Targeted disruption of the galectin-3 gene results in attenuated peritoneal inflammatory responses

Abstract

Galectin-3 is a member of a growing family of beta-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-kappaB 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.

Figure 1.

Vector construction and homologous recombination in the galectin-3 gene. A: The targeting vector was constructed by replacing the 0.5-kb XbaI-XbaI segment at the intron 4-exon 5 junction with the Neo cassette from the pMC1NeoPLA vector. B: The homologous recombinant galectin-3 gene shown with various DNA segments used as probes. C: Predicted DNA sizes from wild-type and homologous recombinant genes in Southern blotting using HindIII and XbaI and the specific flanking probe 1. D: Southern blot of two homologous recombinant clones after digestion with HindIII (H) and XbaI (X) and detection with probe 1. The upper band in both lanes for clones 4A2 and 9A4 were detected using probe 4, specific for the Neo gene (data not shown). The profile for the wild-type parental ES cell D3 is also shown.

Figure 2.

Confirmation 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.

Figure 3.

Reduced 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 thioglycollate broth treatment were cytospun onto glass slides and stained with soluble Wright’s stain for differential identification of leukocytes by standard morphologies. Between 100 and 200 total cells were enumerated from random fields in triplicate using a 100× oil immersion objective. Comparisons between genotypes were tested for significance using the Mann-Whitney U test; * indicates significant differences where P < 0.05. Data are means with SE bars from 4 experiments of a total of 11 mice for each genotype.

Figure 4.

Peritoneal inflammatory cells of gal3^−/− mice exhibit a reduced NF-κB response. Nuclear extracts were obtained from peritoneal inflammatory cells and the κB binding activity was determined by EMSA. A: Peritoneal cells from gal3^−/− and gal3^+/+ mice were obtained 2 days after thioglycollate broth treatment. Each lane represents results from a single mouse. Similar results were obtained from three separate experiments. B: Peritoneal cells from untreated or thioglycollate-treated mice (1 or 2 days after the treatment) were obtained by lavage and cells from three mice for each group were pooled. They were incubated in RPMI (5 × 10 cells/ml) at 37°C in the absence or presence of recombinant TNF-α (40 ng/ml) for 40 minutes. The arrowheads mark the positions of DNA-protein complexes specific for NF-κB.

Figure 5.

Cell areas of adherent inflammatory peritoneal macrophages from gal3^−/− mice are different from macrophages of gal3^+/+ mice. A: Representative phase photomicrographs of macrophages obtained from culturing initially nonadherent cells from peritoneal cavities of thioglycollate-treated gal3^+/+ and gal3^−/− mice. B: Mean cell areas of adherent macrophages. Cell areas of adherent cells were measured from digitized files obtained from three random fields of culture wells. Data are means from three separate experiments and are expressed in an arbitrary unit. Over 400 individual cells from each genotype were measured; a P value < 0.0001 was obtained by Mann-Whitney U comparison.

Figure 6.

Peritoneal macrophages from gal3^−/− mice are more sensitive to apoptotic stimuli than cells from gal3^+/+ mice. A: Inflammatory macrophages obtained from peritoneal cavities of mice 3 days after treatment with 1 ml thioglycollate were cultured as adherent cells with 10 μg/ml LPS and 10 U/ml IFN-γ for the indicated periods. Surviving cells were measured by the MTT assay. Data are shown from one representative experiment as means ± SE. Similar results were obtained from five separate experiments, including experiments in which macrophages elicited with 2 ml thioglycollate broth for 4 days were cultured with various amounts of IFN-γ for 6 hours followed by 1 μg/ml LPS (data not shown). A significant difference between genotypes (P < 0.0001) was obtained by analysis of variance at 5% significance with Bonferroni-Dunn post hoc analysis. B: Cells were cultured in Teflon beakers at 5 × 10 cells/ml and treated with 1.7 U/ml IFN-γ for 6 hours, washed, and then activated with 1 μg/ml LPS for 24 hours. The cells were stained with annexin-V-fluorescein isothiocyanate and analyzed by flow cytometry. Percentages of cells positively stained, gated within region R2, were determined, as shown in representative histograms.