Generation of mice deficient for macrophage galactose- and N-acetylgalactosamine-specific lectin: limited role in lymphoid and erythroid homeostasis and evidence for multiple lectins

Abstract

Macrophage receptors function in pattern recognition for the induction of innate immunity, in cellular communication to mediate the regulation of adaptive immune responses, and in the clearance of some glycosylated cells or glycoproteins from the circulation. They also function in homeostasis by initiating the engulfment of apoptotic cells. Evidence has suggested that macrophage receptors function to recognize cells that are destined for programmed cell death but not yet overtly apoptotic. We have examined the function of a macrophage receptor specific for unsialylated glycoproteins, known as the mouse macrophage galactose- and N-acetylgalactosamine-specific lectin (mMGL) (Ii et al., J. Biol. Chem. 265:11295-11298, 1990; Sato et al., J. Biochem. [Tokyo] 111:331-336, 1992; Yamamoto et al., Biochemistry 33:8159-8166, 1994). With targeted disruption, we tested whether mMGL is necessary for macrophage function, controlled thymic development, the loss of activated CD8 T cells, and the turnover of red blood cells. Evidence indicates that mMGL may play a nonessential role in several of these macrophage functions. Experiments are presented that indicate the existence of another galactose- and N-acetylgalactosamine-recognizing lectin distinct from mMGL. This may explain the absence of a strong phenotype in mMGL-deficient mice.

FIG. 1.

Targeted deletion of mMGL exons 2 and 3 results in mice deficient for mMGL. (A) Genomic mapping of the mMGL amino-terminal cytoplasmic and transmembrane domains revealed a ≈1.0-kb HindIII fragment encoding exons 2 and 3 which was targeted for deletion with replacement vector pPGKneo (23). (B) Tail DNA from F₂ mice digested with HindIII and probed with an upstream genomic probe revealed an endogenous 10.5-kb fragment and the recombinant 12-kb fragment. (C) Northern analysis of mMGL mRNA in mMGL^+/− and mMGL^−/− mice. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. H, HindIII; S, SacI; R, EcoRI; Bx, BstXI; B, BamHI; N, NotI.

FIG. 2.

Localized expression of mMGL in mouse tissues. Tissue sections from mMGL^+/+ and mMGL^−/− mice were stained with rat immunoglobulin (Ig) control or monoclonal anti-mMGL antibodies and compared to Mac-1 staining in each tissue. (A) Cytospin of peritoneal exudate cells. (B) Thymus. Note that cells staining for mMGL in wild-type (WT) mice were seen predominantly in the corticomedullary junction and medulla. (C) Spleen, showing sparse staining of mMGL staining compared to that in skin (D) , lymph node (E) , and lung (F) .

FIG. 2.

Localized expression of mMGL in mouse tissues. Tissue sections from mMGL^+/+ and mMGL^−/− mice were stained with rat immunoglobulin (Ig) control or monoclonal anti-mMGL antibodies and compared to Mac-1 staining in each tissue. (A) Cytospin of peritoneal exudate cells. (B) Thymus. Note that cells staining for mMGL in wild-type (WT) mice were seen predominantly in the corticomedullary junction and medulla. (C) Spleen, showing sparse staining of mMGL staining compared to that in skin (D) , lymph node (E) , and lung (F) .

FIG. 3.

mMGL null mice show normal T-cell development and homeostasis. (A) Thymocytes were harvested from C57BL/6 mice and stained with anti-CD4-PE, anti-CD8-TC, and either PNA directly conjugated to FITC (top) or biotinylated E. cristigalli agglutinin (ECA) and streptavidin-FITC (bottom). Cells were gated on PNA^hi (left) or PNA^lo (right) to indicate populations with terminal O-linked Galβ1-3GalNAc or ECA^hi (left) or ECA^lo (right) to indicate terminal N-linked Galβ1-4GlcNAc. (B) Thymocytes from mMGL^+/− and mMGL^−/− littermates were stained with anti-CD4 and anti-CD8 and analyzed by flow cytometry. (C) CD4SP (top) and CD8SP (bottom) populations were analyzed for PNA binding between mMGL^+/+ and mMGL^−/− mice. (D) Thymocytes were cocultured for 16 h with mMGL- or mock-transfected 3T6 fibroblasts, and the live cell recovery of different subsets was determined by multiplying cell count by percentage of annexin V staining on gated populations. (E) Thymocytes from C57BL/6 mice were harvested and cocultured with thioglycollate-elicited macrophages from mMGL^+/+ and mMGL^−/− mice in vitro.

FIG. 3.

mMGL null mice show normal T-cell development and homeostasis. (A) Thymocytes were harvested from C57BL/6 mice and stained with anti-CD4-PE, anti-CD8-TC, and either PNA directly conjugated to FITC (top) or biotinylated E. cristigalli agglutinin (ECA) and streptavidin-FITC (bottom). Cells were gated on PNA^hi (left) or PNA^lo (right) to indicate populations with terminal O-linked Galβ1-3GalNAc or ECA^hi (left) or ECA^lo (right) to indicate terminal N-linked Galβ1-4GlcNAc. (B) Thymocytes from mMGL^+/− and mMGL^−/− littermates were stained with anti-CD4 and anti-CD8 and analyzed by flow cytometry. (C) CD4SP (top) and CD8SP (bottom) populations were analyzed for PNA binding between mMGL^+/+ and mMGL^−/− mice. (D) Thymocytes were cocultured for 16 h with mMGL- or mock-transfected 3T6 fibroblasts, and the live cell recovery of different subsets was determined by multiplying cell count by percentage of annexin V staining on gated populations. (E) Thymocytes from C57BL/6 mice were harvested and cocultured with thioglycollate-elicited macrophages from mMGL^+/+ and mMGL^−/− mice in vitro.

FIG. 4.

Allogeneic cytotoxic T-cell response in mMGL^+/+ and mMGL^−/− mice. (A) P815 targets were cultured with lymphocytes from the spleen, and CTL lysis was determined. mMGL-deficient mice showed cytolytic activity similar to that in the wild type. (B) Expansion of CD8 T cells after the in vivo response was similar between mMGL^+/+ and mMGL^−/− mice. (C) Percentage of annexin V⁺ CD8 T cells was similar between mMGL^+/+ and mMGL^−/− mice.

FIG. 5.

Erythrocyte (RBC) turnover in mMGL^+/+ and mMGL^−/− mice. Mean life span of red blood cells was determined in (A) mMGL^+/+ and (B) mMGL^−/− mice after biotinylation in vivo. The mean erythrocyte life span was 36.67 days in mMGL^+/+ mice, versus 37.27 days in mMGL^−/− mice.

FIG. 6.

Effect of mMGL deficiency in ST3Gal-I-deficient mice. ST3Gal-I mice were crossed to mMGL-deficient mice to examine the role of mMGL in the CD8 T-cell loss in ST3Gal-I-deficient mice. Solid squares represent ST3Gal-I^+/− mice, which show normal CD8 T-cell numbers in the peripheral blood lymphocytes (PBL). Solid circles represent ST3Gal-I^−/− mMGL^+/− mice, which show decreased numbers of CD8 T cells. Open circles show doubly deficient mice. (Top) Female doubly deficient mice show a trend of increased CD8 T-cell numbers compared to ST3Gal-I-deficient mice, reaching statistical significance in female mice aged 3 and 5 weeks. (Bottom) Male mice showed no statistically significant changes.

FIG. 7.

Identification of Gal/GalNAc-specific lectin(s) distinct from mMGL. (A) Sialic acid (SA) binding alone. (B) Galβ1-3GalNAc-PAA. (C to E) Serial sections were probed with (C) Galβ1-3GalNAc-PAA, (D) Galβ1-3GalNAc-PAA plus Galβ1-3GalNAc, and (E) Galα1-3GalNAc plus Galα1-3GalNAc.

FIG. 8.

Gal/GalNAc-specific lectin(s) is macrophage restricted. (A) Splenic sections from mMGL^−/− mice were probed with biotinylated Galβ1-3GalNAc-PAA or anti-Mac-1 antibodies. Localized binding of the Galβ1-3GalNAc-PAA probe was observed in the splenic red pulp, and this correlated with Mac-1 staining. (B) Double staining of splenic sections from mMGL-deficient mice with Galβ1-3GalNAc-PAA and Mac-1 revealed colocalization of the lectin to a Mac-1⁺ cell. Galβ1-3GalNAc-PAA binding was always seen on Mac-1 binding cells but not the converse, suggesting that the lectin(s) is expressed on a subpopulation of macrophages or mature dendritic cells.