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(Journal of Leukocyte Biology. 2005;78:1127-1135.)
© 2005 by Society for Leukocyte Biology

Galectin-3 interacts with naïve and primed neutrophils, inducing innate immune responses

Julie Nieminen, Christian St-Pierre and Sachiko Sato1

Glycobiology Laboratory, Research Centre for Infectious Diseases, Laval University Medical Centre, Faculty of Medicine, Laval University, Québec, Canada

1Correspondence: Glycobiology Laboratory, Centre de Recherche en Infectiologie du CRCHUL, 2705 boul. Laurier, Ste-Foy, Québec, Canada, G1V 4G2. E-mail: Sachiko.Sato{at}crchul.ulaval.ca


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ABSTRACT
 
The neutrophil is the first line of defense against infection. As a part of the innate immune response, neutrophils start to emigrate from blood to an affected site and their state is altered from passively circulating naïve to primed, and then to fully activated. The extent of neutrophil activation and their subsequent response varies depending on the stimuli and environment that neutrophils encounter. Because neutrophils can also induce deleterious effects on host tissues, tight regulation of recruitment and functions of neutrophils is required for efficient recovery. Galectin-3, a soluble ß-galactoside binding protein, of which expression is up-regulated during inflammation/infection, is suggested to be involved in various inflammatory responses. However, the precise roles of this lectin in innate immunity remain unknown, while it has been demonstrated that galectin-3 binds to naïve and primed neutrophils. Here we report that galectin-3 can induce L-selectin shedding and interleukin-8 production in naïve and primed neutrophils. These activities were shown to be dependent on the presence of the C-terminal lectin domain and the N-terminal nonlectin domain of galectin-3, which is involved in oligomerization of this lectin. We also found that, after galectin-3 binds to neutrophils, primed but not naïve neutrophils can cleave galectin-3, mainly through elastase, which results in the formation of truncated galectin-3 lacking the N-terminal domain. Together, these results suggest that galectin-3 activates naïve and primed neutrophils, and galectin-3-activated primed neutrophils have an ability to inactivate galectin-3.

Key Words: inflammation • lectin • elastase • leukocyte


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INTRODUCTION
 
Neutrophils participate in the innate immune response as the first line of defense against infection, as they are one of the dominant phagocytic leukocytes to be recruited from peripheral blood toward affected sites immediately after and throughout the infection. Pathogens and infected tissues release effector molecules, such as bacterial products and cytokines (including chemokines), which reach blood stream and interact with vascular endothelium and circulating neutrophils [1 , 2 ]. As a result, neutrophils successively migrate through the endothelial cell layer, the interstitial space, and the epithelial cell layer to reach the infected tissue. During this emigration process, the state of neutrophils is altered from naïve to primed [3 4 5 6 ]. The emigrated neutrophils are then fully activated in the affected tissues [3 4 5 6 ]. Once activated, neutrophils phagocyte the pathogen and release microbicidal factors, including reactive oxygen intermediates (ROI), defensins, and proteases through degranulation [7 ].

Various agents, such as formyl-Met-Leu-Phe (fMLP) and interleukin (IL)-8 or cytochalasin B, induce neutrophil priming. In contrast to activated neutrophils, respiratory burst and degranulation are not active in primed neutrophils. However, priming makes neutrophils responsive to subsequent activating stimuli and thereby can rapidly induce potentiated responses [3 4 5 6 ]. The degree of priming and activation, including granule mobilization and release of factors, has been suggested to vary depending on the nature of the agonist and the level of expression of the specific receptors [3 4 5 6 ]. In addition, recent study by Tsuda et al. [8 ] suggest that different inflammatory settings lead to the emergence of divergent subsets of neutrophils with distinct cytokine expression patterns. Thus, the activation of neutrophils by various host factors released in the inflammation/infection settings recently became a topic of interest [8 9 10 ].

Some host factors found in proximity to infection/inflammation sites induce up-regulation of E- and P-selectins and ICAM-1 by vascular endothelium and priming of neutrophils; those inductions are necessary for neutrophil migration [3 4 5 6 ]. Through the interaction between selectins and their ligands, neutrophils begin to roll on the endothelium and then interact with chemoattractants, which induces an increase in the avidity of the neutrophil adhesion molecules, ß2-integrins, for ICAM-1 present on the endothelium [1 , 11 , 12 ]. Neutrophils then bind tightly to the endothelium and initiate transmigration toward the affected site [1 , 11 , 12 ]. Although this paradigm is established, studies of neutrophil emigration during some infections, including streptococcal pneumonia and chronic pneumonia infection by Pseudomonas aeruginosa, have provided evidence that neutrophils also emigrate without the aid of the neutrophil adhesion molecules, selectins and ß2-integrins [2 , 13 14 15 ]. Although emigration requires alternative molecules that can initiate endothelium-neutrophil interactions and modulate the inflammatory status of neutrophils, the identities of such molecules have remained unclarified [2 , 13 14 15 ].

We have recently suggested that one soluble carbohydrate binding protein, called galectin-3, binds to naïve neutrophils and acts as an adhesion molecule that can mediate the initial adhesion to the endothelium [16 ]. The expression of galectin-3 is up-regulated during the development of streptococcal pneumonia, and the release of galectin-3 in the infected alveoli is correlated with neutrophil emigration [16 ]. Thus, we have proposed that galectin-3 may functionally replace some roles played by selectins and ß2-integrins in the pneumonia setting [16 ]. Galectin-3 belongs to a large ß-galactoside-binding protein family (galectin) defined by conserved peptide sequence elements of carbohydrate recognition domain (CRD) [17 18 19 ]. Galectin-3 is actively secreted by inflammatory macrophages, mast cells, and epithelial cells [20 21 22 23 24 25 26 27 28 ]. In addition, the expression and release of galectin-3 are suggested to be up-regulated in various infection/inflammation cases, such as arthritis, hepatic cirrhosis, a murine model of asthma, diabetes, parasitic infection, and streptococcal pneumonia [16 , 29 30 31 32 33 34 ]. Galectin-3 molecules contain a C-terminal CRD and a N-terminal, intrinsically unstructured, nonlectin domain consisting of multiple repeats of a peptide sequence rich in proline, glycine, and tyrosine [17 18 19 ]. Once it binds to ligands by its C-terminal CRD domain, galectin-3 forms a pentamer through its N-terminal repeating domain, and the galectin-3 pentamer cross-links its ligands [35 ]. Through this cross-linking, galectin-3 induces several reactions involved in innate immunity, such as neutrophil-endothelial interaction [16 ], chemoattraction of monocytes and endothelial cells [36 , 37 ], production of ROI by neutrophils [38 , 39 ], and IL-1 by monocytes [40 ]. In vivo, galectin-3 induces proinflammatory reactions, including leukocyte recruitment [36 ], up-regulation of tumor necrosis factor {alpha} (TNF-{alpha}), and chemokines for neutrophils (Isabelle Pelletier, J. Nieminen, C. St-Pierre, S. Sato, S., submitted). In addition, neutrophil emigration into the peritoneal cavity is reduced in galectin-3 null mice [41 , 42 ]. Although the importance of neutrophils in innate immunity has been underlined by its microbicidal activity and by its ability to produce different arrays of cytokines/chemokines, which affect successive immune responses [4 , 5 , 43 ], information on the effects of galectin-3 on neutrophils is relatively limited.

In this regard, the group of Karlsson and others has suggested that neutrophils from peripheral blood (naïve) are nonresponsive to galectin-3, while primed neutrophils produce ROI in response to this lectin [38 , 39 ]. However, others and we have suggested that galectin-3 binds to naïve neutrophil, acting as an adhesion molecule [16 , 44 ]. Thus, it remains to be determined whether galectin-3 can modulate inflammatory responses by directly interacting with different states of neutrophils during the innate immune response. In this report, we have investigated the effect of galectin-3 on naïve and primed neutrophils. Our data suggest that neutrophils in naïve and primed states are responsive to galectin-3, inducing L-selectin shedding and secretion of IL-8. Both the C-terminal lectin domain and the N-terminal repeating domain that is responsible for the oligomerization of this lectin were required for neutrophil activation by galectin-3. Galectin-3 was also deactivated through its cleavage by primed neutrophils after binding to neutrophils. Naïve neutrophils could not efficiently cleave galectin-3 despite its binding to the neutrophil surface. Thus, our data suggest that galectin-3 activates both naïve and primed neutrophils, inducing inflammatory responses, while galectin-3-activated primed neutrophils can deactivate this lectin.


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MATERIALS AND METHODS
 
Reagents, antibodies, and galectin-3
Chemicals and other reagents were obtained from Sigma Chemical Co. (St. Louis, MO) unless specified otherwise. A rat monoclonal antibody against galectin-3 (Mac-2), which binds to the N-terminal domain of galectin-3, was purified from culture medium of the hybridoma M3/38.1.2.8. Anti-CD62L was purchased from BD PharMingen (San Diego, CA). A polyclonal antibody against the C-terminal CRD of galectin-3 was raised in our laboratory as described [45 ]. Recombinant full-length and truncated human galectin-3 were purified as described previously and were passed through Detoxi-Gel endotoxin-removing gels (Pierce, Rockford, IL) [16 ]. Neutrophil elastase was obtained from Calbiochem (Darmstatdt, Germany). Alexa 546-labeled galectin-3 was prepared following the manufacturer instructions (Molecular Probes, Eugene, OR) with a slight modification as described previously [45 , 46 ].

Neutrophil purification
Neutrophils were purified from heparinized blood of healthy volunteers, as described previously [16 ]. Freshly isolated neutrophils are referred here as unprimed neutrophils. A portion of the neutrophils were primed prior to the experiment by a 5 min preincubation with 5 µM cytochalasin B (referred here as primed neutrophils) [38 , 47 ]. Alternatively, neutrophils were primed by fMLP (10–7 M) or by IL-8 (80 ng/ml) for 30 min.

L-selectin shedding on neutrophil
Freshly isolated unprimed or primed neutrophils (5x106 cells/ml) were resuspended in Hank’s buffer containing the indicated concentrations of galectin-3 or the CRD of galectin-3 and incubated for 30 min at 37°C. For the experiments in the presence of lactose, the concentration of sodium chloride in the buffer was adjusted to maintain appropriate osmolarity, i.e., 317 mosmol/L [16 ]. Positive control for L-selectin shedding was achieved through 5 min incubation with 0.1 µg/ml phorbol myristate acetate (PMA). After incubation, cells were incubated with 20 µl of phycoerythrin (PE)-labeled anti-CD62L for 25 min and washed with phosphate-buffered saline (PBS), 0.2%, and bovine serum albumin, 0.1%, sodium azide and fixed in PBS containing 2% formaldehyde. In duplicate of the samples, an isotypic control, PE-labeled anti immunoglobulin G (BD PharMingen) was added to the incubation as a negative control. Surface L-selectin expression level was estimated by detecting fluorescence with a Beckman-Coulter (Fullerton, CA) flow cytometer.

Production of IL-8 by neutrophils
Neutrophils (5x106 cells/ml) were resuspended as above and incubated with galectin-3 or truncated galectin-3 in the presence or absence of 50 mM lactose for 30 min or 2 h at 37°C. After incubation, cell-free supernatants were collected, and IL-8 concentrations were measured by enzyme-linked immunosorbent assay (ELISA; BD PharMingen).

Cleavage of galectin-3 by neutrophils
Unprimed neutrophils (5x106 cells/ml) were resuspended in Hank’s buffer containing galectin-3 and incubated for the indicated times at 37°C. After incubation, cell-free supernatants were obtained by centrifugation at 500 x g for 5 min. Alternatively, neutrophils were primed prior to the experiment by cytochalasin B or by a 30-min preincubation with 10–7 M fMLP or 80 ng/ml IL-8 (BD PharMingen) and used for the cleavage assays. The supernatants were fractionated by SDS-PAGE, then cleaved and full-length galectin-3 in the gel were detected by Coomassie blue staining or transferred on a nitrocellulose membrane for Western blotting with an anti-Mac-2 or an anti-CRD antibody.

Inhibition of galectin-3 by proteases inhibitors
Different protease inhibitors were added to the in vitro cleavage assays and incubated for 30 min at 37°C. All inhibitors were from Roche Molecular Biochemicals except GM6001, Eglin C, and 1,10-ortho-phenantroline (OPA; Sigma Chemical Co.). The final concentrations of ethanol or DMSO (dissolving agents) in the incubation mixture were less than 0.1%.

Confocal microscopy
Primed or unprimed neutrophils were first labeled with Calcein-acetoxymethyl (Calcein, Molecular Probes) as previously published [16 ] and were incubated with 2 µM galectin-3-Alexa 546 at 4 or 37°C. After unbound galectin-3 was removed by a 5-min centrifugation at 500 x g followed by brief washing, neutrophils were fixed in 2% paraformaldehyde, centrifuged for 5 min at 500 x g, resuspended in 50% glycerol, and mounted on a microscope slide for observation by confocal microscope. Fluorescence images acquired through a 60 x 1.4 NA objective (PlanApo, Olympus) were captured at serial optical sections (~10 sections at 1.0 µm intervals) by the FLUOWVIEW FV300 confocal scanning unit (Olympus). Fluorescence from each channel was captured sequentially to eliminate crosstalk between channels. Calcein and Alexa 546 were exited with a 488 nm Argon ion and 543 nm Helium-Neon laser line, respectively, and the green and red fluorescences were split by a 570 nm beam splitter. The green florescence was captured using a combination of a 510–530 nm bandpass and a 510 nm longpass emission filter, and the red fluorescence was imaged using a 575–630 nm bandpass emission filter. Green, red, and merged color images were created by FLUOWVIEW 300 v 3.3 software (Olympus). Then, color contrasts were adjusted using the PhotoShop v 6.0 software.


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RESULTS
 
Galectin-3 induces L-selectin shedding on neutrophils
L-selectin, which principally plays a role in leukocyte homing, is constitutively expressed on neutrophils [48 ]. L-selectin is shed from the surface of neutrophils upon activation, and this shedding is suggested to be involved in appropriate emigration of neutrophils [49 ]. Galectin-3 being released and present in the inflamed environment [16 , 29 30 31 32 33 34 ], we thus examined whether galectin-3 induces L-selectin shedding. As shown in Figure 1A , incubation of unprimed neutrophils with 1 µM of galectin-3 for 30 min resulted in L-selectin shedding. When neutrophils were incubated with galectin-3 in the presence of its antagonist, lactose (50 mM), which competes for the binding of galectin-3 to neutrophils, surface expression of L-selectins remained similar to PBS-treated neutrophils (Fig. 1A) . This finding suggests that the lectin activity of galectin-3 mediates the L-selectin-shedding. Dose-response experiments revealed that L-selectin shedding induced by galectin-3 was evident in unprimed and primed neutrophils at concentrations as low as 0.5 µM (Fig. 1B 1c and 1h) . Whereas L-selectin shedding is slightly more progressive in unprimed neutrophils than in primed neutrophils, both neutrophil subsets were found to be responsive to galectin-3. At a concentration of 2 µM, the L-selectin shedding induced by galectin-3 in neutrophils was found to be comparable with that induced by PMA, an established neutrophil activator, which suggests that galectin-3 activates unprimed and primed neutrophils (Fig. 1B 1e and 1j vs. 1a and 1f ). Most galectin-3 activities are suggested to be dependent on the oligomerization of galectin-3 molecules through the N-terminal tandem-repeating domain [35 , 50 51 52 ] (see review in [27 , 28 , 53 ]). To investigate whether oligomerization of galectin-3 molecules is also necessary for the induction of L-selectin shedding, truncated galectin-3 (CRD), lacking the N-terminal domain, was used in the assay since the truncated form can no longer oligomerize. As shown in Figure 1B (dotted line in b–e and g–j), truncated galectin-3 failed to induce L-selectin shedding. Together with the evidence that binding of galectin-3 is necessary for the induction of L-selectin shedding (Fig. 1A) , those data suggest that oligomerization of galectin-3 through the N-terminal domain as well as its binding to neutrophils through the C-terminal domain are required for the induction of L-selectin shedding. Time course revealed that L-selectin shedding from unprimed and primed neutrophils was evident as early as after 5 min of incubation and proceeded over the observation period (Fig. 2 ).



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  		Figure 1. L-selectin shedding from neutrophils. (A) Unprimed polymorphonuclear neutrophils (PMN; 5x106cell/ml) were incubated for 30 min at 37°C with galectin-3 (1 µM) in the absence (full line) or presence of 50 mM lactose (dotted line; a) or PMA as a positive control (b). L-Selectin expression was estimated using PE-conjugated anti-CD62L antibody, which fluorescence was detected by a Beckman Coulter flow cytometer. (B) Unprimed (a to e) and cytochalasin B-primed (f to j) neutrophils (5x106cell/ml) were incubated for 30 min at 37°C with PMA or different concentrations of galectin-3 (full line) or truncated galectin-3 (CRD; dotted line). Representative data from 4 separate experiments are shown.



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  		Figure 2. Time course of galectin-3-induced L-selectin shedding. Unprimed or cytochalasin B-primed neutrophils were incubated with galectin-3 (1 µM) for indicated times. Representative data from 2 separate experiments are shown.

Galectin-3 induces IL-8 synthesis by neutrophils
IL-8 is a neutrophil chemokine, which is released during inflammation/infection. When incubated with galectin-3 for 2 h, unprimed and primed neutrophils released significant amount of IL-8 (Fig. 3A ). Although in lower amounts [52.7±1.4 pg/ml by galectin-3 (1 µM)-treated neutrophils compared with 16.8±0.1 pg/ml by PBS-treated neutrophils], the induction of IL-8 release by galectin-3 was detectable as early as after 30 min (data not shown). Galectin-3-induced IL-8 production was significantly inhibited in the presence of 50 mM lactose (data not shown). Again, truncated galectin-3, which lacks the N-terminal domain, did not induce any detectable IL-8 secretion (Fig. 3B) . Thus, those data suggest that like L-selectin shedding, the binding and oligomerization of galectin-3 are required to induce IL-8 production.



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  		Figure 3. Induction of IL-8 secretion by galectin-3. (A) Galectin-3 was incubated either with human unprimed or with cytochalasin B-primed neutrophils (5x106 cells/ml) for 2 h at 37°C. Cell-free supernatants were subjected to human IL-8 ELISA assays (BD PharMingen) following manufacturer’s instructions. Alternatively, neutrophils were incubated with 1 µg/ml LPS as a positive control. (B) Full-length or truncated (CRD) galectin-3 that lacks the N-terminal repeating domain was incubated with unprimed neutrophils for 2 h at 37°C and IL-8 concentrations in the cell-free supernatants were determined. Means and SD of 4 for (A) or 2 for (B) separate experiments are shown.

Galectin-3 is cleaved by primed human neutrophils
Galectin-3 has been suggested to be cleaved by several proteases, including bacterial collagenases, metalloprotease (MMP)-2 and -9 and Leishmania surface protease, gp63 [45 , 50 , 51 , 54 ], resulting in the removal of the N-terminal repeating domain, which is involved in the pentamerization of this lectin [35 ]. As shown in Figures 1 and 3 , truncated galectin-3 molecules could not activate neutrophils. Because some proteases are often extracellularly expressed upon activation of neutrophils as a result of degranulation, we sought to examine whether galectin-3-induced activation of neutrophils leads to the cleavage of galectin-3. As seen in Figure 4A , when incubated with galectin-3 for 30 min at 37°C, primed neutrophils cleaved galectin-3 to produce three fragments of 22, 16, and 14 kDa in size. In contrast, unprimed neutrophils appeared to have a weaker cleavage activity, resulting in partial cleavage of galectin-3. As shown in Figure 4B , cleavage of galectin-3 by primed neutrophils could be detected after 15 min of incubation, and within 60 min most galectin-3 was found to be cleaved, resulting in two bands of 16 and 14 kDa. The two bands were recognized by anti-CRD antibody but not by anti-Mac-2 antibody (Fig. 4B and data not shown). Protein staining of the gels with Coomassie blue also confirmed that the 16 and 14 kDa bands were the major protein bands in the supernatants of primed neutrophils incubated with galectin-3 (data not shown). Because the calculated molecular weight of the CRD is ~15 kDa, those data suggest that cleavage of galectin-3 by primed neutrophils results in the formation of truncated galectin-3 that lacks the major part of the N-terminal repeating domain.



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  		Figure 4. Galectin-3 cleavage by neutrophils. (A) Galectin-3 cleavage by primed neutrophils. Galectin-3 (1 µM) was incubated either with human unprimed or with cytochalasin B-primed neutrophils (5x106 cells/ml) for 30 min at 37°C. Cell-free supernatants were subjected to SDS-PAGE followed by Western blotting using anti-CRD antibody to detect full-length and truncated galectin-3. (B) Time course of galectin-3 cleavage. Galectin-3 (1 µM) was incubated with primed neutrophils for indicated times at 37°C. Truncated galectin-3 was detected in the supernatant using anti-CRD antibody. Representative data from 4 separate experiments are shown (C) Inhibition of neutrophil-mediated galectin-3 cleavage by lactose, a galectin-3 antagonist, and Eglin C (fragment 60-63 methyl ester), a specific elastase inhibitor. Galectin-3 (1 µM) was incubated with cytochalasin B-primed neutrophils (5x106 cells/ml) for 30 min in absence or presence of lactose (50 mM) or protease inhibitors (OPA 2 µM or Eglin C 1 µM) at 37°C. Representative data from 3 separate experiments are shown. (D) Galectin-3 cleavage by neutrophil elastase. Galectin-3 (1 µM) was incubated with purified neutrophil elastase (70 mU; Calbiochem) for 15 min at 37°C. Representative data from 2 separate experiments are shown.

Lactose, a galectin-3 antagonist, inhibits galectin-3 cleavage by primed neutrophils
We next investigated whether galectin-3 binding to neutrophils is required for the cleavage of galectin-3 by neutrophils. In the presence of lactose, which competes with galectin-3 for binding to neutrophils, cleavage by primed neutrophils was significantly inhibited (Fig. 4C , lane 5 vs. lane 7). This inhibition was not observed in the presence of mannose, which does not show any affinity toward galectin-3 (data not shown). Thus, the data suggest that binding of galectin-3 to primed neutrophils is required for the cleavage.

Galectin-3 is cleaved by neutrophil elastase
Neutrophils contain various proteases such as collagenase, elastase and MMPs in their granules. Enzymatically, MMP-9 is suggested to be able to cleave galectin-3, to produce a 22 kDa fragment that contains the CRD and a truncated N-terminal domain [54 ]. To determine whether MMP-9 or any other neutrophil proteases are involved in galectin-3 cleavage by neutrophils, various protease inhibitors were added to the incubation mixtures. A cysteine protease inhibitor (E-64), a cysteine and serine protease inhibitor (leupeptin), an aminopeptidase protease inhibitor (bestatin), an aspartate protease inhibitor (pestatin), a papain and trypsin inhibitor (antipain-dihydrochloride), and a chymotrypsin inhibitor (chymostatin) did not significantly inhibit galectin-3 cleavage by neutrophils (data not shown). Other inhibitors were shown to partially inhibit galectin-3 cleavage by neutrophils; these were EDTA-Na, GM60001 and OPA (MMP inhibitors), suggesting that neutrophil MMP is not actively involved in the cleavage of galectin-3 (data not shown and Fig. 4C , lanes 3 and 4). In contrast, the cleavage of galectin-3 by neutrophils was significantly inhibited by the serine protease inhibitor Pefabloc SC, which suggests that serine proteases are likely to be the main type of neutrophils enzymes that cleave galectin-3 (data not shown).

Neutrophils possess different serine proteases in their granules: proteinase 3, cathepsin G, and elastase [7 ]. The specific elastase inhibitor Eglin C fragment 60-63 methyl ester significantly inhibited galectin-3 cleavage when added into the cleavage assays (Fig. 4C , lanes 5 and 6). In fact, purified neutrophil elastase could also cleave galectin-3 with the same cleavage pattern as primed neutrophils (Fig. 4C , lane 5, and D, lane 2) and Eglin C fragment 60–63 methyl ester at the concentration used for the neutrophil cleavage assay inhibited this cleavage (Fig. 4D) . Together, the data suggest that neutrophil elastase is most likely the enzyme responsible for the cleavage of galectin-3.

Galectin-3 is cleaved by neutrophils primed with fMLP or IL-8
In the previous assays, we used cytochalasin B, a cytoskeleton-disrupting agent as a priming agent [7 ]. We next primed neutrophils using pathogen-derived (fMLP) and endogenous agents (IL-8). Formyl peptides are a cleavage product of bacterial proteins, which are known to be neutrophil chemoattractants involved in the recruitment of neutrophils to infected sites and a priming agent involved in granule mobilization [7 , 55 ]. IL-8, a CXC chemokine, is also known to mobilize neutrophil granules. When galectin-3 was incubated with neutrophils primed either with fMLP or IL-8, cleavage was observed after 5 min of incubation and progressed up to 60-min postincubation, resulting in fragments of 22, 16, and 14 kDa (Fig. 5 ). These data suggest that galectin-3 is cleaved by neutrophils that are primed by natural chemoattractants.



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  		Figure 5. Cleavage of galectin-3 by neutrophils primed by fMLP and IL-8. Galectin-3 (1 µM) was incubated with neutrophils primed with either fMLP or IL-8 for the indicated times at 37°C. Representative data from 2 separate experiments are shown.

Galectin-3 distributions in neutrophils differ depending on neutrophil states
Reports by other groups [38 , 39 ] and our data (Figs. 1 2 3 4 5 and ref. [16 ]) suggest that galectin-3 interacts with primed and unprimed neutrophils. Thus, we next studied the localization of galectin-3 in neutrophils. Alexa 546-labeled galectin-3 was incubated with Calcein-labeled unprimed or primed neutrophils. In neutrophils primed with cytochalasin B, most galectin-3 was found inside neutrophils after 5 min of incubation at 37°C (Fig. 6A ). In contrast, incubation of primed neutrophils with galectin-3 at 4°C resulted in the lamina distribution on the surfaces (Fig. 6D) , suggesting that galectin-3 is internalized by primed neutrophils at 37°C. When incubated with neutrophils that were primed either with IL-8 or fMLP for 15 min, galectin-3 was also internalized in a similar manner than with cytochalasin B-primed neutrophils (Fig. 6F and 6G compared with 6B ). In contrast, even incubated at 37°C for up to 15 min, most galectin-3 in unprimed neutrophils was found to be localized as a layer or a lamina on the cell surface with a concentration of galectin-3 at the junctions of adherent cells (Fig. 6H and 6I) , which suggests that galectin-3 is not rapidly internalized in unprimed neutrophils. As shown in Figure 2 , L-selectin shedding was evident after 5 min of incubation with galectin-3. Thus, together, those data raise the possibility that internalization of galectin-3 is not prerequisite for L-selectin shedding. After 30 min of incubation at 37°C, some internalization of galectin-3 was observed in unprimed neutrophils but with a slightly different distribution than in primed neutrophils (Fig. 6J compared with Fig. 6A 6B 6C ). In unprimed neutrophils, galectin-3 was found as a larger cluster near the membrane of the neutrophils (Fig. 6J) . When primed or unprimed neutrophils were incubated with galectin-3 in presence of Pefabloc SC (serine protease inhibitor), which inhibits galectin-3 cleavage, no internalization of galectin-3 was observed, even after 30 min of incubation (Fig. 6E and 6L compared with 6C and 6J ). Galectin-3 was instead distributed as a lamina on the surfaces of neutrophils (Fig. 6E and 6L) , which was observed only in unprimed neutrophils or neutrophils incubated at 4°C (Fig. 6D 6H 6I , and 6K) . In this condition, efficient cleavage of galectin-3 was not detected either (data not shown), suggesting that Pefabloc SC, by preventing galectin-3 cleavage, inhibits internalization of galectin-3. As galectin-3 binding to neutrophils is also necessary for the cleavage of galectin-3 (Fig. 4C) , these results suggest that galectin-3 binding to neutrophils and its cleavage are coupled to its internalization by primed neutrophils.



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  		Figure 6. Galectin-3 interaction with neutrophils. Calcein-labeled neutrophils, which were primed with cytochalasin B (A–E), IL-8 (F), or fMLP (G) or unprimed (H–L) were incubated with Alexa 546-labeled galectin-3 (2 µM) for the indicated times either at 37°C (A–C, E–J, L), or 4°C (D, K). After removal of unbound galectin-3, neutrophils were fixed and mounted onto slide for observation by confocal microscopy. Fluorescence images acquired through a 60 x 1.4 NA objective were captured at serial optical sections (~20 sections at 1.0 µm intervals) by the FLUOWVIEW 300 confocal scanning unit and a z-stacked image of three optical sections of each sample was shown here. (A, Inset) Instead of z-stacked images, YZ images reconstructed by FLUOWVIEW are shown. Representative data from 3 separate experiments are shown. (E, L) Calcein-labeled neutrophils, either primed (E) or unprimed (L), were incubated with Alexa 546-labeled galectin-3 (2 µM) and Pefabloc SC (2 µM) for 30 min at 37°C. Representative data from 4 separate experiments are shown.


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DISCUSSION
 
Neutrophils play a crucial role in the innate immune response, as they are one of the first and dominant leukocytes to emigrate into the affected lesion and have the ability to clear invading pathogens from tissues by engulfing them and/or releasing microbicidal factors, such as ROI. It has recently become evident that the contribution of neutrophils to host defense extends beyond their role as phagocytes, since they can be induced to express different sets of cytokines and chemokines, orchestrating successive responses [8 9 10 , 43 ]. Recent evidences suggest that galectin-3 is involved in innate immune response (reviewed in [27 , 28 , 56 ]), and is extracellularly released at infection/inflammatory sites [16 , 29 30 31 32 33 34 ]. Recent reports suggest that primed (either by cytochalasin B or other pathogen-derived agents) but not naïve neutrophils are responsive to galectin-3 for ROI production [38 , 57 58 59 60 ]. On the other hand, it has also been shown that galectin-3 binds to naïve neutrophils [16 , 44 ]. Thus, it remains elusive whether binding of galectin-3 to neutrophils of different states can induce distinct responses, even though the state of neutrophils evolves from naïve, to primed, to fully activated during the innate immune response. In this report, we demonstrated that galectin-3 induces L-selectin shedding and IL-8 production in naïve and primed neutrophils, suggesting that galectin-3 activates both states of neutrophils. L-selectin shedding was detectable as early as 5 min after exposure to galectin-3 (Fig. 2) . While IL-8 release by neutrophils was evident after incubation for 30 min, the amount of released IL-8 was at least 10 time less than the dose required for neutrophil priming [61 ]. Thus, L-selectin shedding is likely to be directly induced by galectin-3 rather than indirectly by other factors, such as IL-8. When interacting with primed neutrophils, galectin-3 was cleaved, leading to deactivation of galectin-3. In contrast, naïve neutrophils could not cleave galectin-3. Therefore, the deactivation of galectin-3 by primed neutrophils introduces the possibility of a regulation mechanism for some activities induced by galectin-3.

Emigration involves multiple layers of regulations in the adhesiveness of neutrophils through regulation of several adhesion molecules of neutrophils, such as L-selectin, ß2-integrins, and galectin-3. L-selectin is constitutively expressed on the surface of nonactivated leukocytes, including naïve and primed neutrophils, and is involved in homing ([62 , 63 ] and Figs. 1 and 2 ). Neutrophil activation induces L-selectin shedding, which is indicated to be critically involved in appropriate direction of the migration pattern of neutrophils into affected lesions [49 ]. In addition, activation leads to conformational changes in ß2-integrin, increasing its avidity for its ligands, which is required for transmigration across the vascular endothelium [1 , 2 ]. Our preliminary results suggest that galectin-3 also induces the conformational activation of ß2-integrin (C. St-Pierre, S. Sato, unpublished observation). In addition, galectin-3-induced activation initiates production of IL-8, a classical chemokine for neutrophils. Thus, the presented data and galectin-3’s intrinsic ability to act as an adhesion molecule of naïve [16 , 44 ] and primed neutrophils (J. Nieminen, S. Sato, unpublished observation) suggest that by interacting with neutrophils, galectin-3 modulates neutrophil emigration.

As galectin-3 acts as an immunomodulator involved in the immune response (reviewed in refs. [27 , 28 , 56 ]), we also investigated how these activities of galectin-3 could be regulated by neutrophils. In the absence of ligands, galectin-3 is found in the monomer form; yet upon binding to its ligands, galectin-3 molecules oligomerize through the N-terminal nonlectin tandem-repeat domain [35 , 50 , 51 ]. Most of the activities of galectin-3, including promotion of neutrophil adhesion, IL-8 synthesis, and L-selectin shedding as demonstrated here (Figs. 1 and 3 and ref. [16 ]), are dependent on both binding to ligands through the C-terminal lectin (CRD) domain and on oligomerization of the galectin-3 molecules through the N-terminal tandem repeat domain [27 , 28 , 53 , 64 65 66 ]. Therefore, one way to regulate the activity of galectin-3 is to remove the N-terminal repeating domain of galectin-3. Results of this study indicate that primed neutrophils remove the N-terminal domain of galectin-3, thus deactivating galectin-3 in vitro. Our unpublished results, obtained using the lungs of mice intranasally infected with Streptococcus pneumoniae for 24 or 48 h, indicate the formation of truncated galectin-3, lacking the N-terminal domain, in the lungs of infected mice (J. Nieminen, S. Sato, unpublished observation). Thus, those data suggest that the inflammatory activity of galectin-3 is also regulated by neutrophil proteases during infection in vivo.

In contrast to the process of migration, which can be modulated by galectin-3 in primed and naïve neturophils, galectin-3 induces ROI production only in primed neutrophils [38 , 39 ]. Karlsson and co-workers [60 ] have suggested that CD66a and b are the major ligands of galectin-3 in primed neutrophils, being involved in ROI production, while they are absent on the plasma membrane of naïve neutrophils [57 ]. Thus, it is possible that the process for the cleavage of galectin-3 is also triggered by binding of galectin-3 to CD66a and b in primed neutrophils. Recent studies have suggested the critical roles of neutrophil serine proteases in the homeostasis of several cytokines, including TNF-{alpha} and IL-6 in neutrophil-dominated inflammatory process in vivo [67 ]. Thus, proteolytic cleavage of galectin-3 is also likely to be a part of serine protease dependent negative feedback loops, which limit neutrophil activation.

Even though it has already been suggested that, enzymatically, MMP-9 could cleave galectin-3 [54 ], our data indicate that elastase is rather involved in galectin-3 cleavage by neutrophils. An elastase inhibitor, Pefabloc, prevents galectin-3 from cleavage by primed neutrophils as well as from rapid internalization (Figs. 4 and 6) . Rather, the protected galectin-3 forms lattices at the surface of primed as well as naïve neutrophils. Those data argue for recent biochemical findings that pentamers of full-length galectin-3 form heterogeneous, cross-linking complexes with its glycoconjugate ligands [35 ]. Such heterogeneous complexes have a tendency to form robust lectin lattice on the cell surface as were visualized in Figure 6 [35 , 65 , 68 , 69 ]. The biological significance of the galectin-3 lattice in regulation of immune response has been recently underlined by the work suggesting that internalization of transforming growth factor-ß is delayed by the lattice [70 ]. It has also been indicated that the galectin-3 lattice reduces the lateral movement of T cell receptor (TCR), restricting TCR recruitment to the site of antigen presentation [71 ]. Importantly, formation of the lattice requires the N-terminal domain of galectin-3 and we have also suggested previously that the removal of this domain by a protease called gp63 of the parasite Leishmania major leads to disappearance of galectin-3 lattice present on macrophages [45 ]. Since the present work suggests that galectin-3-activated primed neutrophils also cleave the N-terminal domain of galectin-3, how those activated neutrophils are involved in the regulation of galectin-3 lattice formation and functions of leukocytes that are recruited in the affected site is an intriguing question for future investigations.


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ACKNOWLEDGEMENTS
 
This work was supported by Canadian Institutes of Health Research (CIHR), the Fonds de la Recherche en Santé du Québec (FRSQ), and partly by the Canadian Foundation for Innovation (CFI). S. S. is a scholar of FRSQ. We deeply thank our laboratory colleague Isabelle Pelletier (CRCHUL, Canada) for discussion.

Received December 3, 2004; revised June 1, 2005; accepted July 1, 2005.


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S. L. Farnworth, N. C. Henderson, A. C. MacKinnon, K. M. Atkinson, T. Wilkinson, K. Dhaliwal, K. Hayashi, A. J. Simpson, A. G. Rossi, C. Haslett, et al.
Galectin-3 Reduces the Severity of Pneumococcal Pneumonia by Augmenting Neutrophil Function
Am. J. Pathol., February 1, 2008; 172(2): 395 - 405.
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J. Nieminen, A. Kuno, J. Hirabayashi, and S. Sato
Visualization of Galectin-3 Oligomerization on the Surface of Neutrophils and Endothelial Cells Using Fluorescence Resonance Energy Transfer
J. Biol. Chem., January 12, 2007; 282(2): 1374 - 1383.
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 J. Immunol.Home page
P. Barrionuevo, M. Beigier-Bompadre, J. M. Ilarregui, M. A. Toscano, G. A. Bianco, M. A. Isturiz, and G. A. Rabinovich
A Novel Function for Galectin-1 at the Crossroad of Innate and Adaptive Immunity: Galectin-1 Regulates Monocyte/Macrophage Physiology through a Nonapoptotic ERK-Dependent Pathway
J. Immunol., January 1, 2007; 178(1): 436 - 445.
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