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Am J Physiol Endocrinol Metab 285: E403-E411, 2003. First published March 11, 2003; doi:10.1152/ajpendo.00576.2002
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Acute inflammation increases selective uptake of HDL cholesteryl esters into adrenals of mice overexpressing human sPLA2

Uwe J. F. Tietge,1 Cyrille Maugeais,1 Willliam Cain,2 and Daniel J. Rader1

1Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104; and 2Department of Biology, University of Delaware, Newark, Delaware 19716

Submitted 30 December 2002 ; accepted in final form 18 February 2003


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
The acute-phase protein secretory phospholipase A2 (sPLA2) influences the metabolism of high-density lipoproteins (HDL). The adrenals are known to utilize HDL cholesterol as a source of sterols. The aim of the present study was to test the hypothesis that sPLA2 enhances the selective uptake of HDL into the adrenals in response to acute inflammation as a possible physiological role for the sPLA2-HDL interaction. Human sPLA2-transgenic mice, in which sPLA2 expression is upregulated by inflammatory stimuli, were used. Ten hours after induction of the acute-phase response (APR) by injection of bacterial lipopolysaccharide (LPS), plasma levels of HDL cholesterol decreased significantly in sPLA2-transgenic mice (-18%, P < 0.05) but remained unchanged in wild-type mice. The fractional catabolic rates of both 125I-labeled tyraminecellobiose (TC)-HDL and [3H]cholesteryl ether increased significantly in the sPLA2-transgenic mice after induction of the APR (0.18 ± 0.01 vs. 0.21 ± 0.01 pool/h, P < 0.05, and 0.31 ± 0.02 vs. 0.42 ± 0.05 pool/h, P < 0.05, respectively) but remained unchanged in the wild-type mice (0.10 ± 0.01 vs. 0.22 ± 0.02 pool/h, respectively). After induction of the APR, in both groups HDL holoparticle uptake by the liver was increased (P < 0.001). sPLA2-transgenic mice had 2.4-fold higher selective uptake into the adrenals after induction of the APR than wild-type mice (156 ± 6 vs. 65 ± 5%/µg tissue protein, P < 0.001). In summary, upregulation of sPLA2 expression during the APR specifically increases the selective uptake of HDL cholesteryl ester into the adrenals. These data suggest a novel metabolic role for sPLA2: modification of HDL during the APR to promote increased adrenal uptake of HDL cholesteryl ester to serve as source for steroid hormone synthesis.

apolipoproteins; lipoproteins; kinetics; acute-phase response; lipopolysaccharide

DURING THE ACUTE-PHASE RESPONSE (APR), plasma levels of HDL cholesterol are markedly decreased (3, 15). Among the acute-phase proteins that might have an impact on plasma HDL cholesterol metabolism is the secretory group IIa phospholipase A2 (sPLA2). The family of phospholipase genes with a potential impact on lipoprotein metabolism is growing, with the latest discovered being phosphatidylserine-specific phospholipase A1 (PS-PLA1) (1) and lipase H (18). sPLA2 is a 14-kDa Ca2+-dependent enzyme that hydrolyzes phospholipids at the sn-2 position (8). sPLA2 levels in plasma increase dramatically upon acute inflammation (27), and plasma sPLA2 activity has been localized to the HDL fraction in humans (9). We recently reported that transgenic overexpression of sPLA2 in mice in the absence of systemic inflammation has profound effects on HDL metabolism: mice overexpressing human sPLA2 have significantly lower plasma levels of HDL cholesterol due to increased catabolism of HDL apolipoprotein A-I (apoA-I), as well as HDL cholesteryl esters (CEs) (34). However, the physiological significance of sPLA2 expression during acute inflammation and its effects on HDL metabolism remain unclear.

Transgenic mice overexpressing human sPLA2 survive challenges with both Staphylococcus aureus (23) and Escherichia coli (22) better than wild-type mice, suggesting that sPLA2 somehow protects against acute bacterial challenge. However, the mechanism(s) of this protection is unknown. We hypothesized that the metabolic effects of sPLA2 on HDL metabolism might be related to its protective effects. For example, it is well established that human apoA-I-transgenic mice are relatively protected against endotoxin challenge (24). In acute inflammation, the need for adrenal steroid hormone production is increased, and HDL-CEs are a source of cholesterol for steroid hormone synthesis (28, 30, 33, 41, 43). In vitro studies (6) have suggested that sPLA2 modification of HDL makes the CE component of the particle more accessible to selective uptake via the scavenger receptor class B type I (SR-BI) receptor. In separate experiments, we found that hepatic overexpression of SR-BI enhanced selective uptake of HDL-CE to a greater extent in sPLA2/apoA-I-double transgenic mice than in apoA-I-transgenic mice (Tietge UJF, unpublished data). Thus one possible function of sPLA2 might be to enhance the uptake of HDL-CEs into the adrenals to meet the requirements for increased steroid hormone synthesis.

Therefore, the purpose of our study was to test the hypothesis that sPLA2 induction during the APR increases the selective uptake of HDL-CE into the adrenals in vivo. The human sPLA2-transgenic mouse model we utilized in our previous study (34) represents an ideal tool for addressing this question, because the sPLA2 transgene expression is controlled by its own promoter and thereby confers upregulated sPLA2 expression in response to inflammatory stimuli (23). Therefore, we used human sPLA2-transgenic and C57BL/6 mice, which lack endogenous sPLA2 (19), to perform a series of HDL kinetic studies after induction of the APR with bacterial lipopolysaccharide (LPS) to assess plasma catabolism and tissue uptake of HDL apoproteins as well as HDL-CEs. Our data indicate that, in human sPLA2-transgenic mice, upregulation of sPLA2 in response to acute inflammation results in significantly increased selective uptake of HDL-CE into the adrenals, suggesting a potentially important novel physiological function of sPLA2.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Animal studies. The human group II sPLA2-transgenic mice used in this study have been described previously (13, 34). Briefly, a 6.2-kb HindIII restriction fragment, containing all five exons and 1.6 kb of nucleotides upstream from the RNA initiation site and 0.35 kb of nucleotides downstream of the polyadenylation signal sequence of the human sPLA2 gene, was used to generate transgenic mice by standard procedures. The sPLA2-transgenic line has been backcrossed to the C57BL/6 background. Nontransgenic littermates from further breeding of sPLA2-transgenic mice with wild-type C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) were used as controls. The animals were caged in animal rooms with alternating 12-h periods of light (7 AM-7 PM) and dark (7 PM-7 AM) and with ad libitum access to water and mouse chow diet.

The APR was induced by intraperitoneal injection of 40 µg of LPS (E. coli 0111:B4, Difco Laboratories, Detroit, MI) (16) or sterile saline. Blood was obtained by retroorbital bleeding before injection and 10 h after injection for determination of plasma lipid and lipoprotein levels. At the 10-h time point, mice were injected with the radiotracers, and the HDL kinetic studies were carried out as described below.

Plasma lipid and lipoprotein analysis. Mice were bled from the retroorbital plexus by use of heparinized capillary tubes. Blood was drawn into tubes containing 2 mM EDTA, 0.2% NaN3, and 1 mM benzamidine. Aliquots were stored at -20°C until analysis. Plasma total cholesterol, HDL cholesterol, triglycerides, phospholipids, and human apoA-I levels were determined on a Cobas Fara (Roche Diagnostics Systems, Nutley, NJ) by use of Sigma Diagnostics reagents (Sigma Diagnostics, St. Louis, MO).

Western blotting. Western blot analysis for human group II sPLA2 was performed, as previously described (34), with a monoclonal mouse anti-human sPLA2 primary antibody at a concentration of 2 µg/ml (Boehringer Mannheim, Mannheim, Germany) and the enhanced chemiluminescence (ECL) detection system (Amersham, Arlington Heights, IL).

HDL metabolic studies. Autologous HDL was prepared from pooled mouse plasma by sequential ultracentrifugation (density: 1.063 < d < 1.21). After extensive dialysis against sterile PBS containing 0.01% EDTA, one part of the HDL was labeled with 125I-tyramine-cellobiose (TC), as previously described (34). Unbound iodine was removed by passing the solution over a Sephadex G25 desalting column (Amersham Pharmacia Biotech). Finally, the HDL was reisolated by ultracentrifugation (density: 1.063 < d <1.21), extensively dialyzed, sterile filtered, and stored at 4°C until use (34). The other part of the isolated mouse HDL was labeled with cholesteryl hexadecyl ether (cholesteryl-1,2-3H, NEN Life Sciences Products) according to a previously published methodology (34). After labeling, the HDL was reisolated by ultracentrifugation (density: 1.063 < d <1.21), extensively dialyzed, sterile filtered, and stored at 4°C until injection.

To assess the rates of plasma catabolism and to measure the organ uptake of the different tracers, 1 µCi of the 125I-TC-HDL and 1 million dpm of the [3H]CE-HDL were coinjected via the tail veins into mice from the different experimental groups. Blood samples were drawn by retroorbital bleeding at 5 min, 1 h, 3 h, 6 h, 11 h, and 24 h (~25 µl at each time point). An aliquot of 10 µl of plasma from each time point was counted using a Cobra II {gamma}-counter (Packard Instruments, Downers Grove, IL). These data were used to generate the plasma disappearance curves for HDL apolipoproteins. After {gamma}-counting, the same samples were extracted with the method described by Dole (7). The lipid phases were harvested, and aliquots were counted on a scintillation counter (Beckman LS6500; Beckman Instruments, Palo Alto, CA). From these data, plasma disappearance curves for HDL-CEs were calculated. The recovery of [3H]cholesteryl ether after lipid extraction was 97.5 ± 1.9%. Contamination of the extracted 3H-labeled lipids with 125I was <0.5%. Plasma decay curves for both tracers were generated by dividing the plasma radioactivity at each time point by the radioactivity at the initial 5-min time point after tracer injection. Fractional catabolic rates (FCRs) were determined from the area under the plasma disappearance curves fitted to a bicompartmental model by use of the SAAM II program (2).

To measure the organ uptake of HDL apolipoproteins and HDL-CEs, mice were anesthetized with an intraperitoneally injected ketamine-xylazine mixture and perfused extensively with cold PBS by cardiac puncture (34). Then liver, spleen, kidney, and adrenals were harvested. First, radioactivity uptake (125I) into the different organs was assessed by {gamma}-counting to determine uptake of HDL apolipoproteins. Then, the different organs were extracted using the Dole method, and the amount of [3H]cholesteryl ether taken up into each organ was determined on a scintillation counter (Beckman LS6500). Organ uptake for each tracer was expressed as a percentage of the injected dose. The injected dose was calculated by multiplying the initial plasma counts (5-min time point) with the estimated plasma volume (3.5% of total body wt). Selective uptake into each organ was determined by subtracting the percentage of the injected dose of 125I-HDL recovered in each organ from the percentage of the injected dose of [3H]HDL-CE and correcting the value for tissue weight. Values were expressed as micrograms of tissue weight for each organ.

Statistical analysis. Values are presented as means ± SE unless otherwise indicated. Results were analyzed by ANOVA and Student's t-test using the GraphPad Prism Software (GraphPad, San Diego, CA). Statistical significance for all comparisons was assigned at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 DISCLOSURES
 REFERENCES
 
In sPLA2-transgenic mice, injection of saline did not result in any detectable change in plasma sPLA2, whereas plasma sPLA2 was increased 10 h after induction of the APR by injection of LPS (Fig. 1). These data, consistent with a previous report (23), demonstrate that the sPLA2 in these transgenic mice is upregulated by acute inflammation. At baseline, sPLA2-transgenic mice had significantly lower plasma levels of total cholesterol (61 ± 3 vs. 85 ± 7 mg/dl, P < 0.01, Fig. 2A and Table 1), HDL-cholesterol (HDL-C; 44 ± 3 vs. 64 ± 5 mg/dl, P < 0.01, Fig. 2B, Table 1), and phospholipids (116 ± 9 vs. 179 ± 4 mg/dl, P < 0.01, Fig. 2C and Table 1) compared with wild-type mice, consistent with our previous report (34). In both saline-injected groups, plasma lipid levels remained relatively unchanged compared with baseline values (Fig. 2, A—C). After LPS challenge in wild-type mice, plasma total and HDL-C levels did not change significantly compared with baseline values (Fig. 2, A and B), but plasma phospholipid levels decreased significantly by 19 ± 10% (P < 0.05 compared with baseline values, Fig. 2C). In contrast, in sPLA2-transgenic mice, plasma levels of total cholesterol and HDL-C decreased significantly after LPS injection compared with baseline values (-12 ± 5%, P < 0.05, Fig. 1A; -18 ± 6%, P < 0.01, Fig. 1B, respectively), and phospholipids also decreased by 11 ± 6% (Fig. 1C, P < 0.05 compared with baseline values). Thus absence of sPLA2 is associated with a lack of HDL response to endotoxin, whereas expression of inducible sPLA2 confers responsiveness to LPS injection with regard to reduction of HDL-C levels.



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  		Fig. 1. Western blot analysis for human secretory phospholipase A2 (sPLA2) in plasma from sPLA2-transgenic mice at baseline (0 h) and 10 h after injection with either saline or LPS as indicated. Samples were resolved by SDS-PAGE under nondenaturing conditions. Human sPLA2 was visualized using a monoclonal mouse anti-human sPLA2 antibody, as described in EXPERIMENTAL PROCEDURES.

 


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  		Fig. 2. Percent change of plasma lipids and lipoproteins 10 h after either the intraperitoneal injection of saline or the induction of the acute-phase response (APR) by injection of LPS in human sPLA2-transgenic mice and C57BL/6 controls. A: total cholesterol; B: HDL cholesterol; C: phospholipids.

 

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  		Table 1. Plasma lipid and lipoprotein profiles in human sPLA2-transgenic mice and wild-type nontransgenic littermates

 

To assess the metabolic basis for the changes in HDL, we performed a series of HDL metabolism studies simultaneously tracing the catabolism of both HDL apolipoproteins and HDL-CEs in wild-type and sPLA2-transgenic mice in reponse to endotoxin injection. We determined the rate and sites of HDL apolipoprotein catabolism by using 125I-TC-HDL as a tracer. Saline-injected sPLA2-transgenic mice had significantly faster plasma catabolism of HDL apolipoproteins compared with saline-injected wild-type controls, (0.18 ± 0.01 vs. 0.10 ± 0.01 pool/h, P < 0.001, Fig. 3, A and B, and Table 2), consistent with our previous observations (34). LPS injection had no significant effect on the catabolic rate of HDL apolipoproteins in wild-type control mice (Table 2). On the other hand, in sPLA2-transgenic mice, the FCR of HDL apolipoproteins increased significantly in response to LPS (0.21 ± 0.01 pool/h, P < 0.05, Fig. 3B and Table 2). These data demonstrate that upregulation of the sPLA2 transgene is required to mediate the effect of LPS injection on HDL apolipoprotein catabolism.



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  		Fig. 3. Plasma kinetics of 125I-labeled tyramine-cellobiose (125I-TC)-labeled autologous HDL injected 10 h after the administration of either saline ({square}) or LPS ({diamond}). After tracer administration, blood samples were drawn at the time points indicated and analyzed for radioactivity by {gamma}-counting. Values are the fraction of injected dose remaining at each time point. Curves were analyzed using a bicompartmental model on the SAAMII program, and fractional catabolic rate (FCR) values were calculated. A: wild-type C57BL/6 control mice; B: sPLA2-transgenic mice. FCR values of saline-injected sPLA2-transgenic mice were significantly higher than those of saline-injected controls (P < 0.001). FCR values of LPS-injected sPLA2-transgenic mice were significantly increased compared with saline-injected sPLA2 mice (P < 0.05).

 

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  		Table 2. HDL FCRs in sPLA2-transgenic mice and wild-type nontransgenic littermates after saline or LPS injection

 

Use of the trapped ligand 125I-TC-HDL allowed us to determine the tissue sites of HDL apolipoprotein uptake. Uptake of HDL apolipoproteins into the liver was not significantly different between saline-injected sPLA2-transgenic mice and wild-type controls. However, after LPS administration, hepatic uptake of the 125I-TC-HDL tracer increased in wild-type mice (18 ± 2 to 27 ± 2% of injected dose, P < 0.001, Fig. 4A) and in sPLA2-transgenic mice (15 ± 3 to 33 ± 3% of injected dose, P < 0.001, Fig. 4A). There was no difference in HDL apolipoprotein uptake into the spleen between the saline-injected groups of experimental mice. After induction of the APR, there was a significant increase in HDL apolipoprotein uptake into the spleen in wild-type (0.7 ± 0.2 to 2.3 ± 0.3% of injected dose, P < 0.001, Fig. 4B) and sPLA2-transgenic mice (1.0 ± 0.2 to 2.1 ± 0.3% of injected dose, P < 0.01, Fig. 4B). HDL apolipoprotein catabolism by the kidneys was significantly higher in saline-injected sPLA2-transgenic mice than in the wild-type mice (8 ± 1 to 5 ± 1% of injected dose, P < 0.001), as we previously reported (34), but did not change significantly in either group after injection with LPS (Fig. 4C). Uptake of HDL apolipoproteins into the adrenals was extremely low relative to liver and kidney (Fig. 4D).



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  		Fig. 4. Tissue sites of catabolism of 125I-TC-labeled autologous HDL in sPLA2-transgenic (tg) and control mice after the injection of either saline or LPS. Labeled HDL was prepared as described in EXPERIMENTAL PROCEDURES and injected via the tail vein into sPLA2-transgenic and control mice 10 h after induction of the APR or injection of saline. After 24 h, mice were killed and thoroughly perfused with PBS, and the respective tissues were harvested. Uptake of radioactivity into the respective organs was determined by {gamma}-counting. Data represent percentages of injected tracer dose recovered in liver (A), spleen (B), kidneys (C), and adrenals (D). Statistically significant differences (P < 0.05) are as assessed by independent sample t-test: *between saline-injected controls and LPS-injected mice, and #between sPLA2-transgenic and wild-type mice.

 

We also investigated the plasma catabolism of HDL-CE by using as a tracer HDL labeled with [3H]cholesteryl ether in the same mice used for the HDL apolipoprotein kinetic studies described above. Human sPLA2-transgenic mice injected with saline had significantly faster plasma catabolism of HDL-CE than saline-injected control mice (0.31 ± 0.02 vs. 0.22 ± 0.02 pool/h, P < 0.001, respectively; Fig. 5, A and B, and Table 2), consistent with our previous report (34). LPS injection had no significant effect on plasma catabolism of HDL-CE in wild-type mice (Fig. 5A, Table 2). However, in sPLA2-transgenic mice, LPS injection resulted in a significant increase in the FCR of HDL-CE (0.42 ± 0.05 pool/h, P < 0.05, Fig. 5B, Table 2). This indicates that upregulation of the sPLA2 transgene is required to mediate the effects of LPS injection on HDL-CE catabolism.



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  		Fig. 5. Plasma kinetics of [3H]cholesteryl ether-labeled autologous HDL injected 10 h after administration of either saline ({square}) or LPS ({diamond}). After tracer administration, blood samples were drawn at time points indicated, samples were extracted according to the Dole method, and radioactivity in the lipid phase was determined by scintillation counting. Values are fractions of injected dose remaining at each time point. Curves were analyzed using a bicompartmental model on the SAAMII program, and FCR values were calculated. A: wild-type C57BL/6 control mice; B: sPLA2-transgenic mice. FCR values of saline-injected sPLA2-transgenic mice were significantly higher than those of controls (P < 0.001). FCR values of LPS-injected sPLA2-transgenic mice were significantly increased compared with saline-injected sPLA2 mice (P < 0.05).

 

The use of the nonhydrolyzable CE analog [3H]cholesteryl ether enabled the determination of sites of tissue catabolism of HDL-CEs. LPS injection significantly increased hepatic HDL-CE uptake in wild-type mice (44 ± 3 to 52 ± 4% of injected dose, P < 0.05, Fig. 6A); in sPLA2-transgenic mice, tracer uptake into the liver was nonsignificantly increased (70 ± 4 to 78 ± 4% of injected dose, P = 0.06, Fig. 6A). Uptake of HDL-CE into the spleen was increased in both groups of LPS-injected mice, although only in wild-type mice did this increase reach the level of statistical significance (P < 0.05, Fig. 6B). There were no significant changes in the small amount of HDL-CE uptake into the kidneys between the different experimental groups of mice (Fig. 6C). Saline-injected sPLA2-transgenic mice had significantly higher uptake of HDL-CE into their adrenals compared with wild-type controls (0.82 ± 0.06 vs. 0.51 ± 0.06% of injected dose, P < 0.001, Fig. 6D), consistent with our previous report (34). HDL-CE uptake into the adrenals increased significantly in both LPS-injected sPLA2-transgenic mice (1.43 ± 0.11% of injected dose, P < 0.001, Fig. 6D) and LPS-injected wild-type mice (0.82 ± 0.06% of injected dose, P < 0.001, Fig. 6D).



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  		Fig. 6. Tissue sites of catabolism of [3H]cholesteryl ether-labeled autologous HDL in sPLA2-transgenic and control mice after injection of either saline or LPS. Labeled HDL was prepared as described in EXPERIMENTAL PROCEDURES and injected via the tail vein into sPLA2-transgenic and control mice 10 h after induction of APR or injection of saline. After 24 h, mice were killed and thoroughly perfused with PBS, and the respective tissues were harvested. Organs were extracted according to the Dole method, and radioactivity in the lipid phase was determined by scintillation counting. Data represent percentages of injected tracer dose recovered in liver (A), spleen (B), kidneys (C), and adrenals (D). Statistically significant differences (P < 0.05) are as assessed by independent sample t-test: *between saline-injected controls and LPS-injected mice; #between sPLA2-transgenic and wild-type mice.

 

Because the HDL apolipoprotein and HDL-CE kinetic experiments were carried out in the same mice, we used both measurements to determine the selective uptake of CE from HDL into respective organs in response to LPS injection. sPLA2-transgenic mice injected with saline had significantly higher HDL-CE selective uptake into the liver (34 ± 2 vs. 25 ± 3%/µg tissue protein, P < 0.05, Fig. 7A) compared with saline-injected wild-type controls, but LPS injection had no significant effect on hepatic selective uptake of HDL-CE in either group (Fig. 7A). Likewise, there was little effect of LPS on selective uptake of HDL-CE in the spleen in either group. In the kidneys, there was significantly higher "negative selective uptake" (i.e., lipid-poor protein uptake) in saline-injected sPLA2-transgenic mice compared with wild-type mice (-23 ± 2 vs. -17 ± 2%/µg, P < 0.01, Fig. 7C), and this was relatively unaffected by LPS injection (Fig. 7C). In the adrenals, HDL-CE selective uptake after LPS injection was 2.4-fold higher in sPLA2-transgenic mice compared with wild-type mice (156 ± 6 vs. 65 ± 5%/µg tissue protein, P < 0.001, Fig. 7D).



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  		Fig. 7. Selective uptake of HDL cholesteryl ester into tissues of sPLA2-transgenic and control mice after injection of either saline or LPS. For calculation, data shown in Figs. 4 and 6 were used. 125I-TC-labeled HDL uptake (% of injected dose) was subtracted from [3H]cholesteryl ether-labeled HDL uptake (% of injected dose) into the respective organs [liver (A), spleen (B), kidneys (C), and adrenals (D)] to calculate selective uptake, and data were referred to as µg of tissue wt. Statistically significant differences (P < 0.05) are as assessed by independent sample t-test: *between saline-injected controls and LPS-injected mice; #between sPLA2-transgenic and wild-type mice.

 


   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
DISCLOSURES
 REFERENCES
 
The results of this study demonstrate that upregulation of sPLA2 expression by endotoxin injection significantly enhances the plasma catabolism of HDL, results in altered tissue uptake of HDL apolipoproteins and HDL-CE, and specifically increases the selective uptake of HDL-CE into the adrenals, suggesting a novel and important metabolic in vivo role of sPLA2.

The line of sPLA2-transgenic mice used in the present study was made with a genomic construct that results in relatively high baseline sPLA2 expression but also preserves the inducibility of the transgene in response to inflammatory stimuli (23). This allowed us to study the effects of acute inflammation on HDL metabolism in human sPLA2-transgenic mice on a C57BL/6 genetic background compared with wild-type C57BL/6 mice lacking the endogenous mouse sPLA2 enzyme due to a frameshift mutation in the respective gene (19). At baseline, sPLA2-transgenic mice have lower plasma levels of HDL-C compared with wild-type mice (34). The underlying metabolic mechanism is a significantly increased rate of plasma catabolism of HDL apolipoproteins as well as HDL-CE (34), a finding that we confirmed in the current studies. In these studies, induction of the APR in wild-type C57BL/6 mice neither altered the steady-state plasma concentrations of HDL-C nor affected the catabolic rates of HDL apolipoproteins or HDL-CE. In contrast, in sPLA2-transgenic mice, induction of the APR resulted in increased sPLA2 expression and a significant decrease in HDL-C levels. The FCRs of both HDL apolipoproteins and HDL-CE increased significantly in sPLA2-transgenic mice in response to LPS injection. These results indicate that the presence of an inducible and functional sPLA2 gene is required to mediate the effects of acute inflammation on HDL metabolism in mice. In parallel and separate experiments, we crossed human sPLA2-transgenic mice with human apoA-I-transgenic mice and found, consistent with the present results, that upregulation of sPLA2 expression was required for an effect of LPS on plasma HDL-C and apoA-I levels (35).

There are three possible pathways for the cellular uptake of HDL-C: 1) holoparticle uptake of HDL, 2) selective uptake of HDL-C mediated by the SR-BI receptor, and 3) transfer to LDL by CE transfer protein (CETP) and uptake via the LDL receptor. The latter pathway is not active in mice, because this species is lacking CETP. Holoparticle uptake of HDL is a known mechanism for removal of HDL from the circulation (11, 12, 32, 42). One possible receptor for this process is cubilin (26), which may have a role in the renal and placental uptake of HDL (14, 20). In the liver, other poorly characterized mechanisms appear to exist that mediate holoparticle catabolism of HDL. Our data demonstrate that, during the APR, a substantial increase in the hepatic uptake of HDL apolipoprotein and CE occurs, with holoparticle uptake being the most likely mechanism. This process appears to be relatively unaffected by sPLA2. Likewise, there was no significant change in HDL apolipoprotein uptake by the kidneys in sPLA2-transgenic mice after induction of the APR. Therefore, our data suggest that sPLA2 has relatively little, if any, effect on holoparticle HDL uptake by the liver or kidney.

SR-BI has been extensively studied as a receptor that mediates the selective uptake of HDL-C (21, 36, 40). Liver and steroidogenic organs are tissues with high SR-BI expression (21, 31). Previous in vivo studies have established that liver, and especially the adrenals, have high rates of selective uptake of HDL CE (4, 10–12). By simultaneously using trapped ligands for HDL apolipoproteins (125I-TC) as well as HDL-CE ([3H]cholesteryl ether) in the same mice, we quantitated selective HDL-CE uptake in specific tissues in sPLA2-transgenic mice in response to the APR. Transgenic expression of sPLA2 significantly increased the selective uptake of HDL-CE in both liver and adrenals at baseline. Induction of the APR did not significantly increase selective HDL-CE uptake in the liver in either wild-type or sPLA2-transgenic mice. In contrast, induction of the APR induced a significant increase in selective HDL-CE uptake in the adrenals in wild-type mice and especially in sPLA2-transgenic mice. The rate of selective HDL-CE uptake in LPS-injected sPLA2-transgenic mice was 2.4-fold that in LPS-injected wild-type mice, indicating that sPLA2 expression and upregulation by inflammation markedly increase cholesterol uptake from HDL by the adrenals. The most striking metabolic difference between sPLA2-transgenic mice and wild-type littermates after the induction of the APR is the magnitude of selective uptake into the adrenals. Although not specifically explored in this study, there is also the possibility of sPLA2-mediated targeting of HDL CE to monocytes/macrophages during the APR, because SR-BI is expressed on these cells (36).

Expression of sPLA2 confers protection of mice against bacterial infections, which has been demonstrated for gram-positive (23) as well as gram-negative (22) bacterial strains. This has been ascribed at least in part to a direct bactericidal effect of the enzyme (37, 38), possibly due to degradation of bacterial surface phospholipids (39). However, although sPLA2 is able to kill gram-positive bacteria in vitro (29, 38), an efficient defense against gram-negative bacteria requires additional cofactors, such as components of the complement system (25, 39). Furthermore, neither serum nor peritoneal lavage fluid from sPLA2-transgenic mice was bactericidal against E. coli in vitro (22), suggesting that other properties of sPLA2 may have resulted in the increased survival of sPLA2-transgenic mice challenged with bacteria. During acute inflammation, there is a requirement of increased adrenal steroid hormone synthesis, for which HDL-CE is a known source (17, 28, 30, 41).

Our results suggest that one possible physiological role of the sPLA2 enzyme during inflammation is to modify HDL to make HDL CE more accessible for selective uptake into the adrenals. There are three main families of steroid hormones synthesized by the adrenal cortex: mineralocorticoids in the zona glomerulosa, glucocorticoids in the zona fasciculata, and androgens in the zona reticularis (5, 41). Both mineralocorticoids and glucocorticoids could be helpful in the response to acute inflammation. Although there might be species differences in the preferential usage of LDL- or HDL-CE for steroid hormone synthesis (36), a significantly higher expression of SR-BI in the zona fasciculata of the adrenal cortex in mice compared with the zona glomerulosa has been reported (30). SR-BI-mediated uptake is most likely the major route for the delivery of HDL-CE to the steroidogenic pathway in the adrenals (36, 41). It is therefore probable that, during the APR, CE from sPLA2-modified HDL is preferentially taken up by zona fasciculata cells and used for glucocorticoid synthesis. This extends the role of sPLA2 in the host response to bacterial infections beyond a direct antibacterial effect and might contribute to the significantly increased survival of sPLA2-transgenic mice in response to a bacterial challenge.

In summary, we characterized changes in HDL catabolism during the APR in wild-type and sPLA2-transgenic mice. Both groups of mice had increased holoparticle catabolism by the liver and increased selective HDL-CE uptake by the adrenals in response to LPS injection. The sPLA2-transgenic mice demonstrated an especially robust increase in adrenal selective HDL-CE uptake in response to induction of the APR. We suggest that one physiological in vivo role of sPLA2 is to significantly increase selective HDL-CE uptake by the adrenals to help meet the requirements of increased steroid hormone synthesis during acute systemic inflammation. This metabolic effect might be beneficial in the response to bacteremia and other acute inflammatory insults and might contribute to improved host survival in this setting.


   DISCLOSURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 DISCLOSURES
REFERENCES
 
The sPLA2-transgenic mice were produced by Xenogen Biosciences (formerly DNX Transgenic Sciences). This work was supported by National Heart, Lung, and Blood Institute Grant HL-55323 (to D. J. Rader). D. J. Rader is an Established Investigator of the American Heart Association and a recipient of the Burroughs Wellcome Foundation Clinical Scientist Award in Translational Research. U. J. F. Tietge was a recipient of a reasearch fellowship from the Deutsche Forschungsgemeinschaft and the American Heart Association, Pennsylvania-Delaware Affiliate. C. Maugeais was supported by a research fellowship from the American Heart Association, Pennsylvania-Delaware Affiliate.

Current address for U. J. F. Tietge: Dept. of Medicine, Charité Campus Mitte, Humboldt University, Schumannstr. 20/21, D-10117 Berlin, Germany.


   ACKNOWLEDGMENTS
 
We are indebted to Anthony Secreto, Anna Lillethun, and Linda Morrell for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. J. Rader, Univ. of Pennsylvania Medical Center, 654 BRB II/III, 421 Curie Blvd., Philadelphia, PA 19104-6160 (E-mail: rader{at}mail.med.upenn.edu).

Submitted 30 December 2002

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

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