P-Selectin Glycoprotein Ligand-1 Regulates Adhesive Properties of the Endothelium and Leukocyte Trafficking Into Adipose TissueNovelty and Significance
Rationale: Adhesive interactions between endothelial cells and leukocytes affect leukocyte trafficking in adipose tissue. The role of P-selectin glycoprotein ligand-1 (Psgl-1) in this process is unclear.
Objective: The goal of this study was to determine the effect of Psgl-1 deficiency on adhesive properties of the endothelium and on leukocyte recruitment into obese adipose depots.
Methods and Results: A genetic model of obesity was generated to study the effects of Psgl-1 deficiency on leukocyte trafficking. Leukocyte-endothelial interactions were increased in obese leptin receptor mutant mice (Leprdb/db,Psgl-1+/+) but not obese Psgl-1–deficient mice (Leprdb/db,Psgl-1−/−), when compared with lean mice (Lepr+/+,Psgl-1+/+). This effect of Psgl-1 deficiency was due to indirect effects of Psgl-1, because Psgl-1+/+ adoptively transferred leukocytes did not exhibit enhanced rolling in Lepr db/db,Psgl-1−/− mice. Additionally, circulating levels of P-selectin, E-selectin, monocyte chemoattractant protein-1, and macrophage content of visceral adipose tissue were reduced in Leprdb/db,Psgl-1−/− compared with Leprdb/db,Psgl-1+/+ mice. Reduced leukocyte-endothelial interactions and macrophage content of visceral adipose tissue due to Psgl-1 deficiency was also observed in a diet-induced obese mouse model. Psgl-1−/− mice were resistant to the endothelial effects of exogenous IL-1β, suggesting that defective cytokine signaling contributes to the effect of Psgl-1 deficiency on leukocyte-endothelial interactions. Mice deficient in the IL-1 receptor also had reduced levels of circulating P-selectin, similar to those observed in Psgl-1−/− mice.
Conclusions: Deficiency of Psgl-1 is associated with reduced IL-1 receptor-mediated adhesive properties of the endothelium and is protective against visceral fat inflammation in obese mice.
Obesity has been characterized as a chronic, low-grade inflammatory disease that is marked by an increase in macrophage content and macrophage activity in adipose tissue.1–4 Adhesive interactions between leukocytes and endothelial cells play a critical role in leukocyte trafficking during inflammatory diseases.5 Animal models of obesity have demonstrated an increase in leukocyte-endothelial interactions in visceral adipose tissue that is mediated by increased endothelial expression of P-selectin (P-sel) and E-selectin (E-sel).6 The mechanism by which the endothelium is activated in obese adipose tissue is unclear.
P-selectin glycoprotein ligand-1 (Psgl-1) is the primary leukocyte ligand for P-sel7 and an important ligand for E-sel.8 Leukocytes from Psgl-1−/− mice show reduced selectin-dependent rolling9,10 Thus, interactions between Psgl-1 and endothelial selectins may contribute to the influx of macrophages into adipose tissue, which may in turn trigger the chronic low-grade inflammatory state associated with obesity.2,4
To determine the contribution and mechanism(s) of Psgl-1 toward leukocyte trafficking into adipose tissue, we analyzed leukocyte-endothelial interactions and fat inflammation in genetically and diet-induced obese mice with and without Psgl-1. Our findings indicate that Psgl-1 affects leukocyte trafficking, but that this occurs indirectly via Psgl-1–mediated regulation of endothelial adhesive properties.
Animals and Breeding
Psgl-1−/− mice were from Jackson Laboratory (Bar Harbor, Maine) and backcrossed to the C57BL6/J strain for a total of 10 generations before use in cross-breedings. Leprdb/+ mice on the C57BL6/J strain background were purchased from Jackson Laboratory. To generate Leprdb/db,Psgl-1−/− mice, Leprdb/+ males were crossed to Psgl-1−/− females. Leprdb/+,Psgl-1± were then crossed to Psgl-1−/− mice to produce Leprdb/+,Psgl-1−/− offspring. These offspring were then intercrossed to generate Leprdb/db,Psgl-1−/− mice. Leprdb/db,Psgl-1+/+ mice were generated from an intercross between Leprdb/+,Psgl-1+/+ parents. IL-1R−/− and C57BL6/J control mice were from Jackson Laboratory.
Mice were fed either a standard laboratory rodent diet (No. 5001, TestDiet, Richmond, Ind) or a high-fat, high-sucrose rodent diet (D12451, 45 kcal% fat, Research Diets Inc, New Brunswick, NJ) in specific pathogen-free facilities. For the diet-induced obesity study, Psgl-1−/− and Psgl-1+/+ mice were started on a high-fat, high-sucrose diet at 5 weeks of age and remained on it for 10 weeks. All procedures complied with the Principles of Laboratory and Animal Care established by the National Society for Medical Research and were approved by the University of Michigan Committee on Use and Care of Animals.
Analysis of Serum Factors
Serum samples were collected by retroorbital bleeding with nonheparinized capillary tubes (Fisher Scientific, Pittsburgh, Pa). Circulating levels of soluble P-selectin (sP-sel), soluble E-selectin (sE-sel), monocyte chemoattractant protein (MCP-1), and soluble Intercellular adhesion molecule-1 (sICAM-1) were assayed with murine ELISA kits (R&D systems, Minneapolis, Minn) according to manufacturers' instructions. An Ultra Sensitive Rat Insulin Elisa kit (Crystal Chem Inc, Downers Grove, Ill) was used to measure overnight fasted insulin levels, and overnight fasted glucose levels were determined using an Ascensia Contour Blood Glucose Meter and Ascensia Contour test strips (Bayer HealthCare LLC, Tarrytown, NY).
Weight and DEXA Measurement
Body weight and percent body fat were measured using a Dual-Energy X-ray Absorptiometry (DEXA) scanner Lunar PIXImus2 (GE Healthcare Bio-Sciences Corp, Piscataway, NJ) in diet-induced and genetically obese mice at 15 weeks of age.
For details, please see the expanded Methods section, available in the Online Data Supplement at http://circres.ahajournals.org. Macrophages in adipose cross-sections were identified with a rat anti-mouse Mac-3 monoclonal antibody (1:100 dilution; BD Biosciences, San Jose, Calif.
Real-Time Polymerase Chain Reaction
For details, please see Supplemental Materials and Methods. P-sel, E-sel, MCP-1, and tumor necrosis factor α (TNFα) Real-time polymerase chain reaction (RTPCR) primers were used. 7000 System SDS Software and the 2−ΔΔCT method11 were used to analyze the results.
For details, please see Supplemental Materials and Methods. For adoptive transfer experiments, leukocytes were isolated from whole blood of Psgl-1+/+ mice with Histopaque-1077 (Sigma-Aldrich, St Louis, Mo) according to the manufacturer's instructions. The isolated leukocytes were resuspended in 1 mL of PBS and then incubated with 100 μL of 10X stock rhodamine 6G for 30 minutes. Leukocytes were centrifuged at 250g for 10 minutes. Cells were washed twice in PBS and ultimately resuspended in 1 mL PBS. 1x106 leukocytes were then injected into Leprdb/db,Psgl-1−/− and Leprdb/db,Psgl-l+/+ mice.
To determine the type of leukocytes that were adoptively transferred into mice using this protocol, analysis of leukocytes was performed using a Bayer Advia 120 Hematology System.
Bone Marrow Transplantation
For details, please see Supplemental Materials and Methods. Bone marrow transplantation (BMT) was performed as previously described.12 8- to 10-week-old Leprdb/db,Psgl-1+/+ mice were used as recipients for BMT from Psgl-1+/+,Psgl-1−/−, or Psel-1−/− donors. Psgl-1−/− mice received marrow from Psgl-1+/+ donors, and Psgl-1+/+ mice received marrow from Psgl-1−/− donors. IL-1R+/+ mice received marrow from IL-1R−/− donors, and IL-1R−/− mice received marrow from IL-1R+/+ donors.
For details, please see Supplemental Materials and Methods. Recombinant IL-1β (Peprotech, Rocky Hill, NJ) was injected to mice via tail vein. Mice were bled for serum 5 hours after IL-1β injection.
For details, please see Supplemental Materials and Methods. The antibodies for mouse IκBα, phospho-IκBα, and p38 were from Cell Signaling Technology (Danvers, Mass).
Values are expressed as means±SEM. The statistical significance of differences between groups was determined by Student t test. probability values <0.05 were considered significant.
Metabolic Profile of Diet-Induced and Genetically Obese Psgl-1–Deficient Mice
Mice deficient in leptin receptor signaling ( Leprdb/db,Psgl-1+/+) develop severe obesity.13 No differences between Leprdb/db,Psgl-1+/+ mice and leptin receptor deficient mice without Psgl-1 ( Leprdb/db,Psgl-1−/−) in body weight (49.6±0.9 g, n=9 versus 47.7±1 g, n=12, respectively, P=NS), or percent fat mass (56.9±1.1% versus 56.5±0.5%, respectively, P=NS) were observed at 15 weeks of age. Similarly, in diet-induced obesity, no differences between Psgl-1+/+ mice and Psgl-1−/− mice in body weight (31.4±1 g, n=5 versus 27.7±2.9 g, n=5, respectively, P=NS) or percent fat mass (30±3% versus 34.2±4.6%, respectively, P=NS) were observed at 15 weeks of age after 10 weeks on high-fat, high-sucrose diet.
Fasting glucose levels were significantly reduced in Leprdb/db,Psgl-1−/− mice (n=19) compared with Leprdb/db,Psgl-1+/+ mice (n=11) at 10 weeks of age (280.2±19.1 versus 430±27.4 mg/dL, respectively, P<0.001), whereas fasting insulin levels were not different (5.8±0.9 (n=15) versus 5.1±1 ng/mL (n=19), respectively, P=NS). In the diet-induced obesity model, glucose and insulin levels were not significantly different between Psgl-1+/+ (n=5) and Psgl-1−/− (n=4) mice at 10 weeks of age (glucose: 266±30.7 versus 227.5±25.1 mg/dL, P=NS; insulin: 0.34±0.1 versus 0.43±0.2 ng/mL, P=NS).
Effect of Psgl-1 Deficiency on Obesity-Induced Leukocyte-Endothelial Interactions in Cremaster Venules
Intravital microscopy was used to analyze leukocyte-endothelial interactions in cremaster venules of 15-week-old Leprdb/db,Psgl-1+/+ mice. Leukocyte rolling was markedly increased in Leprdb/db,Psgl-1+/+ mice (n=4) compared with lean wild-type mice Psgl-1+/+ (n=4) (108.7±14.4 versus 40.8±11.8 cells/mm, respectively, P<0.01). Leprdb/db,Psgl-1−/− mice (n=4) exhibited significantly reduced rolling compared with Leprdb/db,Psgl-1+/+ mice (9.1±2.2 versus 108.7±14.4 cells/mm, respectively, P<0.001). No significant difference in firm attachment was observed in Leprdb/db,Psgl-1−/− mice compared with Leprdb/db,Psgl-1+/+ mice (17.2±2.9 versus 30.6±6.9 cells/mm, respectively, P=0.1).
In diet-induced obesity, Psgl-1−/− mice exhibited significantly reduced rolling and firm attachment compared with Psgl-1+/+ mice (rolling: 5.3±1.2 versus 35.5±8 cells/mm, respectively, P<0.01; firm attachment: 16.4±4.5 versus 54.7±8.9 cells/mm, respectively, P<0.01).
Circulating Levels of P-Selectin, E-Selectin, and MCP-1 in Obese Psgl-1–Deficient Mice
To determine whether reduced leukocyte-endothelial interactions in genetically and diet-induced Psgl-1 deficient mice were associated with reduced circulating levels of soluble adhesion molecules and MCP-1, sP-sel, sE-sel, and MCP-1 were measured from Leprdb/db,Psgl-1−/−, Leprdb/db,Psgl-1+/+ mice, and from Psgl-1−/− and Psgl-1+/+ mice on a high-fat, high-sucrose diet. At 5, 10, and 15 weeks of age, the levels of sP-sel, sE-sel, and MCP-1 were significantly reduced in Leprdb/db,Psgl-1−/− compared with Leprdb/db,Psgl-1+/+ mice (Figure 1A through 1C). Similarly, in diet-induced obesity at 5, 10, and 15 weeks of age, the levels of sP-sel and sE-sel were significantly reduced in Psgl-1−/− compared with Psgl-1+/+ mice (Figure 1D and1 E). MCP-1 was significantly different between Psgl-1−/− and Psgl-1+/+ mice at 5 weeks of age (Figure 1F).
Effect of Psgl-1 Deficiency on Leukocyte Recruitment and Inflammatory Cytokine Expression In Adipose Tissue in Obese Psgl-1–Deficient Mice
To determine if the decreased leukocyte-endothelial interactions observed in Leprdb/db,Psgl-1−/− mice compared with Leprdb/db,Psgl-1+/+ mice were associated with reduced adipose tissue macrophage content, macrophage immunostaining was performed on visceral (perigonadal and pericardial) and subcutaneous adipose depots retrieved from 15-week-old obese Leprdb/db,Psgl-1−/− and Leprdb/db,Psgl-1+/+ mice. The macrophage content of perigonadal and pericardial fat was significantly reduced in Leprdb/db,Psgl-1−/− mice compared with Leprdb/db,Psgl-1+/+ mice (Figure 2A, Online Figure I A-F). No significant difference in macrophage content was observed in subcutaneous fat between Leprdb/db,Psgl-1−/− and Leprdb/db,Psgl-1+/+ mice (Figure 2A, Online Figure I A-F). Similarly, Psgl-1−/− mice on a high-fat, high-sucrose diet for 10 weeks had a significant reduction in macrophage content of perigonadal fat compared with Psgl-1+/+ control mice (Figure 2B through 2F). No significant difference in macrophage content was observed in subcutaneous fat between these mice (Figure 2B through 2F).
To determine the effect that Psgl-1 deficiency in obesity has on macrophage infiltration in different adipose depots compared with lean mice, macrophage content of subcutaneous and perigonadal fat in Leprdb/db,Psgl-1−/− and Leprdb/db,Psgl-1+/+ mice was compared with lean Psgl-1−/− and Psgl-1+/+ mice. There was no difference in macrophage content of perigonadal fat in Psgl-1−/− (n=5) versus Psgl-1+/+ (n=3) lean mice (14.9±1.0% versus 16.4±0.8% mac3+ cells, respectively, P=NS). There was also no difference in macrophage content of subcutaneous fat in Psgl-1−/− (n=4) versus Psgl-1+/+ (n=3) lean mice (9.7±0.6 versus 8.9±0.1% mac3+ cells, respectively, P=NS). Both lean Psgl-1−/− and Psgl-1+/+ had a significantly higher macrophage content in perigonadal fat compared with subcutaneous fat (Psgl-1−/−: 14.9±1.0% versus 9.7±0.6% mac3+ cells, respectively, P<0.01; Psgl-1+/+: 16.4±0.8% versus 8.9±0.1% mac3+ cells, respectively, P<0.001). There was no difference in macrophage content of subcutaneous fat in Psgl-1−/− compared with Leprdb/db,Psgl-1−/− mice (9.7±0.6% versus 14.8±3.2% mac3+ cells, respectively, P=NS) or Psgl-1+/+ compared with Leprdb/db,Psgl-1+/+ mice (8.9±0.1 versus 15.8±3.9% mac3+ cells, respectively, P=NS). There was a significantly higher macrophage content in perigonadal fat in Leprdb/db,Psgl-1−/− compared with Psgl-1−/− mice (32.8±4.7% versus 14.9±1.0% mac3+ cells, respectively, P<0.05) and in Leprdb/db,Psgl-1+/+ compared with Psgl-1+/+ mice (66.1±2.7% versus 16.4±0.8% mac3+ cells, respectively, P<0.001); however, the rise in macrophage infiltration into perigonadal fat with obesity was significantly higher between Leprdb/db,Psgl-1+/+ and Psgl-1+/+ mice compared with Leprdb/db,Psgl-1−/− and Psgl-1−/− mice (49.8±2.7% versus 17.8±4.7% increase in mac3+ cells, respectively, P<0.001).
The expression of MCP-1 and TNFα was measured in perigonadal adipose tissue retrieved from 15-week-old obese Leprdb/db,Psgl-1−/− (n=14) and Leprdb/db,Psgl-1+/+ (n=13) mice. Leprdb/db,Psgl-1−/− mice had reduced expression of MCP-1 and TNFα in perigonadal adipose tissue compared with Leprdb/db,Psgl-1+/+ controls (MCP-1: 2.33±0.3 versus 4.05±0.7 relative expression units, respectively, P<0.05; TNFα: 3.4±0.4 versus 5.84±0.6 relative expression units, respectively, P<0.01).
Effect of Adoptive Transfer of Wild-Type Leukocytes Into Obese Psgl-1–Deficient Mice
To determine whether the effect of Psgl-1 on leukocyte rolling was due to lack of direct, acute Psgl-1 interactions with endothelial selectins or due to an indirect, more chronic effect of Psgl-1 on adhesive properties of the endothelium, adoptive transfer of rhodamine-labeled leukocytes from Psgl-1+/+ donors into Leprdb/db,Psgl-1−/− (n=3) and Leprdb/db,Psgl-1+/+ (n=5) recipients was performed. Transferred leukocytes consisted of 91.5±3.3% lymphocytes, 0.65±0.5% monocytes, and 5.5±3% neutrophils (n=3).
Control adoptive transfer experiments showed that rhodamine-labeled Psgl-1+/+ donor leukocytes infused into Leprdb/db,Psgl-1+/+ mice exhibited similar leukocyte rolling characteristics as endogenous rhodamine-labeled Leprdb/db,Psgl-1+/+ leukocytes (96.8±12.9 versus 108.7±14.4 cells/mm, respectively, P=NS). When rhodamine-labeled Psgl-1+/+ donor leukocytes were infused into Leprdb/db,Psgl-1−/− mice, the donor leukocyte rolling was markedly less than that observed when Psgl-1+/+ donor leukocytes were infused into Leprdb/db,Psgl-1+/+ mice (2.1±0.7 versus 96.8±12.9 cells/mm, respectively, P<0.001). The donor leukocyte firm attachment was also less in Leprdb/db,Psgl-1−/− mice compared with Leprdb/db,Psgl-1+/+ mice (8.8±2.7 versus 18±2.9 cells/mm, respectively, P<0.05).
Effect of Bone Marrow–Derived Psgl-1 on sP-sel Levels and Leukocyte-Endothelial Interactions in Obese Mice
Because the adoptive transfer experiments suggested that Psgl-1 deficiency affects the activation state of the endothelium, BMT experiments were performed to identify the potential source of Psgl-1 responsible for regulating the endothelium. The BMT involved transplanting Psgl-1+/+ or Psgl-1−/− bone marrow into Leprdb/db,Psgl-1+/+ recipients. To identify the contribution of the hematopoietic Psgl-1 pool on the generation of sP-sel in obesity, sP-sel was measured 4 weeks after BMT. sP-sel was markedly reduced in Leprdb/db,Psgl-1+/+ mice receiving Psgl-1−/− marrow (n=6) compared with Leprdb/db,Psgl-1+/+ mice receiving Psgl-1+/+ marrow (n=3) (45.7±3.8 versus 166.5±5.7 ng/mL, respectively, P<0.001).
Ten weeks post-BMT, the extent of leukocyte-endothelial interactions was measured in these mice using intravital microscopy. Leukocyte rolling and firm attachment was markedly reduced in Leprdb/db,Psgl-1+/+ mice receiving Psgl-1−/− marrow (n=5) compared with Leprdb/db,Psgl-1+/+ mice receiving Psgl-1+/+ marrow (n=4) (rolling: 3.6±1.1 versus 87.0±7.7 cells/mm, respectively, P<0.001; firm attachment: 9.0±2.2 versus 36.0±6.3 cells/mm, respectively, P<0.001). These results implicate the hematopoietic Psgl-1 in regulating the activation state of the endothelium.
Because both platelets and endothelial cells are rich sources of P-sel, bone marrow transplants were performed from wild-type (Psel+/+) or P-sel deficient (Psel−/−) donors into Leprdb/db,Psgl-1+/+ recipients to determine the predominant cellular source of sP-sel. Circulating sP-sel levels in obese mice receiving Psel−/− bone marrow (n=6) were not different from mice receiving Psel+/+ marrow (153.2±3.7 versus 166.5±5.7 ng/mL, respectively, P=NS), implicating the endothelium as the major source of sP-sel in obese mice.
Psgl-1 Regulates Response to Recombinant IL-1β
Because IL-1β has previously been shown to induce selectin-dependent leukocyte rolling,14 we explored the possibility that leukocyte Psgl-1 might affect endothelial expression of selectins and MCP-1 in response to IL-1β stimulation. Therefore, either Psgl-1+/+ or Psgl-1−/− mice were stimulated with recombinant IL-1β. As a control, mice deficient in the IL-1 receptor (IL-1R−/−) were also analyzed for potential nonspecific effects of the recombinant IL-1β preparation. Five hours after an intravenous injection of recombinant IL-1β (500 ng), sP-sel, MCP-1, sE-sel, and sICAM-1 levels were measured from serum. IL-1β-induced serum levels of sP-sel, sE-sel, MCP-1, and sICAM-1 were completely blocked in IL-1R−/− mice, as expected (Figure 3A through 3D). Compared with serum from Psgl-1+/+ mice, IL-1β-induced changes in Psgl-1−/− mice were either completely blocked (sP-sel, MCP-1) or reduced (sE-sel and sICAM-1) (Figure 3A through 3D). Surprisingly, basal levels of sP-sel were reduced in IL-1R−/− mice similar to that observed in the Psgl-1−/− mice, indicating that even basal sP-sel levels are regulated via the IL-1 receptor (IL-1R) (Figure 4).
To confirm that Psgl-1 deficiency in the setting of obesity was also associated with resistance to IL-1β, Leprdb/db,Psgl-1−/− mice (n=4) and Leprdb/db,Psgl-1+/+ mice (n=3) were given 500 ng of recombinant IL-1β. Similar to lean mice, there was no increase in sP-sel in Leprdb/db,Psgl-1−/− mice after IL-1β injection (83.3±14.1 pre-IL-1β versus 83.9±3.5 ng/mL after IL-1β, P=NS), whereas there was a marked increase in sP-sel in Leprdb/db,Psgl-1+/+ mice (183.2±3.5 pre-IL-1β versus 533.1±38.3 ng/mL after IL-1β, P<0.001).
To determine the effect of Psgl-1 deficiency on transcriptional regulation of endothelial selectins (P-sel and E-sel) and MCP-1 after stimulation with recombinant IL-1β, lung tissue (as a rich source of endothelial cells) and perigonadal fat were removed for RNA isolation 5 hours after intravenous IL-1β challenge.
Psgl-1+/+ mice injected with IL-1β had a higher expression of P-sel, E-sel, and MCP-1 in the lung compared with Psgl-1+/+ mice injected with PBS (Figure 3E). There was no difference in P-sel, E-sel, and MCP-1 expression in the lung between Psgl-1−/− and IL-1R−/− mice injected with IL-1β compared with Psgl-1−/− and IL-1R−/− mice injected with PBS (Figure 3E). Psgl-1+/+ mice injected with IL-1β also had a higher expression of P-sel and MCP-1 and a trend toward higher expression of E-sel in perigonadal fat compared with Psgl-1+/+ mice injected with PBS (Figure 3F). There was no difference in P-sel, E-sel, and MCP-1 expression in perigonadal fat among Psgl-1−/− and IL-1R−/− mice injected with IL-1β compared with Psgl-1−/− and IL-1R−/− mice injected with PBS (Figure 3F).
Psgl-1−/− mice were also resistant to the effects of recombinant IL-1β on leukocyte rolling compared with Psgl-1+/+ mice (2.8±1 versus 88.5±10.5 cells/mm, respectively, P<0.001).
Relevant IL-1R Pools for Regulation of Endothelial Selectins
To further characterize the cellular pool of IL-1R relevant for the response to IL-1β, chimeric IL-1R−/− mice were generated by BMT. After IL-1β challenge, sP-sel levels increased in IL-1R+/+ mice with IL-1R−/− marrow and did not change in IL-1R−/− mice with IL-1R+/+ marrow, indicating that P-sel, E-sel, and MCP-1 expression is regulated via nonhematopoietic IL-1R signaling (Figure 5A).
To address the cellular Psgl-1 pools relevant for the response to IL-1β, chimeric Psgl-1−/− mice were also generated by BMT. After IL-1β challenge, sP-sel levels increased in Psgl-1−/− mice with Psgl-1+/+ marrow and did not change in Psgl-1+/+ mice with Psgl-1−/− marrow, indicating that hematopoietic Psgl-1 is regulating the endothelial response to IL-1β via endothelial IL-1R signaling (Figure 5B).
Psgl-1 Effects on NFκB Activation
IκBα retains NFκB dimers in the cytoplasm. On stimulation IκBα is rapidly phosphorylated, a signal that promotes its degradation by the proteasome, thus allowing NFκB dimers to shuttle to the nucleus and initiate gene transcription events.15 Therefore, phosphorylation and degradation of IκBα indicates activation of the NFκB signaling pathway.
To determine if resistance to the endothelial effects of exogenous IL-1β in Psgl-1−/− mice was associated with reduced signaling via NFkB activation pathways, immunoblotting of lung tissue lysate was performed at various time points after IL-1β administration. Lung lysates from Psgl-1−/− mice exhibited less phosphorylation of IκBα 60 minutes after IL-1β challenge, while lysates from Psgl-1+/+ mice exhibited more degradation of IκBα 30 and 60 minutes after IL-1β challenge, indicating reduced activation of the NFκB signaling pathway in Psgl-1−/− mice (Figure 6). p38 was measured as a loading control. Taken together, Psgl-1 deficiency is associated with reduced activation of NFκB in response to IL-1β and reduced expression of MCP-1, P-sel, and E-sel.
Adipose tissue inflammation may contribute to comorbidities associated with obesity.2,4 Leukocyte-endothelial interactions are increased in visceral adipose tissue depots in obese mice.6 These increased leukocyte-endothelial interactions are associated with increased expression of P- and E-sel in endothelial cells from visceral adipose depots.6 Furthermore, antibody blockade of P- and E-sel reduced leukocyte rolling and firm attachment in obese visceral adipose tissue.6 Psgl-1 is the primary ligand for P-sel7 and a major ligand for E-sel.8 Leukocytes from Psgl-1−/− mice show reduced selectin-dependent rolling.9,10
To further explore the implications of selectin-ligand interactions in adipose tissue inflammation, we studied Psgl-1 deficiency in genetic and diet-induced mouse models of obesity. Leprdb/db mice lack leptin receptor signaling and become obese and hyperglycemic at an early age.13 Obese mice lacking leptin or a functional leptin receptor have been shown to develop increased macrophage fat infiltration compared with lean mice.2 Thus, this model is suitable for studying factors involved in obesity-related adipose tissue macrophage trafficking.6 In the current study, Leprdb/db,Psgl-1−/− mice and Psgl-1−/− mice on a high-fat, high-sucrose diet developed normally up to 15 weeks of age, with a similar gain in body weight and fat mass, compared with Leprdb/db,Psgl-1+/+ mice and Psgl-1+/+ mice on a high-fat, high-sucrose diet, respectively. To determine if deficiency of Psgl-1 affected leukocyte-endothelial interactions in obesity, we first needed to establish a model where interactions between leukocytes and endothelial cells were increased in the setting of obesity. Nishimura et al6 previously demonstrated that leukocyte-endothelial interactions were increased locally within visceral adipose depots in obese mice. In the current study, we found that obesity also induces marked increases in leukocyte rolling in cremaster venules of obese Leprdb/db,Psgl+/+ mice at 15 weeks of age compared with lean Lepr +/+,Psgl-1+/+ mice. Thus, the effect of obesity on leukocyte-endothelial interactions may be more systemic than previously suggested, although we cannot rule out a local effect of the adipose tissue surrounding the cremasteric vessels. Irrespective of the mechanism, the cremaster vessels appear to be suitable to study leukocyte-endothelial interactions triggered by obesity in mice and are more suitable for analysis using standard intravital microscopy techniques. We found that the effect of genetic and diet-induced obesity on leukocyte-endothelial rolling was neutralized in the setting of Psgl-1 deficiency suggesting that interactions between Psgl-1 and endothelial selectins are important for obesity-induced leukocyte rolling.
To determine if the decreased leukocyte-endothelial interactions observed in genetically or diet-induced obese Psgl-1−/− mice were associated with reduced adipose tissue macrophage content, macrophage immunostaining was performed on visceral and subcutaneous adipose tissue from those mice. Psgl-1 deficiency in both models of obesity resulted in reduced macrophage content in visceral adipose tissue, while no differences were observed in subcutaneous adipose tissue. Macrophage content was also analyzed in visceral and subcutaneous adipose tissue from lean Psgl-1−/− and Psgl-1+/+ mice. Psgl-1 deficiency was found to decrease the obesity induced rise in macrophage infiltration found in visceral fat. Obesity did not result in an increase in macrophage infiltration into subcutaneous fat, and Psgl-1 deficiency had no effect on macrophage content in subcutaneous fat in lean or obese mice, indicating differential regulation of adipose tissue macrophage infiltration between different adipose depots.
MCP-1 and TNFα expression has been shown to increase in visceral fat from obese mice.16,17 Psgl-1 deficiency in obese mice was associated with reduced MCP-1 and TNFα expression in visceral fat, perhaps due to decreased macrophage content in visceral fat.
Because sP-sel levels are positively regulated by leukocyte Psgl-1 in the basal state,18 we determined if sP- and sE-sel levels in both genetic and diet-induced obesity were also reduced in the setting of Psgl-1 deficiency. In this study, sP-sel and sE-sel were lower in Leprdb/db,Psgl-1−/− mice and Psgl-1−/− mice on a high-fat, high-sucrose diet compared with Leprdb/db,Psgl-1+/+ mice and Psgl-1+/+ mice on a high-fat, high-sucrose diet, respectively. MCP-1 was also lower in Leprdb/db,Psgl-1−/− mice compared with Leprdb/db,Psgl-1+/+ mice. Collectively, these data indicate a regulatory role of Psgl-1 toward the generation of these soluble molecules in obesity.
To determine whether the effects of Psgl-1 on leukocyte-endothelial interactions in obesity were due to direct, acute interactions with endothelial selectins or to other, more chronic, effects of Psgl-1 on endothelial adhesive characteristics, adoptive transfer of rhodamine-labeled leukocytes was performed from Psgl-1+/+ mice into obese Leprdb/db,Psgl-1+/+ and Leprdb/db,Psgl-1−/− mice. Unexpectedly, donor Psgl-1+/+ leukocyte rolling was markedly reduced in the Leprdb/db,Psgl-1−/− recipients compared with the Leprdb/db,Psgl-1+/+ recipients. One explanation for these findings is that leukocyte Psgl-1 chronically upregulates endothelial adhesive molecules by an unclear mechanism.
BMT was performed to determine the cellular source of Psgl-1 in obese mice responsible for altering the adhesive properties of the endothelium. Genetically obese mice receiving bone marrow from Psgl-1−/− mice displayed reduced sP-sel levels in addition to reduced rolling and firm attachment compared with genetically obese mice receiving wild-type bone marrow, indicating that hematopoietic Psgl-1 is responsible for chronically altering the adhesive characteristics of the endothelium. BMT with P-sel deficient donors indicated the predominant tissue source of sP-sel in the obese state was nonhematopoietic.
The findings above suggested that hematopoietic Psgl-1 plays a major role in regulating adhesive characteristics of the endothelium in obesity. Because cytokines such as IL-1β have been shown to induce selectin dependent rolling,14,19 we tested the hypothesis that Psgl-1 would positively regulate the endothelial response to IL-1β, while mice deficient in Psgl-1 would be resistant to IL-1β stimulation. Consistent with this hypothesis, sP-sel and MCP-1 levels in wild-type mice markedly increased after administration of recombinant IL-1β, whereas sP-sel and MCP-1 levels in Psgl-1−/− mice were unchanged after IL-1β stimulation. The rises in sE-sel and sICAM-1 were also attenuated in Psgl-1−/− mice after IL-1β treatment. The reduced circulating levels of these proteins were associated with corresponding reduced gene transcription in lung and perigonadal adipose tissue. The cell source of the transcriptional differences were attributed to the endothelial cells within lung and perigonadal adipose tissue because the factors measured were either exclusively or primarily expressed by the endothelium.18,20–23 Lung tissue was used for transcriptional studies because it is a rich source of endothelial cells, and inflammation in the lung is regulated by IL-1β.24 Overall, the response of Psgl-1−/− mice to IL-1β was similar to that of mice with complete deficiency of the IL-1R. To further confirm the critical role of IL-1R signaling for regulation of sP-sel under basal, unstimulated conditions, sP-sel levels were markedly reduced in IL-1R−/− mice.
Although several cell types have been shown to express Psgl-1 and the IL-1R, the BMT experiments between Psgl-1−/− mice and Psgl-1+/+ mice and between IL-1R−/− and IL-1R+/+ mice indicate that hematopoietic Psgl-1 (ie, leukocyte) is regulating the nonhematopoietic IL-1R signaling (ie, endothelium). The precise cellular source and further downstream mechanism(s) by which Psgl-1 regulates the adhesive properties of the endothelium will require additional study. However, it appears that the presence of Psgl-1 facilitates or the deficiency of Psgl-1 induces a block in pathways leading to NFκB activation, which probably are responsible for the subsequent changes in adhesion molecule expression (Figure 7).
The effect of Psgl-1 on glucoregulation in genetically obese mice may be secondary to the reduced macrophage content of visceral adipose depots as preclinical studies have demonstrated that inflammatory fat leads to insulin resistance.25 This effect on glucose was not observed in the diet-induced obese mice suggesting a more severe state of diabetes may be required to reveal this protective effect of Psgl-1 deficiency on hyperglycemia. Other potential mechanisms for improved glucoregulation may involve IL-1R signaling effects on pancreatic beta cell insulin secretion.26 Of clinical interest, trials with IL-1R inhibitors are associated with improved glucoregulation in humans.27
Taken together, these results indicate an important role of hematopoietic Psgl-1 deficiency in the regulation of the adhesive properties of the endothelium via attenuation of endothelial signaling pathways. As a result, Psgl-1 deficiency is protective against visceral adipose tissue inflammation in obesity. Further elucidation of mechanisms by which leukocytes regulate the endothelial response to cytokines may lead to new treatments aimed at reducing the chronic inflammatory state associated with obesity.
Sources of Funding
This study was supported by National Institute of Health grants HL57346 and HL073150 (D.E.) and by a Fellowship from the Arthritis Foundation (L.F.).
In May 2010, the average time from submission to first decision for all original research papers submitted to Circulation Research was 14.6 days.
Non-standard Abbreviations and Acronyms
- bone marrow transplant
- dual energy x-ray absorptiometry
- Interleukin-1 beta
- Interleukin-1 receptor
- monocyte chemoattractant protein-1
- P-selectin glycoprotein ligand-1
- tumor necrosis factor-alpha
- Received February 9, 2010.
- Revision received June 2, 2010.
- Accepted June 3, 2010.
- © 2010 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
P-selectin glycoprotein ligand-1(Psgl-1) is a ligand on white blood cells that mediates binding to adhesion molecules on endothelial cells and other cell types.
Deficiency of Psgl-1 reduces inflammation in some disease states.
Obesity is associated with inflammation in visceral fat depots.
What New Information Does This Article Contribute?
Deficiency of Psgl-1 leads to reduced visceral fat inflammation in obesity.
Psgl-1 plays an important role in controlling adhesive interactions between blood cells and endothelial cells through an indirect mechanism.
Psgl-1 deficiency leads to reduced endothelial activation in response to cytokine stimulation.
Both Psgl-1–deficient mice and mice deficient in the IL-1 receptor have similarly low levels of circulating P-selectin in the basal state
Inflammation in obese visceral fat may contribute to the adverse consequences of obesity. Identification of the factors responsible for fat inflammation may lead to new treatments aimed at reducing the risks associated with obesity. This study indicates that a leukocyte ligand, Psgl-1, plays an important role in visceral fat inflammation. Furthermore, the absence of Psgl-1 on blood cells leads to reduced activation of the endothelial cells after cytokine stimulation. This is associated with reduced transcription of some adhesion molecules and reduced leukocyte rolling on endothelial cells. Therapeutic antagonism of Psgl-1 may be beneficial in reducing the chronic inflammatory state associated with obesity. In addition, elucidation of the precise mechanism by which Psgl-1 affects adhesive properties of endothelial cells may lead to new targets for the treatment of inflammatory disease states.