Inactivation of the F4/80 glycoprotein in the mouse germ line

Abstract

Macrophages play a crucial role in the defense against pathogens. Distinct macrophage populations can be defined by the expression of restricted cell surface proteins. Resident tissue macrophages, encompassing Kupffer cells of the liver and red pulp macrophages of the spleen, characteristically express the F4/80 molecule, a cell surface glycoprotein related to the seven transmembrane-spanning family of hormone receptors. In this study, gene targeting was used to simultaneously inactivate the F4/80 molecule in the germ line of the mouse and to produce a mouse line that expresses the Cre recombinase under the direct control of the F4/80 promoter (F4/80-Cre knock-in). F4/80-deficient mice are healthy and fertile. Macrophage populations in tissues can develop in the absence of F4/80 expression. Functional analysis revealed that the generation of T-cell-independent B-cell responses and macrophage antimicrobial defense after infection with Listeria monocytogenes are not impaired in the absence of F4/80. Interestingly, tissues of F4/80-deficient mice could not be labeled with anti-BM8, another macrophage subset-specific marker with hitherto undefined molecular antigenic structure. Recombinant expression of a F4/80 cDNA in heterologous cells confirmed this observation, indicating that the targets recognized by the F4/80 and BM8 monoclonal antibodies are identical.

FIG. 1.

Generation of F4/80-deficient mice. (A) Targeting strategy used for inactivation of the murine F4/80 gene. EI, EcoRI; EV, EcoRV; NI, NcoI; SI, StuI; KI, KpnI. Parts of the first coding exon were replaced by insertion of a Cre recombinase cDNA cassette at the ATG initiation codon and a neomycin resistance gene cassette. (B) Southern blot analysis of genomic DNA from targeted ES cell clones. EcoRI digest of genomic DNA from E14.1 wild-type and targeted ES cells. Hybridization with the flanking probe yields a 5.9-kb fragment for the wild-type allele and a 3.6-kb fragment for the targeted allele. Sizes are indicated at the left in kilobases (kb). (C) Southern blot analysis of genomic DNA from mouse tail biopsies and, for comparison, from one targeted ES cell clone. EcoRI digest of genomic DNA from E14.1 wild-type and targeted ES cells and mouse tails. For fragment sizes see panel B. (D) Northern blot analysis verifying the lack of F4/80 RNA in mice homozygous for the targeted F4/80 allele. Total RNA from F4/80^+/+, F4/80^+/Cre, and F4/80^Cre/Cre (i.e., F4/80^−/−) livers was hybridized with a full-length F4/80 cDNA and a GAPDH (glyceraldehyde-3-phosphate dehydrogenase) cDNA (loading control).

FIG. 2.

Absence of F4/80 and BM8 antigen in organs of F4/80-deficient mice. Immunohistochemical staining of liver and spleen sections from control mice (top panels) and F4/80-deficient mice (lower panels) is shown. Cryosections were labeled with MAbs as indicated. Original magnification, ×100.

FIG. 3.

The antigen detected by F4/80 and BM8 antibodies is identical. 293T cells were transiently transfected with a pcDNA3-based expression vector containing the complete coding sequence of F4/80. Cells were labeled with the respective MAbs as indicated.

FIG. 4.

Cre-mediated recombination in F4/80^+/Cre mice crossed with IκBα^+/2loxP mice. (A) Schematic structure of the wild-type and floxed murine ikba locus; exons (red), loxP motifs (blue triangle), and PCR primers (number and black triangle, see Materials and Methods) are indicated. (B) Spleen, livers, and kidneys from (F4/80^+/Cre × IκBα^+/2loxP)F₁ (upper panel) and (F4/80^+/+ × IkBα^+/2loxP)F₁ (middle panel) mice wereanalyzed for Cre recombinase activity. In DNA isolated from spleen, liver, and kidney tissues of (F4/80^+/Cre × IκBα^+/2loxP)F₁ mice, a PCR product that is characteristic for the Cre-mediated deletion at the ikba locus was amplified, whereas in DNA isolated from spleen, liver, and kidney tissues of (F4/80^+/+ × IκBα^+/2loxP)F₁ mice only the germ line configuration of the IkBα alleles can be detected. Results of control PCRs using template DNA derived from wild-type ES cells or tail DNA from F4/80^+/+ IκBα^+/1loxP and F4/80^+/+ IκBα^+/2loxP mice are shown in the lower panel as indicated.