Adiponectin modulates inflammatory reactions via calreticulin receptor–dependent clearance of early apoptotic bodies (original) (raw)
To determine whether adiponectin contributes to the clearance of apoptotic particles in vivo, adiponectin-deficient (APN-KO) and WT mice were injected with dexamethasone to induce massive thymocyte apoptosis (28). At 24 hours after dexamethasone treatment, the thymi of APN-KO and WT mice were assessed for apoptotic cells in histological sections by TUNEL staining. Few TUNEL-positive cells were observed in the thymi of untreated APN-KO and WT mice. However, the thymi of dexamethasone-treated APN-KO mice showed 1.8-fold greater remnant apoptotic cells than those of similarly treated WT mice (Figure 1A); this was accompanied by a 2.7-fold increase in caspase-3 activity in tissue homogenates (data not shown). The i.v. preadministration of an adenoviral vector expressing adiponectin (Ad-APN) but not a control protein (Ad–β-gal) significantly diminished the level of TUNEL-positive cells in APN-KO thymi. This method of delivery results in transduction of liver, and the production of circulating adiponectin oligomers by liver is similar to that found in WT mice (11). Plasma adiponectin levels under these conditions were 13.1 ± 0.6 μg/ml in WT/Ad–β-gal, 19.0 ± 1.4 μg/ml in WT/Ad-APN, less than 0.05 μg/ml in APN-KO/Ad–β-gal, and 13.7 ± 0.5 μg/ml in APN-KO/Ad-APN. The elevation of plasma adiponectin by Ad-APN transduction in WT mice also led to a statistically significant decrease in the number of cell corpses in the thymus.
APN deficiency leads to the accumulation of apoptotic debris. (A) WT and APN-KO mice were treated with dexamethasone (DEX), and thymi were stained for apoptotic cells with TUNEL. In some experiments, mice received an i.v. infusion of Ad-APN or Ad–β-gal 2 days prior to injection of dexamethasone. The number of TUNEL-positive cells per microscopic field for the different experimental conditions is reported. *P < 0.05 versus WT; †P < 0.05 versus Ad–β-gal for WT or APN-KO (n = 3–6). (B) Adiponectin stimulates macrophage engulfment of TUNEL-positive apoptotic debris in thymi of dexamethasone-treated mice. Histological sections were stained with TUNEL (green) and anti-CD11b antibody (red). Scale bar: 20 μm. Colocalization is indicated by yellow in the merged images. ††P < 0.01 versus Ad–β-gal for WT or APN-KO (n = 3–6). M, macrophage.
Immunohistological analysis revealed that adiponectin accumulated in the thymi of WT mice following treatment with dexamethasone (Supplemental Figure 1A; supplemental material available online with this article; doi:10.1172/JCI29709DS1), suggesting that this protein enters the damaged tissue via leakage from the vascular compartment. Thymi sections from APN-KO mice showed no signal, except in the case of Ad-APN treatment. Ad-APN–treated APN-KO mice also showed accumulation of adiponectin in the thymi of dexamethasone-treated mice. Immunofluorescent analysis of thymi sections revealed that adiponectin colocalized with the macrophage marker CD11b, suggesting that adiponectin is recruited to phagocytic cells within this tissue (Supplemental Figure 1B).
Additional histological analyses were performed on the thymi of APN-KO and WT mice that had been treated with dexamethasone to assess the percentage of macrophages containing apoptotic debris (29). The percentage of macrophages with ingested TUNEL-positive material was significantly lower in the thymi of APN-KO mice than in those of WT mice (Figure 1B). Adenovirus-mediated expression of adiponectin reversed the observed deficit in macrophage phagocytosis in APN-KO mice and increased the frequency of macrophages containing apoptotic debris in WT mice. In contrast, treatment with Ad-APN or adiponectin deficiency had no effect on the total number of macrophages present in the histological sections of thymi from either strain of mice (data not shown). To explore whether adiponectin action in the thymus could result from its ability to inhibit dexamethasone-induced apoptosis, thymocytes were isolated from WT mice and incubated with 1 μM dexamethasone in the presence or absence of recombinant adiponectin. While dexamethasone increased thymocyte apoptosis approximately 4-fold in vitro (P < 0.05), the inclusion of 50 μg/ml adiponectin had no detectable effect on basal or dexamethasone-induced cell death (Supplemental Figure 2). Collectively, these data suggest that the adiponectin-mediated reduction in apoptotic cells in the thymus results from the increased clearance of cell corpses rather than the inhibition of thymic cell death.
To directly determine whether adiponectin deficiency affects phagocytic activity of macrophages in vivo, early apoptotic Jurkat T cells were labeled with 5- and 6- carboxytetramethylrhodamine, succinimidyl ester (TAMRA, SE), a rhodamine fluorescent amino-reactive marker, and injected into the intraperitoneal cavity of APN-KO and WT mice (Figure 2A). These mice were pretreated with thioglycollate to recruit inflammatory macrophages, and Ad-APN or the control vector Ad–β-gal was delivered via the jugular vein of WT or APN-KO mice 3 days prior to injection of apoptotic bodies. At 30 minutes after the injection of apoptotic T cells, peritoneal cells were recovered and the uptake of apoptotic bodies was assessed by flow cytometric analysis of macrophages that were dual-labeled with F4/80, a macrophage marker, and TAMRA, SE. APN-KO mice were significantly impaired in the phagocytic uptake of labeled apoptotic cells compared with WT mice (16.2% ± 1.6% versus 35.1% ± 1.4%, TAMRA, SE–positive macrophages, respectively) (Figure 2B). Adenovirus-mediated overexpression of adiponectin rescued the impaired apoptotic cell uptake by macrophages in the peritoneum of APN-KO mice and increased the phagocytic activity of macrophages in WT mice. Phagocytic activity could be stimulated to a similar degree if recombinant human adiponectin protein, produced in a baculovirus expression system, was injected in the intraperitoneal cavity with the apoptotic cells (Supplemental Figure 3). In these assays, adiponectin deficiency did not affect the recruitment of macrophages by thioglycollate treatment, nor did it affect peritoneal levels of TNF-α (Supplemental Figure 4).
Systemic delivery of adiponectin promotes the uptake of apoptotic debris by peritoneal macrophages. (A) Strains of mice were injected with Ad–β-gal or Ad-APN on the same day as thioglycollate treatment. Circulating adiponectin levels at the time of sacrifice were 12.5 ± 1.6 μg/ml in B6.lpr/Ad–β-gal and 19.5 ± 2.4 μg/ml in B6.lpr/Ad-APN. TAMRA, SE–labeled apoptotic Jurkat T cells were injected into the peritoneum of the indicated strains of mice 3 days after the administration of thioglycollate. After 30 minutes, peritoneal cells were removed by lavage and subjected to flow cytometry. (B) Phagocytosis was scored as the percentage of F4/80-positive macrophages that also stained positive for TAMRA, SE. Scatter plots show representative flow cytometry data for 3 experimental conditions. Dual-labeled cells are represented in the upper right quadrant. **P < 0.01 versus Ad–β-gal; ††P < 0.01 versus WT (n = 6).
The lpr mice harbor a mutation in the gene encoding Fas, and they exhibit impaired clearance of dying cells (30). This deficiency contributes to systemic inflammation and lymphadenopathy in C57BL/6 mice whereas lpr in the MRL/Mp+/+ background produces more severe inflammation and animals develop autoimmune phenotypes (31, 32). Similarly to what is observed in APN-KO mice, both strains of lpr mice displayed an impaired clearance of apoptotic cells that were injected into the peritoneum (Figure 2A). Conversely, adenovirus-mediated overexpression of adiponectin stimulated apoptotic cell phagocytosis by macrophages in both B6.lpr and MRL.lpr mice (Figure 2B).
B6.lpr and MRL.lpr mice accumulated apoptotic debris in lymph nodes, with higher levels occurring in the MRL/Mp+/+ background. Basal plasma adiponectin levels were 13.1 ± 0.6 μg/ml in B6.WT, 12.5 ± 1.6 μg/ml in B6.lpr, and 10.6 ± 0.4 μg/ml in MRL.lpr mice. Ad-APN administration led to plasma adiponectin levels of 19.5 ± 2.4 and 23.4 ± 3.8 μg/ml in B6.lpr and MRL.lpr mice, respectively, at 3 days after infection and decreased levels of TUNEL-positive material in submandibular lymph nodes of both strains relative to those of control mice that were treated with the equivalent titer of Ad–β-gal (Figure 3A). Both B6.lpr and MRL.lpr mice developed autoreactive anti-nuclear antibodies (ANA), although levels were higher in the MRL background. Fourteen days after Ad-APN treatment, ANA levels were reduced by factors of 8.9 and 3.6 in B6.lpr and MRL.lpr mice, respectively (Figure 3B). ANA levels were not detectable in WT mice (data not shown). MRL.lpr but not B6.lpr mice also develop anti–double-stranded DNA (anti-dsDNA) antibodies, which are found in patients with systemic lupus erythematosus. Adiponectin overexpression lowered the mean level of anti-dsDNA antibodies by a factor of 4.6 in MRL.lpr mice. The effects of adiponectin overexpression on renal function and morphology were also evaluated because this organ is damaged by the autoimmune disease in MRL.lpr mice. Adiponectin overexpression led to a modest but reproducible reduction in glomerular tuft volume at 14 days following Ad-APN delivery (Figure 3C) and reduced the accumulation of immune complex deposition in the kidney as assessed by immunofluorescence (data not shown). Consistent with these findings, adiponectin overexpression suppressed urinary albumin excretion (Figure 3C). Additional studies showed that MRL.lpr mice displayed normal glucose sensitivity relative to MRL/Mp+/+ mice and that adenovirus-mediated overexpression of adiponectin in MRL.lpr mice did not affect body weight or circulating insulin levels (Supplemental Figure 5, A–C). Thus, adiponectin’s actions in this model were largely antiinflammatory rather than metabolic. Consistent with this notion, adiponectin overexpression led to a reduction in circulating TNF-α in MRL.lpr mice (Supplemental Figure 5D).
Systemic delivery of adiponectin decreases remnant apoptotic levels and inflammation in lpr strains of mice. (A) Adenovirus-mediated overexpression of adiponectin decreases apoptotic cells in lymph nodes of B6.lpr mice at the age of 12 weeks and MRL.lpr mice at the age of 20 weeks. Lymph node sections were stained with TUNEL and quantified by microscopy (n = 6–8). (B) ANA titers and anti-dsDNA antibody titer in the serum of B6.lpr or MRL.lpr mice at 14 days after Ad-APN or Ad–β-gal administration. **P < 0.01 versus Ad–β-gal. (C) Adiponectin overexpression influences renal function and morphology. Photographs show representative glomeruli of MRL.lpr mice treated with Ad–β-gal or Ad-APN 14 days prior to sacrifice. Glomerular tuft volume of control is 3.1 × 105 ± 0.1 × 105 μm3. Scale bar: 50 μm. Urinary albumin is reported as μg protein/d. *P < 0.05 versus Ad–β-gal (n = 8).
APN-KO/lpr mice were generated on the C57BL/6 background so that we could evaluate whether adiponectin deficiency would further impair apoptotic cell clearance and promote inflammation relative to the B6.lpr and APN-KO parental strains. Macrophage uptake of apoptotic cells in the peritoneum of APN-KO/lpr mice was significantly less than in either parental strain as assessed by flow cytometric analysis of TAMRA, SE and F4/80 dual-positive macrophages at 10 weeks of age (Figure 4A). At this age, plasma levels of TNF-α are similar between APN-KO, lpr, and APN-KO/lpr strains, but by 20 weeks of age, APN-KO/lpr mice display much higher levels of TNF-α than the other strains (Supplemental Figure 6). B6.lpr mice develop age-dependent lymphadenopathy and autoimmunity starting at approximately 10 weeks of age (32, 33). Lymphadenopathy was markedly increased in APN-KO/lpr compared with lpr mice at 20 weeks of age, and a statistically significant increase in submandibular lymph node size could be detected in the APN-KO/lpr strain as early as 12 weeks of age (Figure 4B). The lymph nodes from APN-KO/lpr mice displayed higher levels of TUNEL-positive cells compared with those of lpr mice at both 12 and 20 weeks of age whereas the lymph nodes of APN-KO and WT mice showed few or no TUNEL-positive cells (Figure 4C). Adiponectin deficiency also produced an increase in ANA titers by 2.7- and 4.1-fold in lpr mice at 12 and 20 weeks of age, respectively (Figure 4D). Furthermore, adiponectin deficiency led to detectable anti-dsDNA antibody titers in lpr mice at 20 weeks of age whereas the parental B6.lpr strain showed little or no dsDNA immunoreactive material. No anti-dsDNA antibodies could be detected at 12 weeks of age. Both APN-KO and WT mice showed no evidence of autoreactive antibodies (Figure 4D). Finally, the APN-KO/lpr mice displayed features of kidney dysfunction associated with autoimmune disease, including glomerular tuft enlargement and proteinuria, whereas WT, APN-KO, and lpr mice in the C57BL/6 background did not (Figure 4E). These data show that adiponectin deficiency in B6.lpr mice leads to further reductions in apoptotic cell clearance and exacerbates systemic inflammation. Furthermore, these data show that a detectable impairment in apoptotic cell clearance coincides with or precedes the development of the autoimmune and lymphoproliferative phenotypes.
B6/lpr mice deficient in adiponectin display impaired clearance of apoptotic cells and increased systemic inflammation. (A) Impaired clearance of apoptotic cells by peritoneal macrophages in APN-KO/lpr mice compared with APN-KO and lpr mice. Experiments were conducted with the indicated strains of mice. Phagocytosis of apoptotic Jurkat cells was assessed by flow cytometric analysis of F4/80- and TAMRA, SE–positive cells as described in the Figure 1C legend. *P < 0.05 versus B6/lpr APN-KO (n = 4–6). (B) APN-KO/lpr mice have larger submandibular lymph nodes compared with lpr mice. Inset shows a representative photograph of submandibular lymph nodes from each strain of mouse at the age of 20 weeks. Scale bar: 5 mm. Submandibular lymph nodes were excised from the indicated strains of mice at 12 or 20 weeks and weighed. *P < 0.05 versus lpr (n = 6–14). (C) Adiponectin deficiency increases the frequency of apoptotic cells in lymph nodes of B6.lpr mice. Mice were sacrificed at 12 or 20 weeks of age, and TUNEL-positive cells in sections of submandibular lymph nodes were assessed. ND, not detectable. **P < 0.01 versus lpr (n = 6–14). (D) Adiponectin deficiency increases autoreactive antibody titer in B6.lpr mice. ANA titers and anti-dsDNA antibody titers in the sera of each strain of mouse were determined at 12 or 20 weeks of age. **P < 0.01 versus lpr (n = 3–14). (E) Adiponectin deficiency promotes kidney disease in B6.lpr mice. Glomerular tuft volume was determined in histological sections of kidney from the indicated strains of 20-week-old mice. Photographs show representative glomeruli. The calculated glomerular tuft volume of control is 1.1 × 105 ± 0.1 × 105 μm3. Scale bar: 50 μm. **P < 0.01 versus lpr. The daily excretion of urinary albumin was determined immediately prior to sacrifice. *P < 0.05 versus lpr (n = 4–10).
To investigate the mechanism by which adiponectin affects early apoptotic body clearance in vitro, FITC-labeled macrophages from different sources were incubated with viable or apoptotic Jurkat T cells labeled with TAMRA, SE in the presence or absence of adiponectin. Recombinant human adiponectin, produced from a baculovirus expression system, stimulated the uptake of apoptotic Jurkat T cells by 32.8% ± 1.0% by human monocyte-derived macrophages and 23.6% ± 0.4% in differentiated monocytic THP-1 cells in vitro, as determined by flow cytometric analysis of dual-labeled and total macrophages (Figure 5). In agreement with these data, microscopic analyses also revealed that adiponectin stimulated the ingestion of TAMRA, SE–labeled apoptotic cells by human and THP-1 macrophages (Supplemental Figure 7). Recombinant adiponectin was either equally effective or more effective at promoting phagocytosis than C1q (Figure 5), which promotes apoptotic cell uptake by macrophages and is implicated in the development of autoimmune disease (34, 35). Stimulation of apoptotic cell uptake was dependent on the dose of adiponectin in the phagocytosis assay, and this effect appeared to saturate at a final concentration of 4 μg/ml adiponectin (Supplemental Figure 8). Recombinant adiponectin also stimulated the uptake of early apoptotic neutrophils by THP-1 and human macrophages (Supplemental Figure 9A) but had no effect on the uptake of microbeads opsonized with BSA, IgG, or C3b (Supplemental Figure 9B). Adiponectin did not stimulate the uptake of viable Jurkat T cells by human or THP-1 macrophages (Supplemental Figure 9C). Furthermore, adiponectin did not stimulate the uptake of late apoptotic bodies by human or THP-1 macrophages (Supplemental Figure 9D).
Adiponectin promotes the phagocytosis of apoptotic bodies by macrophages in vitro. Apoptotic Jurkat T cells were preincubated for 1 hour with recombinant adiponectin from baculovirus-insect (APN) (50 μg/ml), human C1q (50 μg/ml), or vehicle. Macrophages were then incubated for 30 minutes with TAMRA, SE–labeled Jurkat cells that were either viable or apoptotic due to UVB exposure. Upon mixing apoptotic cells with macrophages, adiponectin and C1q were diluted to a final concentration of 10 μg/ml. Phagocytosis was assessed by flow cytometry. Macrophages were stained with FITC-conjugated anti-human macrophage antibody, and the percentage of phagocytic macrophages was calculated as TAMRA, SE–positive (+) macrophages/total macrophages × 100%. Phagocytic macrophages of control were 32.8% ± 1.0% (human) and 23.6% ± 0.4% (THP-1). **P < 0.01 versus vehicle; †P < 0.05 versus C1q (n = 6–7).
To determine whether adiponectin promotes phagocytosis through an opsonization mechanism, we examined the binding of adiponectin to early apoptotic and viable Jurkat T cells using standard protocols (25, 35). Cells were incubated with FITC-conjugated recombinant human FITC-APN for 60 minutes. Fluorescence microscopy revealed that adiponectin bound to blebs on Jurkat T cells that were morphologically apoptotic following exposure to UVB irradiation with a high, locally intense signal (Figure 6A). Adiponectin also bound to viable Jurkat T cells, but the fluorescence intensity was weak and more uniformly distributed on the cell surface. Flow cytometry was performed to quantify adiponectin binding to viable and apoptotic Jurkat cells (Figure 6B). This analysis revealed greater binding of adiponectin to apoptotic cells compared with viable cells. These findings were corroborated by analyzing adiponectin binding to viable and apoptotic cells using a microplate reader and FITC-labeled adiponectin (Supplemental Figure 10A). More adiponectin was bound to apoptotic cells than viable cells over a wide range of adiponectin concentrations. Collectively, these data show that the expression of an adiponectin receptor is upregulated on the cell surface of the dying cell.
Apoptosis increases the binding of adiponectin to Jurkat T cells. (A) Fluorescence microscopy of FITC-APN to viable (left panel) and apoptotic (right panel) Jurkat T cells. Binding to viable cells was diffuse whereas binding to apoptotic cells was intense and uneven within individual cells. Scale bar: 5 μm. (B) Left panels: representative flow cytometric analyses of FITC-APN to viable (blue) and apoptotic Jurkat T (red) cells. Control (black) represents cells incubated with FITC-conjugated human serum albumin. Right panel: Quantitation of FITC-APN binding to viable and apoptotic cells. **P < 0.01 versus viable cells (n = 3).
To investigate the mechanism by which adiponectin modulates the clearance of apoptotic cells, detergent-solubilized membrane fractions from THP-1 macrophages were incubated with or without polyhistidine-tagged adiponectin protein, precipitated with nickel resin, and subjected to SDS-PAGE. One unique protein band was detected when membrane fractions were treated with nickel resin in the presence of adiponectin but not in its absence (data not shown). This region of SDS-PAGE gel was excised, treated with trypsin, and analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Peptide mass fingerprints were analyzed with the protein prospector program MS-Fit (University of California, San Francisco, Mass Spectrometry Facility: http://prospector.ucsf.edu/prospector) using the database NCBInr.2005.01.06, and 13 peptide masses were found to correspond to calreticulin and cover 33% of the protein sequence. To confirm the presence of calreticulin in the nickel resin precipitate, Western immunoblot analysis was performed with anti-calreticulin antibody. Calreticulin was only detected in the THP-1 membrane fraction when the precipitation with nickel resin was performed in the presence of polyhistidine-tagged adiponectin (Figure 7A). Adiponectin binding to calreticulin was also demonstrated by immunoprecipitating the solubilized THP-1 membrane fraction with anti-calreticulin antibodies followed by the detection of adiponectin by Western immunoblot analysis (Figure 7B). To determine whether adiponectin binds to calreticulin on the cell surface, competition assays between recombinant adiponectin and anti-calreticulin antibody were performed using intact THP-1 macrophages (35). Preincubation with adiponectin for 60 minutes significantly reduced the binding of anti-calreticulin antibody to THP-1 macrophages as determined by flow cytometric analysis of FITC-conjugated secondary antibody to the anti-calreticulin antibody (Figure 7C).
Adiponectin interacts with calreticulin on the macrophage cell surface. (A) Calreticulin is immunoprecipitated by histidine-tagged adiponectin (APN) from detergent-solubilized THP-1 membranes. Membrane fractions were incubated in the presence or absence of polyhistidine-APN and then precipitated with nickel resin. The resin was then treated with a molar excess of histidine to release precipitated proteins, and this material was subjected to SDS-PAGE followed by immunoblot analysis with anti-calreticulin (anti-CRT) and anti-adiponectin (anti-APN) antibodies. WB, Western blot. (B) Adiponectin prepared from E. coli (E-APN) was immunoprecipitated from detergent-solubilized THP-1 membranes by anti-calreticulin antibodies. THP-1 membrane fractions were incubated in the presence or absence of polyhistidine APN and then subjected to immunoprecipitation with anti-CRT or control IgG. Immunoprecipitated material was then subjected to SDS-PAGE, and Western blot analysis was performed with anti-APN or anti-CRT antibodies. (C) Adiponectin inhibits the binding of anti-calreticulin antibody to macrophages. THP-1 macrophages were preincubated with 200 μg/ml adiponectin (red) or vehicle (blue) for 60 minutes followed by incubation with anti-calreticulin antibody (10 μg/ml) for 60 minutes. Cells incubated with chicken IgY followed by treatment with FITC-conjugated secondary antibody served as control. Cells were then incubated with FITC-conjugated secondary antibody to anti-calreticulin antibody and analyzed by flow cytometry. *P < 0.05 versus vehicle (n = 3).
Recent studies have shown that calreticulin is upregulated on the surface of the apoptotic cell, where it acts as a general recognition ligand in the phagocytic process (36). Accordingly, more calreticulin could be detected on apoptotic Jurkat T cells than viable cells by flow cytometric analysis (Supplemental Figure 10B). Preincubation with anti-calreticulin antibody partially but significantly inhibited the binding of adiponectin to apoptotic cells (Supplemental Figure 10C). Finally, adiponectin could be colocalized with calreticulin on apoptotic thymic cells in mice that were treated with dexamethasone (Supplemental Figure 10D).
The binding of FITC-labeled adiponectin to the phagocytic cell surface was also analyzed. Recombinant human adiponectin binding to THP-1 macrophages was saturable, with half-maximal binding occurring at approximately 0.7 μg FITC-APN/ml (Supplemental Figure 11A). Coincubation with 6 μg/ml FITC-APN and anti-calreticulin antibody led to a 37.1% ± 7.2% (P < 0.05) reduction in APN binding compared with control antibody (Supplemental Figure 11B), suggesting that adiponectin interacts with calreticulin in addition to other adiponectin receptors, such as AdipoR1, AdipoR2, and T-cadherin, on the macrophage cell surface. Finally, adiponectin colocalized with calreticulin on the surface of THP-1 cells (Supplemental Figure 11C).
To determine whether adiponectin increases macrophage-mediated removal of early apoptotic bodies through a calreticulin-dependent pathway in vitro, macrophages from different sources were incubated with recombinant adiponectin or vehicle in the presence or absence of anti-calreticulin antibody, and the uptake of apoptotic Jurkat T cells was assessed by flow cytometry (Figure 8A). Preincubation with anti-calreticulin antibody blocked adiponectin-stimulated phagocytosis of apoptotic bodies. This treatment had no effect on basal phagocytosis in human macrophages or THP-1 cells, consistent with a previous report (25). Calreticulin interacts with endocytic receptor protein CD91 to phagocytose apoptotic cells (35). Accordingly, adiponectin-mediated uptake of apoptotic cells was also blocked by incubation with anti-CD91 antibody. To corroborate these data, knockdown experiments were performed with siRNA targeting calreticulin and CD91. Calreticulin expression was reduced by 60.6% ± 2.5%, and CD91 expression was reduced by 92.6% ± 1.8% by treatment with their corresponding siRNA polynucleotides (Supplemental Figure 12), leading to 68% and 95% reductions, respectively, in adiponectin-stimulated apoptotic cell uptake by THP-1 macrophages (Figure 8B).
Calreticulin and CD91 are essential for adiponectin-stimulated uptake of apoptotic cells. (A) Anti-calreticulin antibody and anti-CD91 antibody inhibit adiponectin-stimulated apoptotic cell phagocytosis by macrophages. Each type of macrophage was preincubated with anti-CRT antibody or control chicken IgY or with anti-CD91 antibody or control IgG for 60 minutes. TAMRA, SE–labeled apoptotic cells were preincubated with recombinant adiponectin or vehicle, and uptake of apoptotic debris was determined by flow cytometric analysis. Data are expressed relative to control from human and THP-1 monocytes. Control human and THP-1 macrophages (anti-human macrophage antibody-positive) were 34.1% ± 0.8% and 24.9% ± 1.1% dual-positive for TAMRA, SE, respectively. **P < 0.01 versus IgY or IgG (n = 6–7). (B) Adiponectin-stimulated apoptotic cell phagocytosis by macrophages was inhibited by downregulation of calreticulin or CD91 with siRNA but not siRNA targeting the putative adiponectin receptors. The in vitro phagocytosis assay analyzed dual-positive by cells flow cytometry. Control THP-1 macrophages were 24.3% ± 1.0% positive for TAMRA, SE. **P < 0.01 versus unrelated siRNA with adiponectin (n = 6–7).
AdipoR1, AdipoR2, and T-cadherin are also reported to function as adiponectin receptors (12, 13). To assess whether these receptors are associated with the removal of apoptotic debris by adiponectin, AdipoR1, AdipoR2, and T-cadherin expression was knocked down by RNA interference methods. AdipoR1 was reduced by 69.5% ± 6.1%, AdipoR2 was reduced by 51.6% ± 5.0%, and T-cadherin was reduced by 74.1% ± 11.7%, as determined by flow cytometric analysis of cell-surface protein expression (Supplemental Figure 12). However, reductions of AdipoR1, AdipoR2, or T-cadherin had no influence on adiponectin-stimulated phagocytosis (Figure 8B). Collectively, these data suggest that adiponectin can increase the removal of apoptotic bodies via calreticulin/CD91 but not through the previously identified adiponectin receptors.