Increased susceptibility of thymocytes to apoptosis in mice lacking AIM, a novel murine macrophage-derived soluble factor belonging to the scavenger receptor cysteine-rich domain superfamily

Abstract

Apoptosis of cells must be regulated both positively and negatively in response to a variety of stimuli in the body. Various environmental stresses are known to initiate apoptosis via differential signal transduction cascades. However, induction of signals that may inhibit apoptosis is poorly understood, although a number of intracellular molecules that mediate inhibition of apoptosis have been identified. Here we present a novel murine macrophage-specific 54-kD secreted protein which inhibits apoptosis (termed AIM, for apoptosis inhibitor expressed by macrophages). AIM belongs to the macrophage scavenger receptor cysteine-rich domain superfamily (SRCR-SF), members of which share a highly homologous conserved cysteine-rich domain. In AIM-deficient mice, the thymocyte numbers were diminished to half those in wild-type mice, and CD4/CD8 double-positive (DP) thymocytes were strikingly more susceptible to apoptosis induced by both dexamethasone and irradiation in vivo. Recombinant AIM protein significantly inhibited cell death of DP thymocytes in response to a variety of stimuli in vitro. These results indicate that in the thymus, AIM functions in trans to induce resistance to apoptosis within DP cells, and thus supports the viability of DP thymocytes before thymic selection.

Figure 1

(a) Amino acid sequence of mouse (Balb/c strain) AIM. Dash-underlined sequence corresponds to the potential leader sequence. N-glycosylation sites (three sites) are shaded. Three SRCR domains are indicated by boxes and denoted by SRCR-1, -2, and -3. ▾, exon–intron junctions. The aspartic acid at position 197 (circled) is serine in the 129/sv strain, based on the nucleotide substitution of adenine in the Balb/c strain to guanine in the 129/sv mouse strain. The cDNA sequence of AIM is available from EMBL/GenBank/DDBJ under accession no. AF011428. (b) Peritoneal macrophages were lysed in 0.5% Triton-X buffer, and AIM protein was immunoprecipitated. Immunoprecipitating Ab was anti-AIM polyclonal rabbit Ab, which was generated by immunizing rAIM into the animal. Precipitated protein (right) as well as conditioned medium (5 μl) from either AIM-transfected CHO cells (left) or nontransfected CHO cells (middle) were size-fractionated on 10% SDS-PAGE, blotted on a membrane, and stained by anti-AIM mAb (clone 3G14). Signals were developed by the ECL system (Amersham Pharmacia Biotech).

Figure 1

(a) Amino acid sequence of mouse (Balb/c strain) AIM. Dash-underlined sequence corresponds to the potential leader sequence. N-glycosylation sites (three sites) are shaded. Three SRCR domains are indicated by boxes and denoted by SRCR-1, -2, and -3. ▾, exon–intron junctions. The aspartic acid at position 197 (circled) is serine in the 129/sv strain, based on the nucleotide substitution of adenine in the Balb/c strain to guanine in the 129/sv mouse strain. The cDNA sequence of AIM is available from EMBL/GenBank/DDBJ under accession no. AF011428. (b) Peritoneal macrophages were lysed in 0.5% Triton-X buffer, and AIM protein was immunoprecipitated. Immunoprecipitating Ab was anti-AIM polyclonal rabbit Ab, which was generated by immunizing rAIM into the animal. Precipitated protein (right) as well as conditioned medium (5 μl) from either AIM-transfected CHO cells (left) or nontransfected CHO cells (middle) were size-fractionated on 10% SDS-PAGE, blotted on a membrane, and stained by anti-AIM mAb (clone 3G14). Signals were developed by the ECL system (Amersham Pharmacia Biotech).

Figure 2

(a) Tissue distribution of AIM gene expression. 10 μg of total RNA from various mouse (B6) tissues and from adherent PECs induced by thioglycollate from RAG-2^−/− mouse was hybridized with either AIM cDNA or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA fragment. Weak background signals derived from 18S rRNA, which is slightly shorter than AIM rRNA, can be detected in every tissue. In this experiment, adherent PECs were prepared from RAG-2^−/− mice to avoid any contamination of T and B (B-1 and B-2) cells. However, almost comparable expression was detected in the adherent PECs from wild-type B6 mice (data not shown). (b–i) In situ hybridization analysis of AIM expression (violet/dark blue signals in b, d, f, and h) and its comparison with immunolocalization of pan-macrophage antigen, F4/80 (dark brown signals in c, e, g, and i), in mouse granulomas in the liver (b and c), thymus (d and e), spleen (f and g), and liver (h and i). (b and c) AIM is expressed in a subset of macrophages in the peripheral areas of granulomas (gp) but not in the central areas (gc). g, granulomas; lv, liver tissue. (d and e) AIM is expressed by a subpopulation of cortical macrophages as indicated by arrows (d), although macrophages exist in both cortex and medulla (e). c, cortex; m, medulla. (f and g) AIM is expressed predominantly by a subset of macrophages in the marginal zones. r, red pulp; w, white pulp; mz, marginal zone. (h and i) AIM is expressed by a subpopulation of Kupffer/macrophage cells. Original magnifications: b, ×400; c, e, g, h, and i, ×100; d and f, ×200. (j) No AIM expression was detected in cultured macrophages. Adherent PECs were prepared from intraperitoneal thioglycollate-injected RAG-2^−/− mice. A portion of the cells were cultured on plastic dishes for 16 h in complete DMEM containing 10% FCS. Total RNA was prepared from freshly isolated and cultured macrophages, and hybridized with either AIM or GAPDH cDNA fragment.

Figure 2

(a) Tissue distribution of AIM gene expression. 10 μg of total RNA from various mouse (B6) tissues and from adherent PECs induced by thioglycollate from RAG-2^−/− mouse was hybridized with either AIM cDNA or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA fragment. Weak background signals derived from 18S rRNA, which is slightly shorter than AIM rRNA, can be detected in every tissue. In this experiment, adherent PECs were prepared from RAG-2^−/− mice to avoid any contamination of T and B (B-1 and B-2) cells. However, almost comparable expression was detected in the adherent PECs from wild-type B6 mice (data not shown). (b–i) In situ hybridization analysis of AIM expression (violet/dark blue signals in b, d, f, and h) and its comparison with immunolocalization of pan-macrophage antigen, F4/80 (dark brown signals in c, e, g, and i), in mouse granulomas in the liver (b and c), thymus (d and e), spleen (f and g), and liver (h and i). (b and c) AIM is expressed in a subset of macrophages in the peripheral areas of granulomas (gp) but not in the central areas (gc). g, granulomas; lv, liver tissue. (d and e) AIM is expressed by a subpopulation of cortical macrophages as indicated by arrows (d), although macrophages exist in both cortex and medulla (e). c, cortex; m, medulla. (f and g) AIM is expressed predominantly by a subset of macrophages in the marginal zones. r, red pulp; w, white pulp; mz, marginal zone. (h and i) AIM is expressed by a subpopulation of Kupffer/macrophage cells. Original magnifications: b, ×400; c, e, g, h, and i, ×100; d and f, ×200. (j) No AIM expression was detected in cultured macrophages. Adherent PECs were prepared from intraperitoneal thioglycollate-injected RAG-2^−/− mice. A portion of the cells were cultured on plastic dishes for 16 h in complete DMEM containing 10% FCS. Total RNA was prepared from freshly isolated and cultured macrophages, and hybridized with either AIM or GAPDH cDNA fragment.

Figure 2

(a) Tissue distribution of AIM gene expression. 10 μg of total RNA from various mouse (B6) tissues and from adherent PECs induced by thioglycollate from RAG-2^−/− mouse was hybridized with either AIM cDNA or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA fragment. Weak background signals derived from 18S rRNA, which is slightly shorter than AIM rRNA, can be detected in every tissue. In this experiment, adherent PECs were prepared from RAG-2^−/− mice to avoid any contamination of T and B (B-1 and B-2) cells. However, almost comparable expression was detected in the adherent PECs from wild-type B6 mice (data not shown). (b–i) In situ hybridization analysis of AIM expression (violet/dark blue signals in b, d, f, and h) and its comparison with immunolocalization of pan-macrophage antigen, F4/80 (dark brown signals in c, e, g, and i), in mouse granulomas in the liver (b and c), thymus (d and e), spleen (f and g), and liver (h and i). (b and c) AIM is expressed in a subset of macrophages in the peripheral areas of granulomas (gp) but not in the central areas (gc). g, granulomas; lv, liver tissue. (d and e) AIM is expressed by a subpopulation of cortical macrophages as indicated by arrows (d), although macrophages exist in both cortex and medulla (e). c, cortex; m, medulla. (f and g) AIM is expressed predominantly by a subset of macrophages in the marginal zones. r, red pulp; w, white pulp; mz, marginal zone. (h and i) AIM is expressed by a subpopulation of Kupffer/macrophage cells. Original magnifications: b, ×400; c, e, g, h, and i, ×100; d and f, ×200. (j) No AIM expression was detected in cultured macrophages. Adherent PECs were prepared from intraperitoneal thioglycollate-injected RAG-2^−/− mice. A portion of the cells were cultured on plastic dishes for 16 h in complete DMEM containing 10% FCS. Total RNA was prepared from freshly isolated and cultured macrophages, and hybridized with either AIM or GAPDH cDNA fragment.

Figure 3

(a) Knockout strategy. Restriction maps are shown for the wild-type AIM gene locus (top), targeting vector (middle), and recombinant (bottom) gene locus. Exons, white boxes. neo^r, neomycin resistance gene (black box). pBluescript vector sequence, dashed line. Restriction sites: B, BamHI; N, NcoI; S, SpeI; Xb, XbaI; Xh, XhoI. Probe DNA fragment for Southern blotting is indicated, as are the 2.1- and 3.5-kb NcoI-XbaI hybridizable fragments in wild-type and mutant DNA, respectively. (b) Thymocyte suspensions from AIM^−/− (−/−) and AIM^+/+ (+/+) mice were stained for CD3 and then analyzed by flow cytometry. Histograms of CD3 expression of total thymocytes are displayed. Relative percentage of CD3^high population (which corresponds to mature SP thymocytes) in each type of mice is represented. (c) Apoptosis was induced in vivo in AIM^+/+ (+/+, white boxes) and AIM^−/− (−/−, black boxes) mice by (c) injection of 0.1 or 0.2 mg i.p. of dexamethasone in PBS, or PBS alone, or (d) 0, 2, or 4 Gy of irradiation (¹³⁷Cs). 48 h after stimulation, mice were killed and the number of surviving DP thymocytes was determined by trypan blue exclusion and CD4/CD8 staining. Values represent the average viability of DP cells from two or three mice of each genotype and are normalized to the percentage of viable cells remaining in nonstimulated (PBS-injected, or nonirradiated) mice. Bars, SD. Three sets of experiments were performed for each stimulation, and similar results were obtained.

Figure 3

(a) Knockout strategy. Restriction maps are shown for the wild-type AIM gene locus (top), targeting vector (middle), and recombinant (bottom) gene locus. Exons, white boxes. neo^r, neomycin resistance gene (black box). pBluescript vector sequence, dashed line. Restriction sites: B, BamHI; N, NcoI; S, SpeI; Xb, XbaI; Xh, XhoI. Probe DNA fragment for Southern blotting is indicated, as are the 2.1- and 3.5-kb NcoI-XbaI hybridizable fragments in wild-type and mutant DNA, respectively. (b) Thymocyte suspensions from AIM^−/− (−/−) and AIM^+/+ (+/+) mice were stained for CD3 and then analyzed by flow cytometry. Histograms of CD3 expression of total thymocytes are displayed. Relative percentage of CD3^high population (which corresponds to mature SP thymocytes) in each type of mice is represented. (c) Apoptosis was induced in vivo in AIM^+/+ (+/+, white boxes) and AIM^−/− (−/−, black boxes) mice by (c) injection of 0.1 or 0.2 mg i.p. of dexamethasone in PBS, or PBS alone, or (d) 0, 2, or 4 Gy of irradiation (¹³⁷Cs). 48 h after stimulation, mice were killed and the number of surviving DP thymocytes was determined by trypan blue exclusion and CD4/CD8 staining. Values represent the average viability of DP cells from two or three mice of each genotype and are normalized to the percentage of viable cells remaining in nonstimulated (PBS-injected, or nonirradiated) mice. Bars, SD. Three sets of experiments were performed for each stimulation, and similar results were obtained.

Figure 4

(a) Freshly isolated thymocytes (10⁶/well) from AIM^−/− mice (adherent cells, including macrophages, were depleted by cell incubation on culture plates for 30 min) were incubated with 5 or 10 nM dexamethasone in the presence of conditioned medium of AIM transfectants at indicated concentrations of rAIM (final) for 20 h. To normalize any nonspecific effect potentially caused by conditioned medium, the amount of conditioned medium was equalized by adding conditioned medium from nontransfected CHO cells into wells which contained low or no amounts of conditioned medium of AIM transfectants. (b) Thymocytes were 50 or 100 cGy irradiated and then incubated in the presence of various concentrations of rAIM as above. Surviving DP cells were calculated by trypan blue exclusion and CD4/CD8 staining. Values represent the average viability of DP cells from two independent wells and are normalized to the percentage of the DP cell numbers before culture. Bars, SD. Two sets of experiments were performed for each stimulation, and similar results were obtained.

Figure 5

(a) AIM binding capacity of J774A.1 cells is presented as log fluorescence by black histograms, and the control protein bindings that are regarded as backgrounds are represented by white histograms. (b) CD95/Fas expression on the surface of J774A.1 cells. Cells were incubated with 50 μg/ml of mouse IgG for 15 min on ice to block nonspecific binding of Abs via Fc receptors. Cells were washed once, stained with FITC-conjugated anti-CD95/Fas (clone Jo2), and then analyzed by flow cytometry. White histogram, unstained cells as a control. (c) J774A.1 cells (10⁴/well) were cultured with various concentrations of hamster anti-CD95/Fas mAb plus rabbit mAbs cocktail anti–hamster IgG and 0.1 μg/ml of cycloheximide, in the presence (∼1 ng/ml; white boxes, +) or absence (black boxes, −) of rAIM. After 16 h of culture, [³H]thymidine (1 μCi/well) was pulsed, and cells were further incubated for 4 h and then harvested. Values represent the average [³H]thymidine incorporation from three independent wells and are normalized to the percentage of the incorporation observed without Abs. Bars, SD. Three sets of experiments were performed for each stimulation, and similar results were obtained. (d) J774A.1 cells (10⁴/well) were cultured for 16 h in the presence or absence of rAIM, but without anti-CD95 mAb and cycloheximide. Cells were pulsed with [³H]thymidine (1 μCi/well), further incubated for 4 h, and then harvested. Values represent the average [³H]thymidine incorporation from three independent wells. Bars, SD.