Mice lacking tartrate-resistant acid phosphatase (Acp 5) have disordered macrophage inflammatory responses and reduced clearance of the pathogen, Staphylococcus aureus

Abstract

Tartrate-resistant acid phosphatase (TRAP) is a lysosomal di-iron protein of mononuclear phagocytes and osteoclasts. Hitherto, no role for the enzyme in immunity has been identified; however, knockout mice lacking TRAP have a skeletal phenotype caused by an intrinsic osteoclast defect. To investigate a putative function for TRAP in macrophages (Mphi), we investigated proinflammatory responses and systemic microbial clearance in knockout mice compared with age- and gender-matched congenic wild-type mice. Phorbol 12-myristate 13-acetate (PMA)-stimulated and interferon-gamma (IFN-gamma)-induced superoxide formation was enhanced in peritoneal Mphi lacking TRAP; nitrite production in response to stimulation with lipopolysaccharide (LPS) and IFN-gamma was also increased. In addition, secretion of the proinflammatory cytokines, tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta and IL-12, was significantly greater in TRAP-deficient Mphi when stimulated with LPS, with or without addition of either TNF-alpha or IFN-gamma. The activity of tartrate-sensitive (lysosomal) acid phosphatase was increased in Mphi from the knockout mice but activities of the lysosomal hydrolases N-acetyl beta-glucosaminidase and acid beta-glucuronidase were unchanged, indicating selective activation of compensatory acid phosphatase activity. Evidence of impaired Mphi function in vivo was obtained in TRAP knockout mice, which showed delayed clearance of the microbial pathogen, Staphylococcus aureus, after sublethal intraperitoneal inoculation. After microbial challenge, peritoneal exudates obtained from TRAP knockout mice had a reduced population of Mphi. As peritoneal Mphi and neutrophils lacking TRAP were able to phagocytose and kill S. aureus normally in vitro, TRAP may directly or indirectly influence recruitment of Mphi to sites of microbial invasion. Our study shows that TRAP participates in the inflammatory response of the Mphi and influences effector signalling pathways in innate immunity.

Figure 1

Immunophenotyping of murine macrophage (Mφ). F4/80-positive peritoneal Mφ (a) from wild-type mice express abundant intracellular tartrate-resistant acid phosphatase (TRAP) protein when incubated with lipopolysaccharide (LPS) for 48 hr (b). As expected, Mφ isolated from TRAP-deficient mice do not express TRAP under these conditions (c). Bone marrow Mφ behaved identically (results not shown). Peritoneal Mφ were stimulated in chamber slides and fixed in acetone/methanol (1 : 1), as described in the Materials and Methods. Cells were incubated with immunopurified uteroferrin antibody for 12 hr and intracellular TRAP antigen was visualized using a peroxidase-conjugated secondary antibody and developed with diaminobenzidine. Cells were counterstained with Harris hematoxylin.

Figure 2

Macrophage (Mφ) ultrastructure and staining reactions for acid phosphatase activities in situ. Resting peritoneal Mφ from wild-type (a) and tartrate-resistant acid phosphatase (TRAP)-deficient mice (b) had similar morphologies. Lipopolysaccharide (LPS)-stimulated wild-type Mφ showed abundant TRAP activity, as exemplified by arrows (c). The acid phosphatase activity in wild-type Mφ was principally caused by TRAP as in the absence of tartrate no additional Gomori product was detectable (e). Mφ from TRAP-deficient mice had no TRAP activity (d) but showed increased reactions with the Gomori stain (arrowed) in the absence of tartrate, which represents the tartrate-sensitive lysosomal acid phosphatase isoenzyme (f).

Figure 3

Nitrite production is increased in stimulated macrophages (Mφ) from tartrate-resistant acid phosphatase (TRAP)-deficient mice. Mφ obtained from peritoneum (a) and bone marrow (b) were stimulated with lipopolysaccharide (LPS) (1 µg/ml), with or without interferon-γ (IFN-γ) (100 U/ml) or tumour necrosis factor-α (TNF-α) (500 U/ml). Nitrite was determined in cell supernatants by the Griess reaction, as described in the Materials and Methods. The nitric oxide synthase inhibitor, N-monomethyl-l-arginine (l-NMMA) (300 µm), was added to ensure that the nitrite measured was derived from nitric oxide synthase. The data are representative of at least four experiments and expressed as mean ± SEM values. *P < 0·05.

Figure 4

Tartrate-resistant acid phosphatase (TRAP)-deficient macrophages (Mφ) have an abnormal proinflammatory cytokine secretion profile. Peritoneal Mφ (a), (c) and (e), and bone marrow Mφ (b), (d) and (f), from age- and gender-matched mice, were stimulated with lipopolysaccharide (LPS) (1 µg/ml), interferon-γ (IFN-γ) (100 U/ml) or tumour necrosis factor-α (TNF-α) (500 U/ml), alone or in combination, for 48 hr. Supernatants were assayed for interleukin (IL)-12 (a) and (b), IL-1β (c) and (d), and TNF-α (e) and (f). The data shown are representative of two experiments with peritoneal Mφ (using five to 11 determinations for each condition) and two experiments with bone marrow Mφ (three to six determinations for each condition). Results are expressed as mean ± SEM. *P < 0·05.

Figure 5

Reduced peritoneal clearance of viable Staphylococcus aureus in tartrate-resistant acid phosphatase (TRAP)-deficient mice. Wild-type and TRAP-deficient mice (n = 8–11 at each time-point) were innoculated intraperitoneally with 2 × 10⁷ cells of the S. aureus, pathogenic strain, AKC 25923. Viable bacterial counts were quantified by determining colony-forming units (CFU) on the peritoneal lavage obtained at each time-point, as described in the Materials and Methods. The figure depicts logarithmically transformed mean± SEM counts/ml of lavage fluid. The two clearance curves were shown to be different by analysis of variance, with a significance value of P < 0·001.

Figure 6

Reduced monocyte/macrophage (Mφ) complement in peritoneal exudates from tartrate-resistant acid phosphatase (TRAP)-deficient mice after challenge with Staphylococcus aureus in vivo. Peritoneal exudates were obtained as described in the legend to Fig. 5 (and the Materials and Methods). Cells were analysed for their side- and forward-scatter properties (SSC and FSC, respectively), and gated accordingly. These gatings were confirmed by fluorescence staining with F7/4 and F4/80 antibodies for neutrophils and monocyte/Mφ, respectively. Cells not staining for these surface markers and with appropriate forward- and side-scatter parameters were categorized as lymphocytes. (a) Representative dot-plots of exudate cells 72 hr after infection. (b) Cell subset analysis at 72 hr showing data from three to four mice of each genotype, determined in duplicate and expressed as a percentage of live cells ± SEM. *P < 0·05.