluids were homogenated with 500 μl of buffer containing 1% Triton X-100, 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and protease inhibitors mixture (buffer A; Sigma, Oakville, Ontario, Canada). The homogenates were then centrifuged at 35,000 × g for 15 min to obtain the cell extracts. Ten microliters of cell extracts were used for the estimation of galectin-3.

Leukocytes

Mouse resident alveolar macrophages were purified from the BAL fluid of noninfected mice. BAL fluid was prepared as described above. After spinning down of cells and removal of contaminating RBCs by hypotonic lysis, the leukocytes in BAL fluid were resuspended in RPMI 1640 supplemented with 10% FCS (HyClone Laboratories, Logan, UT) and incubated at 37°C for 1 h on a tissue culture dish. Unbound cells were removed by washing three times with PBS. More than 95% of adherent cells showed an appearance characteristic of macrophages when examined by light microscopy.

Exudated neutrophils were purified from leukocytes that emigrated to air pouches on the dorsum of CD1 mice as described by Tessier et al. (35, 36). Briefly, an air pouch was raised on the dorsum by s.c. injection of 2 ml of sterile air. On day 6 after two injections of air 3 days apart, inflammation was induced by injecting 1 ml of 10 μg/ml LPS in PBS into the air pouch (35, 36). Six hours after the induction, emigrated leukocytes were obtained by washing the air pouches with PBS supplemented with 1 mM EDTA (2 ml, three times). Leukocytes were resuspended in 10% FCS-MEM and incubated at 37°C for 2 h on tissue culture dishes and nonadherent cells were collected. More than 95% of the nonadherent cells showed an appearance characteristic of neutrophils when Diff-Quick-stained and examined by light microscopy.

Alveolar macrophages or exudated neutrophils were homogenized with buffer A as described above, and the levels of galectin-3 in those cells were estimated by Western blotting with anti-galectin-3 Ab.

Secretion of galectin-3 from alveolar macrophages

The membrane fractions of S. pneumoniae were prepared as follows. S. pneumoniae was washed with PBS three times and resuspended in ice-cold buffer containing 20 mM Tris-HCl (pH 7.5). After freezing and thawing twice, insoluble membrane fractions were separated by centrifugation at 5,000 × g for 10 min and the membrane fractions were further washed with PBS twice. Mouse resident alveolar macrophages were purified as described above and were incubated overnight in fresh 10% FCS-RPMI 1640. Alveolar macrophages (1 × 10⁵ cells) were incubated in the presence or absence of LPS (10 μg/ml) or the membrane fraction of S. pneumoniae (serotype 3) at macrophage:S. pneumoniae cell ratio of 1:50 for 2 h. After incubation, culture media were removed and centrifuged to obtain cell-free supernatants. Aliquots (40 μl) of the supernatants were used for the analysis of galectin-3 secretion.

Immunofluorescence analysis of BAL neutrophils

Exudated neutrophils in BAL fluid of mice infected with S. pneumoniae (4 h postinfection) were purified by brief panning of BAL fluids in a petri dish to remove macrophages. Nonbound neutrophils were then incubated with PBS supplemented with 0.1% sodium azide and 0.1% BSA (PAB) together with either anti-galectin-3 (Mac-2) Ab or control rat IgG followed by anti-rat IgG Ab labeled with Alexa 448 (Molecular Probes, Eugene, OR) as described previously (19, 20). FACS analysis was conducted by FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) and neutrophil populations were selected based on the size and the granularity of cells at the time of data collection.

Immunohistochemistry

Fresh left lungs were perfused and fixed overnight at 4°C in 0.1 M phosphate-buffered 4% paraformaldehyde. Tissues were then dehydrated and embedded in paraffin. After blocking with normal rabbit serum (Vector Laboratories, Burlingame, CA), 5 μm of tissue sections were first incubated with anti-galectin-3 Ab (10 μg/ml) or normal rat IgG for 2 h at 37°C. Bound anti-galectin-3 Ab was visualized by biotinylated anti-rat IgG Ab followed by VECTASTAIN Elite ABC reagent following the manufacturer’s instructions (Vector Laboratories). A 0.5% toluidine blue O solution was used as counterstaining in some tissue sections. Specificity of labeling was tested by 1) omitting the primary Ab in the procedure or 2) replacing the primary Ab by normal rat IgG.

Galectin-3 and its CRD

Recombinant human galectin-3 was purified as described previously, with modification (15). Briefly, E. coli JM109 was transformed with an expression plasmid of human galectin-3 (kindly provided by Drs. J. Hirabayashi and K. Kasai, Teikyo University, Tokyo, Japan). Two liters of this E. coli (JM-HG29-c1.2) overnight bacteria culture medium were incubated with 1 mM isopropyl β-d-thiogalactoside for 3 h at 37°C to induce galectin-3 production and E. coli was pelleted by centrifugation. After sonicating bacteria in buffer B (20 mM Tris-HCl (pH 7.5), 0.15 M NaCl) containing 5 mM EDTA, 1 mM DTT, and protease inhibitor mixture (Sigma), cell homogenates were centrifuged to obtain a soluble fraction. This soluble fraction (∼50 ml) was then applied to 5 ml of asialofetuin-agarose (4 mg asialofetuin/ml gel), which was prepared with AminoLink Plus Coupling Gel (Pierce, Rockford, IL) following the manufacturer’s instructions. After extensive washing of the column (∼20 bed volume) with buffer B, galectin-3 was eluted with buffer B containing 100 mM lactose. The eluate was dialyzed against PBS containing 1 mM EDTA and then against PBS to remove lactose. Typically, 3–7 mg of galectin-3 was purified from 2-liter cultures of E. coli. The purity of galectin-3 was determined by Coomassie blue staining and silver staining of a SDS-polyacrylamide gel. Purified galectin-3 was filter-sterilized, kept at 4°C, and used within 1 mo.

The CRD of galectin-3 was prepared as described by Massa et al. (24). Briefly, galectin-3 was digested with collagenase VII (Sigma) in 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 2 mM CaCl₂ for 37°C for 4 h. The digestion was stopped by adding EDTA (final concentration, 5 mM), and the CRD of galectin-3 was purified with asialofetuin agarose affinity column chromatography as described above.

In vitro neutrophil adhesion assay

Human neutrophils, which do not secrete galectin-3 (26), were purified from blood of healthy volunteers. Briefly, after incubating heparinized blood with 3% dextran, the leukocyte- and erythrocyte-rich fractions were subjected to Ficoll-Hypaque separation (Amersham Pharmacia Biotech) to purify neutrophils. When primed neutrophils were required, neutrophils were treated in vitro with fMLP (27, 37). When differentiated HL-60 cells were required, HL-60 cells were incubated overnight in the presence of all trans retinoic acid (1 μM).

Human endothelial cells ECV304 or HUVECs (Cell Applications) were plated (10⁴ cells/well) to 96-well plates and grown to confluence in 10% FCS-M199 (Life Technologies) or in endothelial cell growth medium (Cell Applications), respectively. The endothelial monolayers were washed gently with serum-free M199 medium twice and 25 μl of fresh serum-free M199 medium was added to each well. Human neutrophils or HL-60 were labeled with calcein-acetoxymethyl (Molecular Probes) by following the manufacturer’s instructions. Labeled neutrophils (10 μl, 10⁵ cells) together with PBS (25 μl) containing various amounts of galectin-3 were added to the wells and plates were incubated for the indicated time at 37°C. At the end of incubation, the plates were immersed in cold PBS and inverted. Excess liquid and nonadherent cells were removed by blotting onto paper towels (repeated three times) (38). Bound neutrophils were lysed by adding 1% Triton X-100-PBS and the associated fluorescence was measured using a fluorometric plate reader (PerSeptive Biosystems, Foster City, CA). When the adhesion assays were performed in the presence of lactose, the sodium chloride concentration in the PBS was adjusted to maintain appropriate osmolarity, i.e., 317 mosmol/L. In the case when endothelial cells were exposed to LPS, confluent endothelial monolayers were preincubated in the 10% FCS-M199 containing 2 μg/ml LPS for 2 h at 37°C. After preincubation, monolayers were washed and then the adhesion assays were conducted as described above in the presence of LPS (2 μg/ml).

Immunofluorescence analysis of human blood neutrophils and human endothelial cells ECV304

Human blood neutrophils were prepared as described above. Monolayers of ECV304 were trypsinized briefly to obtain single cell suspensions. Neutrophils or ECV304 cells (1 × 10⁶ cells) were first incubated with galectin-3 (1 μM) for 10 min and unbound galectin-3 was removed by washing twice with PAB. To analyze the level of galectin-3 that bound to cells, cells were then incubated with anti-galectin-3 Ab or control rat IgG followed by anti-rat IgG Ab labeled with Alexa 448 (Molecular Probes). FACS analysis was conducted as described above.

Accumulation of soluble galectin-3 in BAL fluids of mice infected with S. pneumoniae

To study a role of galectin-3 in the recruitment of neutrophils into the lung tissue during infection with S. pneumoniae, a mouse model was used (4). Mice (CD1) were inoculated with S. pneumoniae (serotype 3; 1 × 10⁷ CFU) intranasally, resulting in induction of typical pneumonia symptoms, including recruitment of leukocytes into alveoli and lung interstitium.

BAL fluids were collected from infected mice and centrifuged to separate the leukocytes that had emigrated to alveolar spaces, from supernatants (BAL sup) containing factors and proteins secreted into the alveolar spaces (4, 34). Galectin-3 content in the BAL sup was estimated by Western blotting with anti-galectin-3 mAb (Fig. 1⇓A). A very low level of galectin-3 was detected in the BAL sup from untreated mice and from mice infected for 2 h. However, 4 h after infection, the amount of galectin-3 in the BAL sup was significantly increased by an average of ∼5-fold compared with those from uninfected mice, reaching a peak (32-fold of those from uninfected mice) 12 h postinfection (Fig. 1⇓B). The level of galectin-3 in the BAL sup remained the same level up to 48 h and then gradually decreased by 72 h when 30% of mice began to die due to pneumonia (data not shown). Thus, these data indicate that galectin-3 is actively released into the alveolar space 4–48 h after infection with S. pneumoniae.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 1.

Accumulation of galectin-3 in BAL fluid from mice infected with S. pneumoniae. A, Mice (CD1) infected with S. pneumoniae (serotype 3) were killed at given times, and ∼2 ml of BAL fluid was collected from each mouse (see Materials and Methods). After centrifugation to obtain cell-free supernatants, levels of galectin-3 were compared by Western blotting. Each lane represents a sample from one mouse. B, Integrated intensities of galectin-3 were obtained by densitometric scanning. Means and SD of the arbitrary units were plotted against time after inoculation.

Correlation of the kinetics of accumulation of galectin-3 in alveoli with the kinetics of neutrophil emigration to alveoli during S. pneumoniae infection

The numbers of neutrophils in BAL fluids were increased during S. pneumoniae-induced inflammation (Fig. 2⇓) as previously reported (4, 34). In BAL fluids from mice treated with PBS (Fig. 2⇓) or from untreated mice (data not shown), negligible number of neutrophils (∼3 × 10⁴ cells/ml) were present. The number of neutrophils in BAL fluids was significantly increased 4 h after S. pneumoniae inoculation (Fig. 2⇓), reaching a peak around 12 h, and was maintained at this level up to the point of death 96 h after infection. In contrast, a small but significant number of resident alveolar macrophages (∼3 × 10⁵ cells/ml BAL fluid) was observed in BAL fluids from uninfected mice. The number of macrophages in BAL fluid was not altered significantly during the first 24 h after infection (Fig. 2⇓). Thus, the accumulation of galectin-3 in BAL sup (Fig. 1⇑) of infected animals correlated closely to the kinetics of neutrophil extravasation into alveolar spaces.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 2.

Leukocyte emigration in BAL fluid during streptococcal pneumonia. BAL fluids were collected from mice (CD1) infected with S. pneumoniae (serotype 3) at the indicated times. Total leukocytes in BAL were counted and leukocyte populations were enumerated from Diff-Quick-stained cytospin preparations.

Endogenous galectin-3 expression in alveolar leukocytes during early streptococcal infection

The levels of endogenous galectin-3 in total leukocytes that were recovered from infected alveoli were next analyzed. Equal proportions of total lysates of cell fractions isolated from BAL fluids were applied to SDS-polyacrylamide gels and the amounts of endogenous galectin-3 were estimated by Western blotting. The levels of galectin-3 associated to BAL leukocytes from infected mice remained similar to those from uninfected mice (Fig. 3⇓A), despite the fact that 26- to 33-fold more neutrophils were present in those BAL fluids from infected alveoli compared with uninfected alveoli (Fig. 2⇑).

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 3.

Galectin-3 expression in leukocytes. A, Endogenous galectin-3 in leukocytes of BAL fluids. Six hours after inoculation of S. pneumoniae (serotype 3), exudated leukocytes in BAL fluids from mice (CD1) were prepared from lavage fluid. After lysis of cells, 10 μl of cell lysates were subjected to SDS-PAGE followed by Western blotting with anti-galectin-3 Ab to analyze the levels of galectin-3 in alveolar exudated leukocytes. Each lane represents a sample from one infected or control mouse. Means and SD of the arbitrary units which were obtained by densitometric scanning are shown. Despite massive accumulation of neutrophils in BAL obtained from infected mice (Fig. 2⇑), the levels of total cellular galectin-3 were not significantly changed. B, Galectin-3 in alveolar macrophages and inflammatory exudated neutrophils. Cell lysates of alveolar macrophages and inflammatory exudated neutrophils were prepared, and levels of endogenous galectin-3 in cellular extracts from indicated numbers of alveolar macrophages and neutrophils were analyzed by Western blotting with anti-galectin-3 Ab. Representative data from two separate experiments are shown.

We next analyzed the level of endogenous galectin-3 in exudated neutrophils and alveolar resident macrophages. Neutrophils were purified from leukocytes that had emigrated to inflamed s.c. dorsal air pouch, and alveolar resident macrophages were prepared from BAL fluids. As Fig. 3⇑B shows, no significant level of galectin-3 was detected in lysates of the mouse neutrophil fraction (2 × 10⁵ cells). By contrast, galectin-3 was readily detected in the extracts from 10 times lower number of alveolar macrophages (2 × 10⁴ cells) (Fig. 3⇑B, lane 2). This result suggests that, unlike human neutrophils that express galectin-3 intracellularly (Ref. 39 and data not shown), murine neutrophils do not express endogenous galectin-3. Together, these data suggest that increased level of galectin-3 in BAL sup obtained from infected mice (Fig. 1⇑) is not due to the accumulation of neutrophils and thus galectin-3 is released from other cells, including alveolar macrophages.

Alteration of tissue distribution of galectin-3 during S. pneumoniae infection

Immunocytochemical analysis was next performed on sections of formalin-fixed lungs from mice infected with S. pneumoniae for 12 h to determine whether the distribution of galectin-3 in the lung was altered during the early stage of streptococcal pneumonia (Fig. 4⇓). In noninfected lungs, positive staining with anti-galectin-3 Ab was scattered (Fig. 4⇓A). The distribution and the shape of the labeled cells suggest that some resident alveolar macrophages were galectin-3 positive, consistent with a previous report suggesting that galectin-3 is localized to alveolar macrophages (40). No significant galectin-3 positive staining was observed in uninfected alveolar wall (Fig. 4⇓A). In striking contrast to the noninfected lung, galectin-3-positive staining in S. pneumoniae-infected lung was not limited to alveolar macrophages in S. pneumoniae-infected lung (Fig. 4⇓, B and D). A greater number of alveolar parenchyma cells particularly located in the vicinity of vascular regions were labeled (Fig. 4⇓, B and D). Dispersed interstitial macrophages, alveolar epithelial cell layer, and vascular endothelium were most likely among the galectin-3-positive cells. At some places, diffuse but distinctive colored deposits were also observed in the alveolar interstitium located in between the alveolar epithelium and the vascular endothelium (Fig. 4⇓B). In contrast, negative staining of infected lung tissue sections was displayed when anti-galectin-3 Ab was replaced with normal rat IgG (Fig. 4⇓C). Thus, the redistribution of galectin-3 we observed at 12 h postinfection occurred within the window of active recruitment of neutrophils that occurs during development of the pneumonia.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 4.

Galectin-3 expression in lung tissues of mice infected with S. pneumoniae. Paraffin sections of lungs of mice (CD1) treated with PBS (A) or S. pneumoniae (serotype 3) for 12 h (B–D) were incubated either with anti-galectin-3 Ab (A, B, and D) or with normal rat IgG (C) and bound Ab was detected by biotinylated anti-rat IgG Ab followed by streptavidin-peroxidase. Some sections were counterstained with 0.5% toluidine blue O (C and D). Magnification: A and B, ×100; C and D, ×1000. Arrowheads indicate some of galectin-3-positive regions, which are likely associated with cells in lungs. Arrow indicates diffused galectin-3-positive region in the alveolar interstitium. A, Alveolus; V, blood vessel. Representative data from three separate experiments are shown.

Secretion of galectin-3 from alveolar macrophages

We have previously reported that peritoneal inflammatory, but not resident and quiescent, peritoneal macrophages release galectin-3 extracellularly: about one-half of newly synthesized galectin-3 is found to be secreted from the activated macrophages within 3 h (19, 20), suggesting that the machinery involved in galectin-3 secretion is regulated in a manner dependent on the differentiation/activation stages of macrophages. Thus, we next investigated whether alveolar macrophages secrete galectin-3, contributing to the accumulation of galectin-3 in BAL fluids during streptococcal pneumonia.

Resident alveolar macrophages were obtained from untreated mice. Fresh medium was added to the cells and the cells were incubated for an additional 3 h in the presence of LPS or S. pneumoniae membrane fraction. Galectin-3 released into the medium was analyzed by Western blotting. As shown in Fig. 5⇓A, a small amount of galectin-3 was secreted from these control macrophages or macrophages incubated in the presence of LPS. In contrast, the release was significantly augmented when macrophages were incubated with the membrane fraction of S. pneumoniae (Fig. 5⇓A), suggesting that S. pneumoniae stimulates the release of galectin-3 from alveolar macrophages.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 5.

Release of galectin-3 from alveolar macrophages and detection of surface-associated galectin-3 on neutrophils which emigrated into infected alveoli. A, Release of galectin-3 from alveolar macrophages. Alveolar macrophages purified from noninfected mice were incubated with S. pneumoniae membrane fraction (cell ratio, macrophage:S. pneumoniae, 1:50) or LPS (10 μg/ml) for 3 h. Galectin-3 secreted into medium was analyzed by Western blotting. Representative data from two separate experiments are shown. B, Association of galectin-3 with the surface of neutrophils recruited into alveoli. Neutrophils which were emigrated to infected alveoli were obtained from BAL fluids. Neutrophils were incubated with anti-galectin-3 Ab followed by Alexa 488-anti-rat IgG Ab to detect surface-associated galectin-3 on exudated neutrophils. Representative data from two separate experiments are shown.

Binding of galectin-3 to the surface of neutrophils which emigrated to infected alveoli

As shown in Fig. 3⇑B, only a negligible amount of endogenous galectin-3 was found in mouse inflammatory neutrophils. However, our results show that galectin-3 accumulated in S. pneumoniae-infected alveoli, into which neutrophils emigrated. Thus, if mouse neutrophils express galectin-3 ligands on their surface, galectin-3 will bind to the surface of those emigrated neutrophils. Interestingly, Feuk-Lagerstedt et al. (41) have suggested that neutrophils express galectin-3 ligands such as CD66. Thus, we next studied whether galectin-3 that was released to the infected alveoli was associated with neutrophils that emigrated to the alveoli. Neutrophils obtained from S. pneumoniae-infected alveoli were treated with anti-galectin-3 Ab, followed by anti-rat IgG-Alexa 448, and the surface expression of galectin-3 was analyzed by flow cytometry. As shown in Fig. 5⇑B, a low but significant level of galectin-3 was found on the neutrophil surface. This result suggests that galectin-3 released into infected alveoli during infection binds to the cell surface of neutrophils, which emigrated to the alveoli.

Lack of accumulation of galectin-3 in alveoli infected with E. coli

Extravasation of neutrophils into the alveolar space can be induced by other bacterial pathogens. Thus, we investigated whether galectin-3 was released into the alveolar space during E. coli infection. Mice (BALB/c, CD1, or C57BL/6) were intranasally inoculated either with E. coli or S. pneumoniae (Harvard strain) as described above. As shown in Fig. 6⇓B, inoculation of BALB/c mice with E. coli induced a robust emigration of neutrophils into alveoli (about twice that induced by S. pneumoniae) and the release of a proinflammatory factor, TNF-α. In contrast, the amount of galectin-3 released into alveolar spaces infected with E. coli remained the same as that of the noninfected controls and substantially less than that found in S. pneumoniae infection (Fig. 6⇓A). We also found levels of galectin-3 in S. pneumoniae (Harvard strain)-infected alveoli of CD1 and C57BL/6 mice or in S. pneumoniae (serotype 3)-infected alveoli of CD1 mice comparable to those found in S. pneumoniae-infected alveoli of BALB/c mice (data not shown). We did not observe any significant increase of galectin-3 in E. coli-infected alveoli of CD1 and C57BL/6 mice (data not shown). Thus, this specific accumulation of galectin-3 induced by S. pneumoniae was observed when different mouse strains and S. pneumoniae strains were used. Together, these data demonstrate that accumulation of galectin-3 is not induced in E. coli infection and thus is specific to S. pneumoniae infection.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 6.

Limited accumulation of galectin-3 in BAL fluids of mice infected with E. coli. Mice (BALB/c) were inoculated intranasally with either E. coli or S. pneumoniae (Harvard strain), and the BAL fluids were collected 6 h after infection. A, The levels of galectin-3 were analyzed by Western blotting. Each lane represents a sample from one mouse. B, The levels of galectin-3 and TNF-α were analyzed by sandwich ELISA and the means and SD are shown. ND, Not detected (under the limit of detection). Total neutrophil counts in the BAL samples are also shown.

The ability of galectin-3 to mediate adhesion of neutrophils to endothelial cell layer in vitro

To investigate whether galectin-3 participates as an adhesion molecule in neutrophil diapedesis, its ability to mediate the adhesion of neutrophils to endothelial cells was analyzed in vitro. Human neutrophils were labeled with a fluorescent dye, calcein-acetoxymethyl, and added to a human endothelial cell line ECV304 monolayer in the presence or absence of galectin-3. As shown in Fig. 7⇓A, adhesion of neutrophils onto the endothelial monolayer was promoted 5-fold in the presence of exogenously added galectin-3 at concentrations >0.66 μM. The adhesion of neutrophils reached to plateau within 10 min (Fig. 7⇓B) in the presence of 0.66 μM galectin-3. When the human promyelocytic cell line, HL-60 cells (undifferentiated or differentiated with retinoic acid), which are suggested to express surface galectin-3 ligands different from those of neutrophils (41), were used instead of neutrophils, no obvious promotion of HL-60 binding to endothelial cells by galectin-3 was found (data not shown). Thus, those data demonstrated that galectin-3 can promote neutrophil adhesion to endothelial cells. Preincubation of endothelial cells or neutrophils with LPS or prepriming of neutrophils with low concentration of fMLP did not cause further increase of galectin-3-mediated neutrophil adhesion (data not shown).

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 7.

Mediation of neutrophil adhesion to endothelial cell layers by galectin-3 in vitro. A, Dose response. Calcein-labeled neutrophils were added to an ECV304 endothelial cell monolayer in the presence of increasing doses of galectin-3. After incubation for 20 min at 37°C, unbound neutrophils were removed and bound neutrophils were calculated based on the fluorescent level of each well. Representative data from four separate experiments are shown (means and SD of triplicate determinations). B, Time course. Calcein-labeled neutrophils were added to ECV cells in the presence of 0.66 μM galectin-3 for the indicated times. Representative data from three separate experiments are shown (means and SD of triplicate determinations). C, Expression of galectin-3 ligands on the surface of ECV304 endothelial cells. Single cell suspensions of ECV304 were first incubated with galectin-3 (1 μM) for 10 min. After removing unbound galectin-3 by washing cells, cells were first incubated with anti-galectin-3 Ab followed by Alexa 488-anti-rat IgG Ab to detect cell surface-associated galectin-3. Representative data from two separate experiments are shown. D, Expression of galectin-3 ligands on the surface of neutrophils. Neutrophils were first incubated with galectin-3 (1 μM) for 10 min. Surface-associated galectin-3 was analyzed as described in C. Representative data from two separate experiments are shown.

We next studied whether ECV 304 endothelial cells as well as human neutrophils express galectin-3 ligands on their cell surface (Fig. 7⇑, C and D). ECV304 cells or neutrophils were first incubated with galectin-3. Then, cells were washed to remove unbound galectin-3 and the levels of cell surface-bound galectin-3 were analyzed by flow cytometry to study the expression of galectin-3 ligands on the cell surfaces. As shown in Fig. 7⇑, C and D, extracellularly added galectin-3 bound to the surface of endothelial cells and neutrophils, respectively, suggesting that both cells express galectin-3 ligands on the surface. Thus, together, the data suggest that peripheral blood neutrophils as well as endothelial cells express galectin-3 ligands on their surfaces to support galectin-3-dependent adhesion and that the adhesion can be achieved without any inflammatory priming of cells.

To study whether this adhesion resulted from the adhesion activity of galectin-3 itself rather than the mechanism in which galectin-3 is involved in the activation of some other adhesion molecules, the adhesion assay was performed at 4°C (Fig. 8⇓A). The presence of galectin-3 also increased the adhesion of neutrophils to the endothelial cells when the adhesion assay was performed at 4°C (nearly 75% of that at 37°C, Fig. 8⇓A), suggesting that galectin-3 directly mediates neutrophil adhesion.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 8.

Cross-linking of neutrophils and endothelial cells by galectin-3 to mediate cell adhesion. A, Temperature independence. Adhesion assays were performed for 20 min at either 4 or 37°C in the presence of the indicated concentrations of galectin-3. Representative data from three separate experiments are shown (means and SD of triplicate determinations). B, Inhibition of galectin-3 mediating neutrophil adhesion by the galectin-3 antagonist, lactose. Adhesion assays were conducted in the presence of 1.25 μM galectin-3. Lactose (100 mM) was also added as indicated. Representative data from three separate experiments are shown (means and SD of triplicate determinations). C, Requirement of the N-terminal tandem repeating domain for neutrophil adhesion. Various concentrations of galectin-3 C-terminal CRD fragment (galectin-3-CRD) were added to the adhesion assays, which were conducted for 20 min at 37°C. Representative data from three separate experiments are shown (means and SD of triplicate determinations). D, Mediation of neutrophil adhesion on HUVEC monolayer by galectin-3. Instead of using ECV304, primary cultured HUVEC monolayers were used for the adhesion assay with various concentration of galectin-3. For the activation of cells with LPS, HUVEC monolayers were first incubated with LPS (2 μg/ml) for 2 h before the adhesion assays, which were also conducted in the presence of LPS (2 μg/ml). Representative data from three separate experiments were shown (means and SD of triplicate determinations).

Galectin-3-mediated adhesion was efficiently inhibited in the presence of lactose (100 mM), which can compete the binding of galectin-3 to their ligands (Fig. 8⇑B), but not by mannose (100 mM), which does not antagonize galectin-3 (data not shown), suggesting that lectin activity of galectin-3 mediates the adhesion of neutrophils to endothelial cells. Galectin-3 has only one CRD located at C-terminal and it is suggested that oligomerization through non-lectin N-terminal domain is essential to exert cross-linking activity of galectin-3 (23, 24). To investigate whether the N-terminal domain of galectin-3 is involved in neutrophil adhesion, truncated galectin-3 lacking N-terminal tandem repeating domain (galectin-3-CRD), instead of full-length galectin-3, was added in the neutrophil adhesion assay. The truncated galectin-3 failed to support neutrophil adhesion (Fig. 8⇑C), suggesting that the full-length lectin mediates neutrophil adhesion to endothelial cells through oligomerization of its N-terminal multiple repeating domain.

We also tested whether galectin-3 can also support human neutrophil adhesion to freshly isolated primary HUVEC. As shown in Fig. 8⇑D, in the presence of galectin-3 (above 1 μM), human neutrophils adhered to HUVEC layer to the same extent as observed with ECV304, suggesting that HUVEC also expresses galectin-3 ligands with which galectin-3 can mediate adhesion of neutrophils to endothelium. Interestingly, pretreatment of endothelial cells with LPS followed by the presence of LPS during the adhesion assay did not augment the number of adherent neutrophils in the presence of galectin-3, suggesting that HUVEC cells express galectin-3 ligand before LPS activation (Fig. 8⇑D).