Tumor Suppressor p53 Inhibits Autoimmune Inflammation and Macrophage Function

Breeding pairs of C57BL/6J (B6) p53^−/+ mice (34) were purchased from The Jackson Laboratory (Bar Harbor, ME). These mice were used to generate p53^+/+ and p53^−/− mice. The p53 gene mutation was tracked by PCR. All mice were housed in the University of Pennsylvania animal care facilities under pathogen-free conditions, and all procedures were approved by the animal care and use committee.

STZ was purchased from Sigma (St. Louis, MO). The following reagents were purchased from BD Pharmingen (San Diego, CA): purified rat anti-mouse IL-1β, IL-6, IL-12, tumor necrosis factor-α (TNF-α), and γ-interferon (IFN-γ) monoclonal antibodies; recombinant mouse IL-1β, IL-6, IL-12, TNF-α, and IFN-γ. Cytokines in the culture supernatant, serum, and organ extracts were measured by quantitative enzyme-linked immunosorbent assay per the manufacturer’s recommendations.

Autoimmune diabetes was induced using multiple low doses of STZ as previously described (35). Briefly, 7- to 9-week-old male mice were injected intraperitoneally once a day for 5 consecutive days with 40 mg/kg body wt STZ dissolved in 25 mmol/l citrate buffer, pH 4.5. Mice were tested once every other day for urinary glucose levels using a Keto-Diastix kit (Bayer, Elkhart, IN). Mice were considered diabetic if the urinary glucose levels were ≥500 mg/dl on two consecutive tests. To determine the degree of insulitis, pancreata were fixed in 10% formalin, sectioned, stained with hematoxylin/eosin, and examined by microscopy.

To prepare macrophages from bone marrow, femurs and tibiae were surgically removed and the marrow flushed with Dulbecco’s modified Eagle’s medium (DMEM). After removing erythrocytes, bone marrow cells were cultured in 30% L-cell–conditioned medium at 10⁶/ml for 7–8 days. Adherent cells were collected and cultured in DMEM containing macrophage colony-stimulating factor (R&D Systems) for 24 h. In some experiments, macrophages were collected directly from peritoneum and cultured at 10⁶/ml, with or without 10 units/ml of IFN-γ and 200 pg/ml of lipopolysaccharide, for different times.

Spleen and pancreas were aseptically removed from the mice and homogenized in RPMI-1640 containing 1% CHAPS(3-[(3-cholamidopropyl)dimethyammonio]-1-propanesulfonate; Calbiochem,La Jolla, CA) at a ratio of 1:10 (wt/vol) using a Dounce grinder. The homogenates were centrifuged at 2000g for 20 min, and the supernatant was collected and stored at −80°C before the cytokine assay.

Bone marrow–derived macrophages were cultured in DMEM with or without 10 ng/ml of recombinant mouse IFN-γ for 4 h followed by an overnight incubation with or without 1 μg/ml of lipopolysaccharide in the presence of 2 μmol/l of GolgiStop (BD PharMingen). Cells were stained with anti–IL-12p40/p70-apc (clone C15.6) and CD11b-Tr-color (clone M1/70.15; Caltag) per BD PharMingen intracellular cytokine staining protocols and analyzed on a FACSCalibur (BD Biosciences) using the Cell-Quest program (BD Biosciences). Data were further processed using FlowJo software (Tree Star, San Carlos, CA).

Cells were first incubated on ice for 20 min in a lysis buffer containing 0.05 mol/l Tris, pH 8.0, 0.15 mol/l NaCl, 0.01 mol/l EDTA, and 1% NP40 in the presence of a protease inhibitor. The cell lysate was centrifuged at 2300g for 10 min at 4°C, and the supernatant was fractionated by electrophoresis on a 12% PAGE and transferred to a nitrocellulose membrane. After blocking with 5% nonfat milk, the membrane was incubated with either rabbit anti–STAT-1 (Upstate Biotechnology, Lake Placid NY) or anti–β-actin and with horseradish peroxidase–labeled secondary antibodies (Amersham, NJ). Color was developed using enhanced chemiluminescence Western blotting detection reagents (Amersham/Pharmacia Biotech, Piscataway, NJ).

The significance of the differences in disease severity and immune parameters was determined by the Mann-Whitney test and ANOVA, respectively.

To determine whether p53 is required for the development of type 1 diabetes, wild-type control mice and mice homozygous for the p53 gene mutation were treated daily with a low dose of STZ (40 mg STZ/kg body wt) for 5 consecutive days (days 0–4) (36,37). Diabetes was monitored by both urine glucose levels and pancreatic islet histochemistry. As shown in Fig. 1, diabetes developed at a low frequency in control B6 mice (∼25%), starting ∼9 days after the first STZ injection. In contrast, the incidence of the disease was markedly increased in p53^−/− mice, reaching 95% by day 25 (Fig. 1). Consistent with these clinical findings, hematoxylin and eosin staining of pancreatic sections of control and p53^−/− mice revealed significant differences. Insulitis, characterized by peri- and intraislet infiltration of inflammatory cells, was observed in most p53^−/− mice (Fig. 2). By contrast, the majority of pancreatic islets in control mice appeared normal, with no signs of inflammation. Therefore, p53 deficiency significantly increased the incidence of STZ-induced diabetes.

It has been reported that some of the p53^−/− mice develop tumors at ∼15 weeks of age (29,34,38). To avoid this complication, we had planned our experiments in such a way that they could be completed before mice reached 13 weeks of age. Furthermore, we routinely performed mouse autopsy at the end of the experiments to search for signs of tumor growth in various organs. To date, we have detected no tumors in mice <13 weeks of age, whereas tumors (principally lymphomas and osteosarcomas) were detected in some of the p53^−/− breeders that were >17 weeks of age.

Low-dose STZ–induced diabetes is an autoimmune disease mediated by inflammatory cells and their cytokines. To determine whether p53 gene mutation alters cytokine production, we examined the levels of cytokines in the serum and spleen as well as the pancreas of p53^+/+ and p53^−/− mice 30 days after STZ injection. We detected IL-1 and -6 in all of the samples and found that both were significantly increased in p53^−/− group compared with p53^+/+ group (Fig. 3). Additionally, the concentration of IL-12 in the serum of p53^−/− mice was also significantly higher than that of p53^+/+ mice (25 ± 1.5 vs. 7 ± 1.1 ng/ml, P < 0.001), although its concentrations in splenic and pancreatic extracts were essentially the same in the two groups (data not shown). By contrast, the levels of IFN-γ in all samples tested were not significantly different in the two groups (data not shown). These results indicate that p53 gene mutation may selectively affect the production of inflammatory cytokines.

Because macrophages are major inflammatory cells that produce IL-1, -6, and -12, we next examined whether p53 gene mutation directly affected the functions of these cells. As shown in Fig. 4, after IFN-γ and lipopolysaccharide stimulation, p53^−/− macrophages produced significantly higher levels of IL-6 and IL-12p40 than control cells. By contrast, the production of TNF-α and nitric oxide was not significantly affected by p53 deficiency (data not shown).

To determine whether the increased IL-12 production resulted from an increase in the number of IL-12–producing cells, we performed flow cytometry analysis of macro-phages. As shown in Fig. 5, the frequency of IL-12–producing cells was significantly increased in the p53^−/− culture. Additionally, the relative IL-12 levels were also higher in p53^−/− (mean fluorescence intensity = 214) than in p53^+/+ (mean fluorescence intensity = 163) cells. Taken together, these results indicate that p53 regulates proinflammatory cytokine production in macrophages.

Both p53 and STAT-1 are latent transcription factors that can be activated by a variety of stimuli, including lipopolysaccharide and IFN-γ. To explore the potential mechanisms of p53 action in IL-6 and -12 cytokine expression after lipopolysaccharide/IFN-γ treatment (Figs. 4 and 5), we examined whether STAT-1 expression and phosphorylation were normal in p53^−/− macrophages. In p53^+/+ macrophages, STAT-1 was expressed at high levels (Fig. 6) but was not phosphorylated without stimulation (Fig. 7, 0 min). Stimulation with lipopolysaccharide increased the basal levels of STAT-1 (Fig. 6) and induced its rapid phosphorylation (Fig. 7). Importantly, in p53^−/− macrophages, both total (Fig. 6, 0 min) and phosphorylated STAT-1 (Fig. 7, 20 min) levels were significantly increased. These results indicate that p53 may act upstream of STAT-1 in macrophages to modulate cytokine production after lipopolysaccharide stimulation.

STZ is a d-glucopyranose derivative of N-methyl-N-nitrosourea endowed with potent alkylation properties. In addition, it can also serve as a nitric oxide donor, promoting the production of reactive oxygen species. At high doses, STZ may directly destroy insulin-producing β-cells, leading to acute diabetes. At low doses (including the dose used in this study), however, STZ-induced β-cell damage may not be sufficient to cause diabetes, and participation of inflammatory cells is essential for the development of the disease (35,39,40). The mechanisms of inflammation in low-dose STZ-induced autoimmune diabetes are not clear. Results reported here clearly establish that the tumor suppressor p53 is crucial for this process. The importance of p53 in autoimmune inflammation can be generalized to other autoimmune disease models such as autoimmune encephalomyelitis in B6 mice (41) and collagen-induced arthritis in DBA/1 mice (10).

If p53 plays an inhibitory role in the development of autoimmune diseases, what are the mechanisms of its actions? Like many transcription factors, p53 is expressed by both the immune system and the nonimmune systems. On the one hand, p53 expressed by lymphocytes and cells of the innate immune system may activate or inactivate genes encoding apoptotic proteins, cell cycle inhibitors, and inflammatory cytokines. This would affect the functions of inflammatory cells. On the other hand, p53 expressed by the nonimmune systems, e.g., pancreatic islets, may also play important roles in the development of autoimmune diseases. Thus, the net effect of p53 in autoimmune diseases is likely determined by a balance between these two types of actions.

Results reported here support the view that p53-mediated gene repression in inflammatory cells plays a decisive role in the development of autoimmune diseases. Thus, it has been reported that p53 may directly repress the IL-4 and -6 promoter activities in certain cell lines (26,27). In the case of arthritis, it has been reported that IL-6 production in the arthritic synovium is upregulated when p53 gene mutation is present (10,31), although the cell type(s) producing IL-6 in this model is not clear. In this study, we discovered that p53 may repress not only IL-6 but also IL-1, and -12 expression in macrophages. Because these cytokines play crucial roles in the pathogenesis of autoimmune diabetes (42,43), their upregulation in p53^−/− mice likely contributes to the exacerbated disease in these animals. Interestingly, IL-12 is upregulated in diabetes-prone NOD mice (44), and injection of recombinant IL-12 into NOD mice accelerated the disease (45). Conversely, blocking the functions of IL-12 using anti–IL-12 antibody or IL-12 antagonists significantly impeded the development of diabetes in NOD mice (43,46,47). In the current study, we found that IL-12 was increased in the serum and macrophages but not in the spleen and pancreas of p53^−/− mice. This indicates that the effect of p53 on IL-12 expression in vivo could be tissue- or cell-specific. Further studies are needed to address this issue. Nonetheless, results reported here support the theory that p53 inhibits autoimmune inflammation by inactivating genes encoding inflammatory cytokines. Additionally, our data also indicate that p53 may regulate type 1 diabetes through a separate mechanism involving STAT-1. STAT-1 is a crucial mediator of inflammatory cytokine signaling, and blocking STAT-1 activity using suppressors of cytokine signaling prevents type 1 diabetes (48). There are several potential mechanisms through which p53 may regulate the expression and activities of STAT-1. First, p53 may directly repress the promoter of STAT-1 gene, reducing its mRNA expression. Second, p53 may regulate the expression of genes important for the phosphorylation of STAT-1. Finally, it has been reported that p53 may directly interact with STAT-1 to enhance its proapoptotic activity (49). Regardless of the mechanisms, our observation that STAT-1 expression is enhanced in p53^−/− macrophages provides a novel link between p53 and STAT-1. Because macrophages play crucial roles in the development of innate immunity, results reported here also provide direct evidence that p53 may be involved in regulating innate immune responses.

As discussed above, development of low-dose STZ-induced diabetes requires the participation of both T-cells and macrophages (35,39,40). The roles of p53 in regulating T-cell functions have also been reported (50). Therefore, enhancement of diabetes in p53-deficient mice may be caused by the loss of p53 function not only in macrophages but also in T-cells. Further investigation is required to clarify this issue.

In summary, we have discovered that p53 deficiency significantly increased the incidence of autoimmune diabetes in mice and that this may be caused by the loss of p53-mediated inhibition of proinflammatory cytokines and STAT-1. These results provide new insights into the molecular mechanisms of autoimmune diabetes that may lead to the development of new strategies for the treatment of the disease.

This work was supported by Grants AI50059, DK065848, and AI053052 from the National Institutes of Health.