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William Harvey Research Institute, Barts, and The London, Charterhouse Square, London, UK
1Correspondence: The William Harvey Research Institute, Barts, and The London, Queen Mary School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ, UK. E-mail: m.perretti{at}qmul.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: small interfering RNA • homing • delayed-type hypersensitivity
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. This latter aspect has so gained interest in recent years that classes of peptidic or nonpeptidic endogenous inhibitors of specific aspects of the inflammatory reaction have been identified and at least partially characterized (2
, 3)
. In most, if not all, cases, their identification has been possible by initial testing of synthetic preparations (pharmacological studies) such that, in the case of a positive inhibitory action, these studies have paved the way to analysis of the patho-physiological impact and role of the endogenous mediator (1
, 3)
.
Galectin-1 (Gal-1) possesses a series of requisites to suggest it belongs to the class of endogenous anti-inflammatory and proresolving factors. Structurally related to a family of lectins that display a high degree of specificity for carbohydrate binding (4)
, human recombinant (hr)-Gal-1 was found to possess anti-inflammatory properties. Treatment of mice with hr-Gal-1 or local overexpression of Gal-1 in the peritoneum of arthritic mice by active release from syngeneic transgenic fibroblast application resulted in a significant anti-arthritic effect (5)
. Mechanistically, these pharmacological actions were linked to induction of T-cell apoptosis, a process thought to play a positive functional role in this disease model. The anti-inflammatory efficacy of Gal-1 in vivo was complemented by a much larger series of in vitro studies where high concentrations of the protein indeed were found to induce T-cell apoptosis (4
, 6
, 7)
. Induction of T-cell apoptosis occurs via the interaction of Gal-1 with putative cell surface receptors such as CD45, CD43, and CD7 (6
, 8
, 9)
. The efficacy of exogenously applied Gal-1 in models of colitis and hepatitis has been attributed also to its proapoptotic function (10
, 11)
. The need to investigate apoptosis-independent effects of endogenous Gal-1 is prompted by the anti-inflammatory actions displayed by hr-Gal-1 in distinct models of acute inflammation, even in the absence of overt involvement of apoptosis phenomena (12
, 13)
. However, despite Gal-1 null mice having been produced more than a decade ago (14)
, no detailed analysis has been conducted to date in the context of the inflammatory response, with preliminary studies indicating a lack of phenotype in these animals (14
, 15)
.
Supported by the notion that: 1) a distinct Gal-1 expression is detected between endothelial cells and blood leukocytes (13)
, with higher degrees in the former cell type (13
, 16)
, and 2) administration of hr-Gal-1 inhibits blood cell trafficking and interaction with inflamed microvascular vessels (13)
, we tested the hypothesis that endothelial Gal-1 could be an important molecular determinant for endogenous anti-inflammation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The following commercial mouse anti-human antibodies were used in this study: ICAM-1 (clone 15.2), VCAM-1 (clone 1.G11B1), E/P-selectin (clone 1.2B6), P-selectin (clone AK-6) (all purchased from AbD Serotec, Raleigh, NC, USA) and β-actin (clone AC-15) (Sigma-Aldrich Corp., St. Louis, MO, USA).
Cell culture and transfection
Human umbilical vein endothelial cells (HUVECs; Cambrex, Walkersville, MD, USA) were seeded 24 h before transfection at a density of 2 x 105 cells in antibiotic-free media. Transfections were performed with nontargeting, scrambled siRNA or a pool of 3 target-specific Gal-1 siRNAs (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) with cells at 60–80% confluence. For each 35 mm2 dish, 80 µl of transfection medium was mixed with 7.2 µl of 10 µM siRNA duplex. In a second tube, 4.8 µl of transfection reagent was diluted with 80 µl of transfection medium. Following 5 min of incubation at room temperature, the two mixtures were combined, gently mixed, and incubated for a further 20 min. After addition of 0.64 ml of transfection medium, the entire mixture was overlaid onto HUVECs and incubated for 5 h at 37°C in a humidified CO2 incubator. Then, 0.8 ml of complete media containing double the amount of FCS was added and cells incubated for a further 24 h before aspirating and replacing with fresh media. Knockdown of Gal-1 protein expression was monitored by Western blotting, and cells were assayed 48 h post-transfection because cells had reached an optimal degree of confluence for the flow chamber experiments. Uptake of siRNA was tested using fluorescein-conjugated scrambled siRNA and measured in the FL1 channel on a FACscan (BD, Franklin Lakes, NJ, USA) using CellQuest software. HUVECs were clearly distinguished by their forward and side-scatter characteristics.
Western blotting
Adherent endothelial cells were detached using 1x trypsin, flasks were rinsed with PBS, and cells were centrifuged at 200 g for 5 min to form a pellet. Following centrifugation, supernatants were aspirated and, for analysis of total cellular proteins, 200 µl per 5 x 106 cells of 1x hot sample buffer (50 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 100 mM DTT, and 0.01% bromphenol blue) was added directly to the pellet and syringed 5x with a 21-gauge needle to fully lyse the cells. To determine the subcellular localization of Gal-1, membrane and cytoplasmic extracts were obtained using a protocol adapted from Hilgenberg et al. (18)
. Flasks were washed with ice-cold PBS, and 100 µl of lysis buffer (20 mM Tris-HCL, pH 7.5; 10 mM EDTA; 10 mM EGTA; and the following protease inhibitors: 10 µM leupeptin, 1 µM pepstatin A, 200 µM phenylmethyl-sulfonyl fluoride, 1 mM sodium fluoride, and 1mM β-glycerolphosphate) was added. HUVECs were harvested using 1x trypsin as above and homogenized through a fine 21-gauge needle 5x to ensure sufficient lysis. Homogenates were centrifuged at 300 g for 2 min at 4°C to remove cell debris, and the supernatants were transferred to a new eppendorf. Then, after centrifugation at 800 g for 45 min at 4°C, the new supernatants were collected as the cytosolic fraction. Then, 1% (v/v) Triton X-100 was added to the lysis buffer, 100 µl of which was added to the pellet, resuspended, and incubated on ice for 15 min to obtain the membrane fraction.
Samples were subjected to electrophoresis on a 12% SDS-polyacrylamide gel and transferred to nitrocellulose membrane. Membranes were blocked overnight at 4°C with 5% nonfat dried milk diluted in Tris-buffered saline (TBS) solution (150 mM NaCl; 2 mM Tris base; pH 7.4) containing 0.1% Tween 20 (TBS-T). Membranes then were incubated for 1 h at room temperature (RT) in TBS-T with the rabbit polyclonal Gal-1 antibody (Ab) (1:5000 dilution), and 1 h with a horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary Ab (1:2000 dilution) (DakoCytomation, Carpinteria, CA, USA). For β-actin detection, membranes were incubated for 1 h at RT in TBS-T with the mouse monoclonal Ab (1:10,000 dilution) and 1 h with an HRP-conjugated rat anti-mouse IgG secondary Ab (1:2000 dilution) (DakoCytomation). Immunoblotting and visualization of proteins by enhanced chemiluminescence (GE Healthcare, formerly Amersham Biosciences, Uppsala, Sweden) were performed according to the manufacturer’s instructions. For each condition, extract equivalents obtained from the same number of cells were used.
Flow chamber assay
HUVECs were stimulated with TNF-
(10 ng/ml; Sigma-Aldrich, Poole, UK) for 4 h and SDF-1 (20 ng/ml; Peprotech, Rocky Hill, NJ, USA) for 15 min (19)
. In some experiments, 0.4 µg/ml hr-Gal-1 (27 nM) was added to HUVECs at the same time as TNF- treatment in the presence or absence of 30 mM lactose. Human lymphocytes were isolated from healthy volunteers using double-gradient density centrifugation as described (20)
. Immediately prior to flow, lymphocytes were diluted to 1 x 106 cells/ml in Dulbecco phosphate-buffered saline supplemented with Ca2+ and Mg2+ and incubated for 10 min at 37°C prior to perfusion over endothelial monolayers. Flow chamber technical conditions were as described previously (21)
.
Flow cytometry
Hr-Gal-1 (27–270 nM) was added to HUVECs concomitantly with TNF-, and adhesion molecule expression was determined with specific mouse anti-human monoclonal antibodies (all at 5 µg/ml: ICAM-1, VCAM-1, E/P-selectin, or P-selectin) for 1 h on ice. After secondary staining with fluorescein isothiocyanate (FITC) -conjugated F(ab')2 goat anti-mouse IgG (1:200; AbD Serotec, Raleigh, NC, USA), fluorescence in the FL1 channel of the FACScan was quantified, and median fluorescence intensity (MFI) units were recorded.
Determination of cell surface binding sites on HUVECs was conducted as described previously (13)
. Briefly, cells (2x105) were incubated with biotinylated Gal-1 (2.7–270 nM) for 1 h on ice. After secondary staining with phycoerythrin (P/E) -conjugated streptavidin (Caltag, Burlingame, CA, USA) for a further 45 min, the extent of binding was analyzed by flow cytometry, with fluorescence expressed as MFI units following acquisition in the FL2 channel.
Animals
Breeding founders of the Gal-1–/– mouse colony were received from the Consortium for Functional Glycomics, and a colony was established at B&K Universal (Hull, UK). These mice were on a homogenous C57/BL6 background, and age- and sex-matched wild-type (WT) controls were purchased from B&K Universal. All experiments were performed with male animals (body weight
25–30 g) strictly following UK Home Office regulations (Guidance on the Operation of Animals, Scientific Procedures Act 1986).
In vivo homing assay
Homing assays were performed as recently described (22)
. Briefly, spleens were collected from 5 WT mice, homogenized, and passed through a 70 µm cell strainer to obtain single-cell suspensions. Contaminating erythrocytes were removed by hypotonic lysis, and unlysed cells were labeled for 20 min at 37°C with 0.5 µM carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Eugene, OR, USA). Cells (1.5x107) were injected i.v. into 5 recipient Gal-1–/– and WT mice. After 4 h, mice were sacrificed by cardiac puncture under halothane (3%) anesthesia, and 500 µl of blood was collected for subsequent analysis. Spleens and mesenteric lymph nodes were homogenized, RBCs lysed, and cell suspensions analyzed by flow cytometry using a FACScan II analyzer (Becton Dickinson, Cowley, UK).
Delayed-type hypersensitivity reaction
Mice were sensitized intradermally in the base of the tail with 100 µl of 10 mg/ml methylated BSA (met-BSA) emulsified in an equal volume of complete Freund’s adjuvant containing 1 mg/ml heat-killed Mycobacterium tuberculosis. For homing experiments, spleens were harvested on day 15 from 5 sensitized WT mice. Cells were labeled as described above and 1.5 x 107 lymphocytes were injected i.v. into 5 donor WT and Gal-1–/– recipient mice 1 h before challenge and allowed to circulate for 5 h. Challenge was performed by injection at day 15 with 50 µg met-BSA in the right hind footpad and 50 µl of vehicle (saline) in the left. At 4 h after challenge, mice were sacrificed and paws removed and processed as described (23)
. Briefly, paws were sliced into 1- to 2-mm pieces and fragments digested in 2 ml digest buffer (RPMI containing 3 mg/ml collagenase type I, 1 mg/ml hyaluronidase, and 50 mM HEPES; all from Sigma) per paw for 1 h at 37°C. Digested tissue was passed through a 70 µm cell strainer and analyzed by flow cytometry. Paw volume was measured using a water displacement plethysmometer at 4 h postchallenge prior to sacrifice of the mice. The volume displaced by the noninflamed left hind paw was subtracted from that of the inflamed paw to give a measure of paw edema.
Statistical analysis
Results are expressed as mean ± SE and statistical differences were determined by analysis of variance followed by the Dunnett test. For in vivo homing experiments, the statistical analysis was carried out using the Student’s t test. Values of P < 0.05 were considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A). Incubation of HUVECs for 1 h with increasing concentrations (2.7–270 nM) of biotinylated hr-Gal-1 resulted in a dose-dependent increase in Gal-1 binding, which was significant using 27 and 270 nM Gal-1: quantified binding was 84 ± 6.2, 615 ± 5.5 and 72 ± 2.1, 630 ± 53.5 MFI units for control and activated HUVECs, respectively (Fig. 1B
). No significant differences were measured depending on whether the HUVECs were activated with a 4 h treatment with TNF- (Fig. 1B
). Pre-incubation of HUVECs for 4 h with these two higher concentrations of Gal-1 caused a significant decrease in the proportion of lymphocytes recruited to the endothelium under flow (Fig. 1C
). These results are consistent with our previous observations showing hr-Gal-1 inhibition of in vivo cell trafficking (17)
. Pretreatment of the monolayers of HUVECs with hr-Gal-1 did not alter adhesion molecule expression following TNF- stimulation, illustrating that such a mechanism is not mediating the effect of Gal-1 on lymphocyte recruitment. To verify whether the effects of Gal-1 on lymphocyte recruitment in the flow chamber were carbohydrate-dependent in nature, 30 mM lactose was added in conjunction with Gal-1 in some experiments. Gal-1 binding to HUVECs was significantly attenuated in the presence of lactose (14±1.3 MFI units) (Fig. 2
A), and this finding was functionally relevant because a complete reversal of the effects of Gal-1 on lymphocyte recruitment in the flow chamber was evident (Fig. 2B
).
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Downregulating Gal-1 in HUVECs augments lymphocyte recruitment
Endothelial Gal-1 expression was reduced efficiently in the presence of a specific siRNA when compared with nontargeting scrambled siRNA (Fig. 3
A). Application of siRNA suppressed endothelial Gal-1 by 50% at 48 h post-transfection without affecting the expression of the housekeeping gene β-actin, as monitored by Western blotting (Fig. 3A
). For verification of siRNA uptake, HUVECs were transfected transiently with fluorescein-conjugated scrambled siRNA and analyzed by flow cytometry. As shown in Fig. 3B
, transfected cells displayed a markedly increased fluorescence intensity and, hence, incorporation of siRNA.
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Attenuated endothelial Gal-1 expression led to a substantial increase (86%) in the initial capture of lymphocytes to the endothelium, which is a prerequisite for further interactions; therefore, a subsequent increase in cell rolling and adhesion also was seen (Fig. 2C
). This phenotype could be reversed by addition of exogenous hr-Gal-1 (27 nM) to endothelial monolayers (90±7% inhibition of the increased lymphocyte recruitment displayed by siRNA-treated HUVECs; n=4). These alterations in leukocyte/HUVEC interactions were not a consequence of altered endothelial cell adhesion molecule expression (Table 1
); basal levels of ICAM-1, VCAM-1, and E-selectin were higher in transfected cells compared with nontransfected HUVECs. TNF- stimulation yielded increased expression of ICAM-1 and E-selectin in all cells; VCAM-1 levels remained unchanged in Gal-1 siRNA-transfected cells. Therefore, the differences observed in adhesion molecule levels post-transfection likely do not account for the functional effects of Gal-1 siRNA on lymphocyte recruitment under flow.
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In vivo lymphocyte trafficking is elevated in Gal-1–/– mice
To assess whether the results seen in vitro would translate to lymphocyte-endothelial cell interactions in vivo, we performed short-term homing assays. We initially examined the trafficking of naive lymphocytes in naive animals. As shown in Fig. 4
A, Gal-1–/– mice displayed a significant increase in the homing of fluorescently-labeled cells to mesenteric lymph nodes compared with WT mice. Homing to spleens also was slightly elevated in Gal-1–/– mice (Fig. 4A
). In contrast to selectin-deficient mice, Gal-1–/– mice show no change in peripheral blood leukocyte counts nor the relative leukocyte subclasses (Table 2
); therefore, increased homing cannot be attributed to hematological changes.
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Next, we addressed the recruitment characteristics in inflammatory conditions whereby mice were sensitized with met-BSA for a subsequent delayed-type hypersensitivity response. Lymphocytes collected from the spleens of sensitized WT mice were fluorescently labeled and injected into WT or Gal-1–/– mice after challenge with the antigen in the paw. Under these conditions, Gal-1–/– mice display a marked and significant increase in the recruitment of labeled cells to the site of inflammation compared with WT equivalents (Fig. 4B
). Paw edema also was significantly greater in the inflamed paw of the Gal-1–/– mice at 4 h postchallenge with met-BSA (68.7±5.7 µl vs. 38.3±10.7 µl for Gal-1–/– and WT mice, respectively; data are from 5 mice per genotype, P<0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Various studies have indicated an anti-inflammatory function for Gal-1 via mainly induction of apoptosis of activated immature and mature, but not resting, T cells (4
, 6
, 7
, 24)
. Relatively high concentrations (10 µM) are required to elicit these effects, which are dependent on the cross-linking of specific cell surface glycoproteins such as CD45, CD43, and CD7 (9)
. However, a recent investigation has shown that induction of phosphatidylserine exposure and apoptosis of activated T cells might be an artifact resulting from the priming action of DTT, a finding that perhaps reduces the validity of previous studies (25)
.
In contrast to in vitro pro-apoptotic effects, anti-inflammatory effects of hr-Gal-1 are observed at nM concentrations, whereby Gal-1 prevents T-cell adhesion to ECM glycoproteins while inhibiting TNF- and IFN-
secretion (26)
and neutrophil (13)
and T-cell transendothelial passage, as determined in static conditions (8)
. The novel results we present here indicate that endogenous Gal-1 acts to modulate lymphocyte-endothelial interactions under flow conditions both in vitro and in vivo.
At variance from leukocytes, endothelial cells expressed promptly detectable levels of Gal-1 (13
, 16)
. Importantly, the lectin is predominantly localized in the membrane pool and could be upregulated on specific experimental conditions (16)
. Additionally, increased Gal-1 expression is detected in high endothelial venules (HEVs) of inflamed human lymph nodes, the site of lymphocyte trafficking in vivo (16)
. Of note, in comparison to HEVs, HUVECs express only minute amounts of endogenous chemokines needed to promote lymphocyte activation and adhesion to the endothelium. Previous studies have demonstrated that immobilized chemokines can augment not only adhesion but also earlier integrin-mediated capture (tethering) of lymphocytes on inflamed endothelium (27)
. Therefore, we briefly overlaid the chemokine stromal cell-derived factor-1 (SDF-1, CXCL12) on TNF--activated HUVECs for more physiological experimental conditions. We now propose a functional role for these observations, demonstrating a potent inhibitory action for Gal-1 on lymphocyte capture in conditions that mimic the inflamed HEVs. Reducing Gal-1 protein levels in HUVECs was associated with a marked increase (90%) of cell recruitment, and this phenotype was rescued by adding a low concentration (27 nM) of hr-Gal-1. In addition, and equally important, addition of the exogenous protein to untreated HUVECs also markedly inhibited lymphocyte capture. Collectively, these data demonstrate an unequivocal inhibitory role for Gal-1 on the early recruitment of lymphocytes on activated endothelial monolayers under flow. This effect is evident also in static conditions, where decreased lymphocyte transmigration was observed when endothelial Gal-1 expression was increased in response to cancer cell conditioned media (8)
. Lymphocyte adhesion using static conditions apparently is unaffected by application of hr-Gal-1 (8)
whereas, under flow conditions, which have been shown to be crucial for the appropriate activation of lymphocytes with subsequent firm adhesion and transmigration (28)
, we could reveal a critical role of Gal-1 in regulating both cell rolling and adhesion.
We demonstrate a significant degree of Gal-1 binding to monolayers of HUVECs, illustrating the possible existence of numerous binding sites for Gal-1 on the endothelium. In addition, results in the flow chamber suggest that Gal-1 is acting in a chemokine-like manner because it requires presentation by the endothelium for its anti-adhesive effects. Short incubation of lymphocytes with hr-Gal-1 prior to flow had no effect on cell recruitment (data not shown), while either depletion of endogenous Gal-1 or co-incubation of endothelial cells with hr-Gal-1 for 30–240 min prior to flow significantly enhanced or reduced lymphocyte capture, adhesion, and rolling, respectively. A number of studies have shown chemokines to be more effective at inducing lymphocyte recruitment when presented by the endothelium than in a soluble state (29
, 30)
. This study suggests that Gal-1 also acts in this manner but as a negative regulator of cell recruitment. Of interest, an unrelated protein, TNF-stimulated gene 6, behaves in a similar fashion and requires addition to the monolayer of HUVECs to inhibit subsequent leukocyte rolling and adhesion under flow (31)
.
Previous studies have described a role for hr-Gal-1 in limiting cell recruitment under inflammatory conditions (12)
; however, we have shown that homing of lymphocytes also is negatively regulated by endogenous Gal-1 in naive mice. Such an effect was not linked to endothelial cell alterations in adhesion molecule expression or in the generation of endothelial-derived autacoids, including nitric oxide or prostacyclin, because the phenotype of hr-Gal-1-treated HUVECs was not rescued by cell treatment with indomethacin or N
-Nitro-L-arginine methyl ester (unpublished results). These data suggest that Gal-1 exerts a tonic inhibitory action on lymphocyte recruitment, aiming at maintaining tissue homeostasis. Therefore, the actions of Gal-1 under normal physiological conditions may function to limit the exposure of T cells to antigens within lymph nodes and, hence, induction of an undesired immune response. This possibility suggests that Gal-1 may have a prominent role in autoimmune diseases, which clearly warrants further investigation. Note that homing in naive mice is significantly increased in the lymph nodes whereas it is not significant to the spleen, an organ in which L-selectin, LFA-1, or 4 integrins are not required for homing (32)
.
We have recently shown rapid modulation of Gal-1 expression in postcapillary venule endothelium in a model of peritonitis (33)
. We now provide functional relevance to these immunohistochemical analyses because increased numbers of T lymphocytes homed to the inflamed paw in a met-BSA-induced delayed type hypersensitivity response in Gal-1–/– mice but not in their WT counterparts. The mechanism by which endogenous Gal-1, and likely hr-Gal-1, is influencing lymphocyte recruitment has yet to be fully elucidated. Of interest, a recent study indicates hr-Gal-1 to modulate Th1 and Th17 lifespan, thereby affecting autoimmune disease, as assessed in an experimental model of multiple sclerosis (34)
. It is highly unlikely that such mechanism, evident at doses of hr-Gal-1 much higher than those used in the present study, would be relevant for the observed marked increase in T-cell recruitment to the inflamed paws, especially in view of the rapid kinetics used here (4 h). However, it is possible that later phases of the delayed-type hypersensitivity response to met-BSA could be altered in Gal-1–/– mice, possibly evoking the recently proposed mechanism of action of this protein on Th1 and Th17 functionality (34)
. A few studies have implicated Gal-1 to modulate T-cell cytokine production, switching from a Th1 to a Th2 phenotype, including inhibition of IL-2 (35)
, IFN-, and TNF- production by IL-2-activated T cells (26)
; this effect is associated with increased expression of IL-10 (36)
. In complex immune conditions, a combination of Gal-1 effects of T-cell modulating cytokines with the inhibition of T-cell recruitment onto the vascular endothelium, which we now report likely, contributes to the overall dampening of the inflammatory response.
In conclusion, we have revealed here a novel endogenous anti-inflammatory pathway centered on endothelial Gal-1 that targets lymphocyte homing under normal physiological conditions and trafficking in an inflammatory context. We thus propose that during inflammation, cell surface Gal-1 levels on endothelial cells are increased in a bid to limit the influx of leukocytes into the surrounding tissues to contain the inflammatory response.
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ACKNOWLEDGMENTS |
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Received for publication August 1, 2007. Accepted for publication September 13, 2007.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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4 integrin clustering by immobilized chemokines stimulates leukocyte tethering and rolling on endothelial vascular cell adhesion molecule 1 under flow conditions. J. Exp. Med. 192,495-506This article has been cited by other articles:
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  L. V Norling, M. Perretti, and D. Cooper Endogenous galectins and the control of the host inflammatory response J. Endocrinol., May 1, 2009; 201(2): 169 - 184. [Abstract] [Full Text] [PDF]
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 M. J. Perone, S. Bertera, W. J. Shufesky, S. J. Divito, A. Montecalvo, A. R. Mathers, A. T. Larregina, M. Pang, N. Seth, K. W. Wucherpfennig, et al. Suppression of Autoimmune Diabetes by Soluble Galectin-1 J. Immunol., March 1, 2009; 182(5): 2641 - 2653. [Abstract] [Full Text] [PDF]
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 S. SAUSSEZ, C. DECAESTECKER, S. CLUDTS, P. ERNOUX, D. CHEVALIER, K. SMETANA Jr., S. ANDRE, X. LEROY, and H.-J. GABIUS Adhesion/Growth-regulatory Tissue Lectin Galectin-1 in Relation to Angiogenesis/Lymphocyte Infiltration and Prognostic Relevance of Stromal Up-regulation in Laryngeal Carcinomas Anticancer Res, January 1, 2009; 29(1): 59 - 65. [Abstract] [Full Text] [PDF]
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 S. D. Liu, C. C. Whiting, T. Tomassian, M. Pang, S. J. Bissel, L. G. Baum, V. V. Mossine, F. Poirier, M. E. Huflejt, and M. C. Miceli Endogenous galectin-1 enforces class I-restricted TCR functional fate decisions in thymocytes Blood, July 1, 2008; 112(1): 120 - 130. [Abstract] [Full Text] [PDF]
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