Galectin-3 is a member of a growing family of β-galactoside-binding animal lectins. Previous studies have demonstrated a variety of biological activities for this protein in vitro, including activation of cells, modulation of cell adhesion, induction of pre-mRNA splicing, and regulation of apoptosis. To assist in fully elucidating the physiological and pathological functions of this protein, we have generated galectin-3-deficient (gal3−/−) mice by targeted interruption of the galectin-3 gene. Gal3−/− mice consistently developed fewer inflammatory cell infiltrations in the peritoneal cavities than the wild-type (gal3+/+) mice in response to thioglycollate broth treatment, mainly due to lower numbers of macrophages. Also, when compared to cells from gal3+/+ mice, thioglycollate-elicited inflammatory cells from gal3−/− mice exhibited significantly lower levels of NF-κB response. In addition, dramatically different cell-spreading phenotypes were observed in cultured macrophages from the two genotypes. Whereas macrophages from gal3+/+ mice exhibited well spread out morphology, those from gal3−/− mice were often spindle-shaped. Finally, we found that peritoneal macrophages from gal3−/− mice were more prone to undergo apoptosis than those from gal3+/+ mice when treated with apoptotic stimuli, suggesting that expression of galectin-3 in inflammatory cells may lead to longer cell survival, thus prolonging inflammation. These results strongly support galectin-3 as a positive regulator of inflammatory responses in the peritoneal cavity. Galectin-3 is a member of a growing family of β-galactoside-binding animal lectins. Previous studies have demonstrated a variety of biological activities for this protein in vitro, including activation of cells, modulation of cell adhesion, induction of pre-mRNA splicing, and regulation of apoptosis. To assist in fully elucidating the physiological and pathological functions of this protein, we have generated galectin-3-deficient (gal3−/−) mice by targeted interruption of the galectin-3 gene. Gal3−/− mice consistently developed fewer inflammatory cell infiltrations in the peritoneal cavities than the wild-type (gal3+/+) mice in response to thioglycollate broth treatment, mainly due to lower numbers of macrophages. Also, when compared to cells from gal3+/+ mice, thioglycollate-elicited inflammatory cells from gal3−/− mice exhibited significantly lower levels of NF-κB response. In addition, dramatically different cell-spreading phenotypes were observed in cultured macrophages from the two genotypes. Whereas macrophages from gal3+/+ mice exhibited well spread out morphology, those from gal3−/− mice were often spindle-shaped. Finally, we found that peritoneal macrophages from gal3−/− mice were more prone to undergo apoptosis than those from gal3+/+ mice when treated with apoptotic stimuli, suggesting that expression of galectin-3 in inflammatory cells may lead to longer cell survival, thus prolonging inflammation. These results strongly support galectin-3 as a positive regulator of inflammatory responses in the peritoneal cavity. Galectins are members of a recently identified family of β-galactoside-binding animal lectins.1Barondes SH Castronovo V Cooper DNW Cummings RD Drickamer K Feizi T Gitt MA Hirabayashi J Hughes C Kasai K Leffler H Liu F-T Lotan R Mercurio AM Monsigny M Pillai S Poirer F Raz A Rigby PWJ Rini JM Wang JL Galectins: A family of animal β-galactoside-binding lectins. (letter to the editor).Cell. 1994; 76: 597-598Abstract Full Text PDF PubMed Scopus (1085) Google Scholar Presently more than ten members have been identified, and additional homologues are likely to be discovered. Existing information suggests that they may be involved in a multitude of biological processes including regulation of cell growth and apoptosis, neoplastic transformation, and inflammatory responses.2Barondes SH Cooper DNW Gitt MA Leffler H Galectins: structure and function of a large family of animal lectins.J Biol Chem. 1994; 269: 20807-20810Abstract Full Text PDF PubMed Google Scholar, 3Kasai K Hirabayashi J Galectins: a family of animal lectins that decipher glycocodes.J Biochem (Tokyo). 1996; 119: 1-8Crossref PubMed Scopus (453) Google Scholar, 4Hughes RC The galectin family of mammalian carbohydrate-binding molecules.Biochem Soc Trans. 1997; 25: 1194-2298Crossref PubMed Scopus (108) Google Scholar, 5Perillo NL Marcus ME Baum LG Galectins: versatile modulators of cell adhesion, cell proliferation, and cell death.J Mol Med. 1998; 76: 402-412Crossref PubMed Scopus (579) Google Scholar One of the best characterized members, galectin-3, is composed of a carboxyl-terminal lectin domain connected to an amino-terminal nonlectin part consisting primarily of short tandem repeats.6Liu F-T Molecular biology of IgE-binding protein, IgE-binding factors and IgE receptors.CRC Crit Rev Immunol. 1990; 10: 289-306Google Scholar, 7Hughes RC Mac-2: a versatile galactose-binding protein of mammalian tissues.Glycobiology. 1994; 4: 5-12Crossref PubMed Scopus (131) Google Scholar, 8Liu F-T Role of galectin-3 in inflammation.in: Caron M Seve D Lectins and Pathology. Harwood Academic Publishers, London2000: 51-65Google Scholar Galectin-3 is widely distributed in tissues and is found in various epithelial cells, dendritic cells, and inflammatory cells.9Flotte TJ Springer TA Thorbecke GJ Dendritic cell and macrophage staining by monoclonal antibodies in tissue sections and epidermal sheets.Am J Pathol. 1983; 111: 112-124PubMed Google Scholar The expression of this lectin is correlated with cell proliferation,10Moutsatsos IK Wade M Schindler M Wang JL Endogenous lectins from cultured cells: nuclear localization of carbohydrate-binding protein 35 in proliferating 3T3 fibroblasts.Proc Natl Acad Sci USA. 1987; 84: 6452-6456Crossref PubMed Scopus (240) Google Scholar, 11Agrwal N Wang JL Voss PG Carbohydrate-binding protein 35: levels of transcription and mRNA accumulation in quiescent and proliferating cells.J Biol Chem. 1989; 264: 17236-17242Abstract Full Text PDF PubMed Google Scholar cell differentiation,12Liu F-T Hsu DK Zuberi RI Kuwabara I Chi EY Henderson Jr, WR Expression and function of galectin-3, a β-galactoside-binding lectin, in human monocytes and macrophages.Am J Pathol. 1995; 147: 1016-1029PubMed Google Scholar, 13Nangia-Makker P Ochieng J Christman JK Raz A Regulation of the expression of galactoside-binding lectin during human monocytic differentiation.Cancer Res. 1993; 53: 1-5PubMed Google Scholar and transactivation by viral proteins.14Hsu DK Hammes SR Kuwabara I Greene WC Liu F-T Human T lymphotropic virus-1 infection of human T lymphocytes induces expression of the β-galactose-binding lectin, galectin-3.Am J Pathol. 1996; 148: 1661-1670PubMed Google ScholarA number of biological activities of galectin-3 have been demonstrated in vitro. This lectin has been shown to activate various cells, including mast cells,15Frigeri LG Zuberi RI Liu F-T ɛBP, a β-galactoside-binding animal lectin, recognizes IgE receptor (FcɛRI), and activates mast cells.Biochemistry. 1993; 32: 7644-7649Crossref PubMed Scopus (167) Google Scholar, 16Zuberi RI Frigeri LG Liu F-T Activation of rat basophilic leukemia cells by ɛBP, an IgE-binding endogenous lectin.Cell Immunol. 1994; 156: 1-12Crossref PubMed Scopus (61) Google Scholar neutrophils,17Yamaoka A Kuwabara I Frigeri LG Liu F-T A human lectin, galectin-3 (ɛBP/Mac-2), stimulates superoxide production by neutrophils.J Immunol. 1995; 154: 3479-3487PubMed Google Scholar monocytes/macrophages,12Liu F-T Hsu DK Zuberi RI Kuwabara I Chi EY Henderson Jr, WR Expression and function of galectin-3, a β-galactoside-binding lectin, in human monocytes and macrophages.Am J Pathol. 1995; 147: 1016-1029PubMed Google Scholar and lymphocytes.14Hsu DK Hammes SR Kuwabara I Greene WC Liu F-T Human T lymphotropic virus-1 infection of human T lymphocytes induces expression of the β-galactose-binding lectin, galectin-3.Am J Pathol. 1996; 148: 1661-1670PubMed Google Scholar, 18Dong S Hughes RC Galectin-3 stimulates uptake of extracellular Ca2+ in human Jurkat T-cells.FEBS Lett. 1996; 395: 165-169Abstract Full Text PDF PubMed Scopus (79) Google Scholar The effects of this lectin in cell adhesion have also been demonstrated.19Kuwabara I Liu F-T Galectin-3 promotes adhesion of human neutrophils to laminin.J Immunol. 1996; 156: 3939-3944PubMed Google Scholar, 20Inohara H Akahani S Koths K Raz A Interactions between galectin-3 and Mac-2-binding protein mediate cell-cell adhesion.Cancer Res. 1996; 56: 4530-4534PubMed Google Scholar, 21Sato S Hughes RC Binding specificity of a baby hamster kidney lectin for H type I and II chains, polylactosamine glycans, and appropriately glycosylated forms of laminin and fibronectin.J Biol Chem. 1992; 267: 6983-6990Abstract Full Text PDF PubMed Google Scholar These extracellular functions suggest a possible role for this protein in modulation of immune reactions and inflammatory responses. In addition, evidence for various intracellular activities of galectin-3 are also available. It has been identified as a component of hnRNP,22Laing JG Wang JL Identification of carbohydrate binding protein 35 in heterogeneous nuclear ribonucleoprotein complex.Biochemistry. 1988; 27: 5329-5334Crossref PubMed Scopus (97) Google Scholar as well as a factor in pre-mRNA splicing,23Dagher SF Wang JL Patterson RJ Identification of galectin-3 as a factor in pre-mRNA splicing.Proc Natl Acad Sci USA. 1995; 92: 1213-1217Crossref PubMed Scopus (355) Google Scholar and shown to have anti-apoptotic activity, possibly through a mechanism involving its interactions with Bcl-2 family members, with which this lectin shares sequence similarity.24Yang R-Y Hsu DK Liu F-T Expression of galectin-3 modulates T cell growth and apoptosis.Proc Natl Acad Sci USA. 1996; 93: 6737-6742Crossref PubMed Scopus (666) Google Scholar, 25Akahani S Nangia-Makker P Inohara H Kim HRC Raz A Galectin-3: a novel antiapoptotic molecule with a functional BH1 (NWGR) domain of Bcl-2 family.Cancer Res. 1997; 57: 5272-5276PubMed Google ScholarGalectin-3 has been found to be overexpressed in certain pathological conditions, including human atherosclerotic lesions.26Nachtigal M Al-Assaad Z Mayer EP Kim K Monsigny M Galectin-3 expression in human atherosclerotic lesions.Am J Pathol. 1998; 152: 1199-1208PubMed Google Scholar The association of this lectin with neoplastic transformation has also been extensively documented. It is overexpressed in some types of cancer for which the normal parental cells do not express the protein; the examples include specific types of lymphoma,14Hsu DK Hammes SR Kuwabara I Greene WC Liu F-T Human T lymphotropic virus-1 infection of human T lymphocytes induces expression of the β-galactose-binding lectin, galectin-3.Am J Pathol. 1996; 148: 1661-1670PubMed Google Scholar, 27Konstantinov KN Robbins BA Liu F-T Galectin-3, a β-galactoside-binding animal lectin, is a marker of anaplastic large-cell lymphoma.Am J Pathol. 1996; 148: 25-30PubMed Google Scholar thyroid carcinoma,28Fernádez PL Merino MJ Gómez M Campo E Medina T Castronovo V Cardesa A Liu F-T Sobel ME Galectin-3 and laminin expression in neoplastic and non-neoplastic thyroid tissue.J Pathol. 1997; 181: 80-86Crossref PubMed Scopus (135) Google Scholar, 29Xu XC El-Naggar AK Lotan R Differential expression of galectin-1 and galectin-3 in thyroid tumors: potential diagnostic implications.Am J Pathol. 1995; 147: 815-822PubMed Google Scholar and hepatocellular carcinoma.30Hsu DK Dowling CA Jeng KCG Chen JT Yang RY Liu FT Galectin-3 expression is induced in cirrhotic liver and hepatocellular carcinoma.Int J Cancer. 1999; 81: 519-526Crossref PubMed Scopus (195) Google Scholar However, down-regulation of this lectin has been observed in other kinds of neoplasms, including colon,31Lotz MM Andrews Jr, CW Korzelius CA Lee EC Steele Jr, GD Clarke A Mercurio AM Decreased expression of Mac-2 (carbohydrate binding protein 35) and loss of its nuclear localization are associated with the neoplastic progression of colon carcinoma.Proc Natl Acad Sci USA. 1993; 90: 3466-3472Crossref PubMed Scopus (226) Google Scholar, 32Castronovo V Campo E van den Brûle FA Claysmith AP Cioce V Liu F-T Fernandez PL Sobel ME Inverse modulation of steady state mRNA levels of two non-integrin laminin binding proteins in human colon carcinoma.J Natl Cancer Inst. 1992; 84: 1161-1167Crossref PubMed Scopus (97) Google Scholar breast,33Castronovo V Van den Brule FA Jackers P Clausse N Liu FT Gillet C Sobel ME Decreased expression of galectin-3 is associated with progression of human breast cancer.J Pathol. 1996; 179: 43-48Crossref PubMed Scopus (185) Google Scholar ovarian,34van den Brûle FA Berchuck A Bast RC Liu F-T Pieters C Sobel ME Castronovo V Differential expression of the 67-kD laminin receptor and 31-kD human laminin-binding protein in human ovarian carcinoma.Eur J Cancer. 1994; 30A: 1096-1099Abstract Full Text PDF PubMed Scopus (108) Google Scholar and uterine carcinoma,35van den Brûle FA Buicu C Berchuck A Bast RC Deprez M Liu FT Cooper DNW Pieters C Sobel ME Castronovo V Expression of the 67-kD laminin receptor, galectin-1, and galectin-3 in advanced human uterine adenocarcinoma.Hum Pathol. 1996; 27: 1185-1191Abstract Full Text PDF PubMed Scopus (149) Google Scholar although it has also been observed that galectin-3 expression correlates positively with the progression of colon carcinoma.36Irimura T Matsushita Y Sutton RC Carralero D Ohannesian DW Cleary KR Ota DM Nicolson GL Lotan R Increased content of an endogenous lactose-binding lectin in human colorectal carcinoma progressed to metastatic stages.Cancer Res. 1991; 51: 387-393PubMed Google Scholar, 37Schoeppner HL Raz A Ho SB Bresalier RS Expression of an endogenous galactose-binding lectin correlates with neoplastic progression in the colon.Cancer. 1995; 75: 2818-2826Crossref PubMed Scopus (213) Google Scholar Studies of cells transfected with galectin-3 cDNA have indeed provided evidence for its role in tumor transformation and metastasis.38Raz A Zhu D Hogan V Shah N Raz T Karkash R Pazerini G Carmi P Evidence for the role of 34-kDa galactoside-binding lectin in transformation and metastasis.Int J Cancer. 1990; 46: 871-877Crossref PubMed Scopus (172) Google ScholarTherefore, functions of galectin-3 appear to be multifaceted, extending to both intracellular and extracellular compartments. Because of its wide tissue distribution and pleiotropic effects in many systems, galectin-3 is likely to be involved in a variety of physiological and pathological processes. In an attempt to elucidate more definitively the biological functions of galectin-3, we have generated a mouse model in which this gene is inactivated through homologous recombination. We initially focused on the study of inflammatory responses in these mice and have found that the galectin-3 deficiency results in significantly altered inflammation elicited in the peritoneal cavity by thioglycollate broth.Materials and MethodsGeneration of Galectin-3-Null MiceA vector used for homologous recombination was constructed from the cloned galectin-3 genomic DNA.39Gritzmacher CA Mehl VS Liu F-T Genomic cloning of the gene for an IgE-binding lectin reveals unusual utilization of 5′ untranslated regions.Biochemistry. 1992; 31: 9533-9538Crossref PubMed Scopus (35) Google Scholar As shown in Figure 1, a segment from exon 4 to exon 5 within the mouse galectin-3 gene was inserted into pMC1Neo (Stratagene, La Jolla, CA) upstream of the thymidine kinase promoter-Neo cassette. Another segment from exon 5 to exon 6 followed downstream from the Neo cassette. Thus, exon 5 is interrupted by the Neo gene in this vector construct. Murine stem cells, D3, were electroporated with this vector, using procedures previously described.40Shier P Otulakowski G Ngo K Panakos J Chourmouzis E Christjansen L Lau CY Fung-Leung W-P Impaired immune responses toward alloantigens and tumor cells but normal thymic selection in mice deficient in the beta2 integrin leukocyte function-associated antigen-1.J Immunol. 1996; 157: 5375-5386PubMed Google Scholar G418 resistant cells were screened for homologous recombination by polymerase chain reaction (PCR) and Southern blotting, using procedures described below. Screening of 894 clones resulted in two with successful homologous recombination.One clone was propagated and injected into 3.5-day-old blastocysts from C57BL/6 mice and the injected blastocysts were implanted into pseudo-pregnant CD1 mothers. Male chimeric mice were bred to C57BL/6 females to produce mice hemizygous for the galectin-3 null mutant (gal3+/−). Interbreeding gal3+/− mice resulted in mice homozygous for the galectin-3-null condition (gal3−/−). Gal3−/− mice were viable and fertile. For experimentation, wild-type (gal3+/+) mice produced by gal3+/− interbreeding were carried in a separate lineage as controls. Both gal3+/+ and gal3−/− mice were propagated and maintained in standard specific-pathogen-free environments.Polymerase Chain Reaction and Southern Blot AnalysisPolymerase chain reactions were performed under standard conditions using Taq DNA polymerase (Promega, Madison, WI. on a Perkin-Elmer 9600 or Ericomp EZ cycler. Southern blotting analyses were performed by capillary transfer of DNA to charged nylon membranes (BioRad, Richmond, CA or Amersham, Arlington Heights, IL). Membranes were probed with an intron 3 fragment corresponding to probe 1, or probe 4 corresponding to the Neo cassette, to determine homologous recombination, as shown in Figure 1B, radiolabeled by random priming with [32P]-dATP.Immunoblot AnalysisTissues were extracted with 20 mmol/L Tris-HCl, pH 7.5 containing 10 mmol/L EDTA, 0.15 mol/L NaCl, 1% Triton X-100 (v/v) and protease inhibitors 0.24 u/ml Aprotinin, 1 μg/ml pepstatin, 1 μg/ml leupeptin, 1 mmol/L phenylmethylsulfonyl fluoride, and 100 μg protein from each extract was then adsorbed with lactosyl-Sepharose 4B.41Hsu DK Zuberi R Liu F-T Biochemical and biophysical characterization of human recombinant IgE-binding protein, an S-type animal lectin.J Biol Chem. 1992; 267: 14167-14174Abstract Full Text PDF PubMed Google Scholar The bound proteins were eluted, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and analyzed by immunoblotting using specific rabbit antibodies as described.12Liu F-T Hsu DK Zuberi RI Kuwabara I Chi EY Henderson Jr, WR Expression and function of galectin-3, a β-galactoside-binding lectin, in human monocytes and macrophages.Am J Pathol. 1995; 147: 1016-1029PubMed Google ScholarInduction of Peritoneal InflammationMice were injected with 1 ml of autoclaved Brewer's thioglycollate broth as described.42Mishell BB Selected Methods in Cellular Immunology. Freeman, San Francisco1980: 3Google Scholar At various intervals, peritoneal cells were harvested by lavage with minimum essential medium containing Earle's salts (MEM). Cells were washed once with culture medium before subsequent manipulations. For quantitation of leukocyte subpopulations, the cells in the lavage fluid were allowed to attach to glass slides by cytospin, treated with soluble Wright's stain (Leukostat, Fisher Scientific, Pittsburgh, PA) and identified as macrophages, eosinophils, neutrophils, and lymphocytes by standard morphology. Cell counts were obtained in triplicate for each sample from 100–200 cells using a 100× oil immersion objective.Electrophoretic Mobility Shift Assay (EMSA)Nuclear extracts were prepared by a previously described method43Dignam JD Lebovitz RM Roeder RG Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei.Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9142) Google Scholar with slight modifications44Kravchenko VV Pan Z Han J Herbert JM Ulevitch RJ Ye RD Platelet-activating factor induces NF-kappa B activation through a G protein-coupled pathway.J Biol Chem. 1995; 270: 14928-14934Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar and EMSA was performed as described.45Browning DD Pan ZK Prossnitz ER Ye RD Cell type- and developmental stage-specific activation of NF-kappaB by fMet-Leu-Phe in myeloid cells.J Biol Chem. 1997; 272: 7995-8001Crossref PubMed Scopus (59) Google Scholar Briefly, the nuclear extracts (2.5 μg protein) in 12 μl of binding buffer (5 mmol/L HEPES, pH 7.8, 5 mmol/L MgCl2, 50 mmol/L KCl, 0.5 mmol/L dithiothreitol, 0.4 mg/ml poly(dI-dC) (Pharmacia, Piscataway, NJ), 0.1 mg/ml sonicated double-stranded salmon sperm DNA, and 10% glycerol) were incubated for 10 minutes at room temperature. Subsequently, approximately 20 to 50 fmoles of 32P-labeled NF-κB-specific oligonucleotide probe (30,000–50,000 cpm) were added and the reaction mixture was incubated for 10 minutes at room temperature. The samples were analyzed on 6% polyacrylamide gels in 50 mmol/L Tris-borate buffer containing 1 mmol/L EDTA or 50 mmol/L Tris/380 mmol/L glycine/2 mmol/L EDTA, at 12 V/cm for 2 to 2.5 hours. Radioactivities were detected by exposure to X-ray film or phosphorimager plates.Culture and Measurement of Adhesion Areas of Peritoneal MacrophagesPeritoneal macrophages were enriched by adherence onto tissue culture-treated plates in Dulbecco's modified Eagle's medium (DMEM), supplemented with 2 mmol/L glutamine and 10% (v/v) fetal bovine serum, at 37°C in an atmosphere of 7.5% CO2. After two hours of incubation, wells were gently pipetted to remove nonadherent cells. More than 90% of the adherent cells were macrophages as evaluated morphologically after processing with Wright's stain. Initially nonadherent cells were obtained after two serial adherence procedures in tissue culture flasks. Culturing of these cells resulted in additional adherent macrophages. The morphology of adherent macrophages cultured for various periods were imaged from three fields of each well using a Hamamatsu XC-77 CCD camera attached to a Nikon microscope with an inverted stage. Image-1 from Universal Imaging Corporation was used to acquire and enhance image contrast, and NIH Image software was used to measure cell attachment areas from three random fields.Induction of Apoptosis of Peritoneal Macrophages in VitroInflammatory peritoneal macrophages were obtained from mice treated with 1 ml thioglycollate broth for 3 days by lavage, and cultured in RPMI/10% fetal bovine serum (RP10F) for 1 hour. Adherent cells were cultured in the presence of 10 μg/ml lipopolysaccharide (LPS, Escherichia coli 0111:B4 List Biologicals, Campbell, CA) and 10 U/ml interferon-γ (IFN-γ, Boehringer/Roche, Indianapolis, IN), and cell viability was measured by the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay46Hansen MB Nielsen SE Berg K Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill.J Immunol Methods. 1989; 119: 203-210Crossref PubMed Scopus (3316) Google Scholar at regular intervals. Alternatively, inflammatory macrophages obtained from peritoneal cavities of mice 4 days after 2 ml thioglycollate treatment, were cultured for 30 minutes in RPMI 1640. The adherent cells were exposed to 0 to 60 U/ml IFN-γ in RP10F for 6 hours washed with RP10F, and then 1 μg/ml LPS in RP10F for 24 hours.47Shnyra A Brewington R Alipio A Amura C Morrison DC Reprogramming of lipopolysaccharide-primed macrophages is controlled by a counterbalanced production of IL-10 and IL-12.J Immunol. 1998; 160: 3729-3736PubMed Google Scholar The cells were washed with phosphate-buffered saline, pH 7.2, fixed in 5. formaldehyde/phosphate-buffered saline, and stained with 0.05% crystal violet in 20% EtOH. Absorbance at 550 nm in methanol due to the stained adherent cells was measured after the wells were extensively washed with deionized water.Detection of Apoptotic Macrophages by Annexin-V BindingInflammatory peritoneal exudate cells (5 × 106/ml) were cultured in Teflon beakers in the presence of IFN-γ for 6 hours, washed, and then incubated with 1 μg/ml LPS as described above. At various time points, cells were removed and stained with annexin-V-fluorescein isothiocyanate (PharMingen, La Jolla, CA) according to manufacturer's directions. Processed cells were analyzed by flow cytometry on a Becton Dickinson (San Jose, CA) Facscan.Statistical AnalysisComparison of data from gal3+/+ and gal3−/− mice were performed with the statistical software Statview. Data were subjected to the Mann-Whitney U test or analysis of variance with Bonferroni-Dunn post hoc analysis.ResultsHomologous Recombination and Production of gal3−/− MiceThe genomic structure of galectin-3 contains six exons39Gritzmacher CA Mehl VS Liu F-T Genomic cloning of the gene for an IgE-binding lectin reveals unusual utilization of 5′ untranslated regions.Biochemistry. 1992; 31: 9533-9538Crossref PubMed Scopus (35) Google Scholar (Figure 1A), with exons 2 and 3 coding for the amino-terminal region and exons 4–6 coding for the carboxyl-terminal carbohydrate-binding domain. Our strategy for inactivating galectin-3 in mice was to interrupt the region coding for the carbohydrate-binding domain with a neomycin resistance gene. Specifically, a short intron 4-exon 5 segment (0.5 kb) was substituted with the antibiotic-resistance gene (Neo). Figure 1B depicts the structure of the homologous recombinant galectin-3 gene and Figure 1C predicts the restriction fragment profiles of both wild type and homologous recombinants using the specific probes shown in Figure 1B. The genomic Southern blot of two homologous recombinant gal3+/− mouse embryonic stem cell clones is shown in Figure 1D, visualized with probe 1. The upper band in each lane for clones 4A2 and 9A4 were detected with probe 4, but no bands were observed in parent D3 (data not shown).Figure 2A shows the Southern blot analysis of galectin-3 gene from gal3−/−, gal3+/+, and gal3+/− mice. The 2.9-kb intron 3-exon 5 fragment of the galectin-3+/+ gene and the corresponding 4-kb homologous recombinant of the galectin-3−/− are evident. Hemizygous mice are characterized by the presence of both DNA fragments. To demonstrate that the galectin-3 gene was indeed inactivated in gal3−/− mice, several organ extracts were prepared and adsorbed with lactosyl-Sepharose 4B, and the adsorbed proteins were analyzed by immunoblotting. As shown in Figure 2B, although gal3+/+ mice expressed large amounts of galectin-3 in lung, spleen, and thymus tissues, gal3−/− mice were deficient in this protein in these various organs, as expected. Gal3−/− mice are viable and fertile and do not exhibit any overt defects. Various organs from gal3−/− mice were further examined histologically and no apparent alterations were detected. Organs examined included adrenal gland, brain, colon, duodenum, esophagus, gall bladder, heart, hypophysis, kidney, knee joint, liver, lung, lymph nodes, mesentery, ovary, pancreas, salivary glands, skeletal muscle, skin, small intestine, stomach, spleen, testis, thymus, thyroid, and urinary bladder. No significant differences were observed in body weights and weights of major organs, and no differences were found in blood cell counts and chemistry profiles between gal3−/− and gal3+/+ mice. Lymphocyte subpopulations of thymus, spleen, and lymph node were also examined and total numbers of lymphocytes, ratios of CD4+/CD8+ cells, and numbers of CD3+ cells, were comparable between gal3−/− and gal3+/+ mice.Figure 2Confirmation of the inactivated galectin-3 gene in homologous recombinant mice. A: Southern blot analysis of DNA fragments from homozygous (gal3−/− and gal3+/+) and hemizygous (gal3+/−. mice digested with HindIII and XbaI, and analyzed using probe 1, as described in Figure 1D. The positions of DNA markers (kb) are shown at the left margin. B: Immunoblot of organ extracts from gal3+/+ and gal3−/− mice adsorbed with lactosyl-Sepharose and probed with a specific rabbit anti-galectin-3 antiserum. Each lane represents galectin-3 from 100 μg total protein. Lu, lung; Li, liver; S, spleen; T, thymus. The positions of protein Mr standards (× 10−3) are shown at the left margin.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Gal3−/− Mice Exhibit Decreased Levels of Peritoneal InflammationAs an initial approach to assess the effect of galectin-3 deficiency on inflammatory responses, we examined the peritoneal inflammation induced by thioglycollate broth. Untreated (Day 0. gal3−/− mice consistently contained fewer leukocytes in the peritoneal cavities, although the differences were not statistically significant (P = 0.1879). Although both types of mice mounted inflammatory responses to thioglycollate broth treatment, gal3−/− mice clearly exhibited reduced inflammation (Figure 3, Table 1). One day after thioglycollate broth stimulation, the yields of inflammatory leukocytes in the peritoneal cavities of gal3−/− mice were significantly lower (P < 0.05) than that of gal3+/+ mice (Figure 3). Similar trends were observed 3 and 6 days after thioglycollate stimulation (Table 1).Figure 3Reduced peritoneal inflammatory responses are observed in gal3−/− mice when induced with thioglycollate broth. A: Mice matched by sex and age were injected intraperitoneally with 1 ml of thioglycollate broth, and inflammatory cells were harvested at various intervals. Viable leukocytes determined by trypan blue exclusion were enumerated in duplicate from individual mice. Means for each time point are shown with SE bars from 9 and 5 separate experiments, with a total of 22 and 14 mice for each genotype for days 0 (untreated. and 1, respectively. The results from gal3+/+ mice are shown in closed circles and those from gal3−/− mice are shown in open circles. B: Inflammatory peritoneal cells obtained 1 day after