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(Journal of Leukocyte Biology. 2002;72:711-717.)
© 2002 by Society for Leukocyte Biology

Endotoxin induces rapid metalloproteinase-mediated shedding followed by up-regulation of the monocyte hemoglobin scavenger receptor CD163

Katharine A. Hintz*, Athos J. Rassias{dagger}, Kathleen Wardwell*, Marcia L. Moss{ddagger}, Peter M. Morganelli§,||, Patricia A. Pioli*, Alice L. Givan*, Paul K. Wallace§, Mark P. Yeager{dagger} and Paul M. Guyre*

Departments of
* Physiology and
§ Microbiology, Dartmouth Medical School, Hanover, New Hampshire;
{dagger} Department of Anesthesiology, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire;
{ddagger} Cognosci, Inc., Research Triangle Park, North Carolina; and
|| Department of Microbiology, Veterans Administration Hospital, White River Junction, Vermont

Correspondence: Dr. Paul M. Guyre, Department of Physiology, Dartmouth Medical School, 1 Medical Center Dr., 740 W. Borwell Bldg., Lebanon, NH 03756-0001. E-mail: Paul.M.Guyre{at}dartmouth.edu


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ABSTRACT
 
CD163, a monocyte and macrophage-specific surface glycoprotein, which is increased by interleukin-10 and glucocorticoids, is a scavenger receptor for hemoglobin/haptoglobin complexes. We report a rapid and highly reproducible rise in soluble CD163 in the plasma of human volunteers given intravenous lipopolysaccharide (LPS). We also show that LPS induces shedding of CD163 from the surface of isolated monocytes, identifying shedding from monocytes and macrophages as a likely mechanism for the endotoxemia-associated rise in plasma CD163 in vivo. Studies using the inhibitor TAPI-0 indicate that a metalloproteinase is responsible for LPS-mediated shedding of CD163. Finally, we demonstrate a marked increase in surface CD163 expression on circulating monocytes 24 h following experimental endotoxemia. These findings show that CD163 is rapidly mobilized in response to bacterial endotoxin. As hemoglobin can bind LPS and enhance its toxicity, it will be important to determine how cell surface and soluble CD163 influence inflammatory processes during sepsis.

Key Words: lipopolysaccharide • glucocorticoid • inflammation • in vivo • sepsis


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INTRODUCTION
 
More than 500,000 patients per year develop septic shock and related complications, with a mortality rate as high as 35–45% [1 ]. Although new approaches such as treatment with recombinant-activated protein C have led to modest declines in sepsis-related mortality [2 ], further progress will require a more complete understanding of all components comprising the host response to infection and of their regulatory mechanisms. Bacterial lipopolysaccharide (LPS; or endotoxin), acting through mechanisms that involve CD14, Toll-like receptors, and other molecules [3 ], is in large part responsible for the extensive, inflammatory response associated with Gram-negative sepsis. In vivo, LPS stimulates the production of many cytokines and acute-phase proteins, including haptoglobin (Hp), which when complexed with hemoglobin (Hb), was recently shown to be a ligand for the monocyte/macrophage-specific glycoprotein CD163 [4 5 6 ]. As Hb can bind LPS and enhance its lethality in mice [7 , 8 ], interactions involving CD163, Hb/Hp, and LPS may play an as yet unidentified role in the monocyte and macrophage response to LPS. It is notable that glucocorticoids and interleukin (IL)-10 increase rapidly in vivo in response to LPS [9 ]. These two mediators cause a pronounced increase in CD163 synthesis [10 11 12 13 ] and are also well-known for reducing LPS toxicity [14 15 16 ]. We and others [17 , 18 ] have identified CD163 as a soluble protein in plasma. We have now used a CD163-specific enzyme-linked immunosorbent assay (ELISA) and flow cytometric analysis to characterize the acute and delayed effects of LPS on plasma and cell surface CD163. As there is recent evidence that soluble CD163 may act as an anti-inflammatory molecule [19 ], our finding that CD163 is an early acute-phase reactant identifies it as a potentially important participant in the systemic response to infection.


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MATERIALS AND METHODS
 
The Dartmouth College Committee for Protection of Human Subjects (Hanover, NH) approved all studies reported in this manuscript, and written, informed consent was obtained as necessary.

Purification of CD163
Anti-CD163 agarose was prepared per the manufacturer’s instructions [ImmunoPure® PROTEIN G IMMUNOGLOBULIN G (IgG) orientation kit, Pierce, Rockford, IL] using monoclonal antibody (mAb) Mac 2-158 (Maine Biotechnology Services, Inc., Portland). Blood (25 mL) was obtained from a healthy donor in a heparinized syringe and spun at 1400 g for 30 min to isolate plasma. Plasma was combined with 2 mL anti-CD163 agarose described above in a 50-mL conical tube, rotated at 4°C for 72 h, and packed into the column provided by the manufacturer. After washing with 16 mL 1.0 M NaCl (wash buffer), protein was eluted with ImmunoPure® elution buffer, pH 2.8 (Pierce), and fractions were neutralized with 1M TRIS, pH 9.4. Protein-containing fractions were pooled and dialyzed to phosphate-buffered saline (PBS). Purity was assessed to be greater than 70% by resolution on a silver-stained 6% sodium dodecyl sulfate-polyacrylamide gel under reducing conditions. Protein concentration was determined using a bicinchoninic acid protein assay kit (Pierce). Purified CD163 was used to calibrate a reference plasma that was used to standardize the measurement of soluble CD163 by ELISA.

In vitro culture of mononuclear cells
Peripheral blood mononuclear cells (PBMC) were isolated from heparinized whole blood by using Ficoll-Hypaque (d=1.077) [20 ]. Monocytes were purified from mononuclear cell fractions as described by Mentzer et al. [21 ]. The cells were cultured in 5% CO2 and 37°C in complete medium consisting of Hepes-buffered RPMI 1640 (Hazelton Biologicals, Lenexa, KS), 20 µg/mL gentamicin (Elkins-Sinn, Cherry Hill, NJ), 10% fetal bovine serum (Hyclone Laboratories, Logan, UT) or pooled human serum, 5 x 10-5 M 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO). In some experiments 0.5 ng/mL IL-1ß (PeproTech, Rocky Hill, NJ) was added to protect monocytes from apoptosis [22 ]. IL-1ß did not affect CD163 expression [11 ] (see Fig. 3a , inset). When indicated, 200 nM dexamethasone (Dex; Steraloids, Wilton, NH) was added during culture to increase CD163 expression. Cells were then treated for 2 h with medium alone, 10-8 M phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich), which has been shown to induce CD163 shedding [23 ], or 5 ng/mL LPS (endotoxin) from Escherichia coli (E. coli 0111:B4; Sigma-Aldrich). After pelleting of cells at 450 g for 5 min, supernatants were collected and analyzed immediately for soluble CD163. Cells were resuspended and stained for CD163 expression with purified or biotinylated mouse IgG1 mAb Mac 2-158 (Maine Biotechnology Services) or an IgG1 isotype control (Caltag Laboratories, Burlingame, CA) followed by a goat anti-mouse F(ab')2 fluorescein isothiocyanate (FITC) secondary antibody (Caltag) or streptavidin R-phycoerythrin (PE; Caltag). Cells were then analyzed by flow cytometry.



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  		Figure 3. LPS causes shedding of monocyte CD163 in vitro. Purified human monocytes from healthy donors cultured for 24 h with medium alone or Dex (200 nM), followed by complete medium, PMA (10-8 M), or LPS (5 ng/mL) treatment for 2 h. (a) Cells stained for monocyte surface CD163 expression; (b) cell culture supernatants analyzed for soluble CD163. (a) Cells were stained for CD163 expression with biotinylated mouse IgG1 mAb Mac 2-158 (Maine Biotechnology Services) followed by streptavidin R-PE (Caltag). (Inset) Cells were stained for CD163 expression with mouse IgG1 mAb Mac 2-158 (Maine Biotechnology Services) followed by goat anti-mouse F(ab')2 FITC secondary antibody (Caltag). Data shown are representative of four comparable experiments, and values are mean ± SD of triplicates. *, P < 0.05 compared with medium alone.

For the inhibitor studies, cells were isolated, cultured as indicated above, and subsequently treated for 1 h with 1 ng/mL LPS from E. coli 0111:B4 (Sigma-Aldrich) in the presence or absence of increasing concentrations of 10 µM tumor necrosis factor {alpha} (TNF-{alpha}) inhibitor (TAPI-0; Peptides International, Louisville, KY). TAPI-0 was preincubated with cells for 30 min prior to the addition of LPS and was included throughout the treatment. Cells were stained for CD163 expression with mouse IgG1 mAb Mac 2-158 (Maine Biotechnology Services) or an IgG1 isotype control (Caltag) followed by goat anti-mouse F(ab')2 FITC secondary antibody (Caltag). Cells were then analyzed by flow cytometry.

Administration of LPS and sample collection
Healthy volunteers were injected intravenously (i.v.) with a bolus dose of LPS (4 ng/kg) in 0.9% normal saline from E. coli (group O:113, Lot EC-5, Center for Biologics Evaluation and Research, FDA, Bethesda, MD). For each subject to serve as his/her own control, on a separate day prior to LPS infusion, each subject received a bolus dose of normal saline of equal volume to what they would receive on the LPS infusion day. Blood (5 mL) was drawn into a heparinized syringe (heparin sodium, 1000 units/mL; American Pharmaceutical Partners, Inc., Los Angeles, CA) from a peripheral indwelling venous catheter at the following time points: three baseline samples drawn during the hour prior to LPS administration and then 0.25, 0.5, 0.75, 1, 2, 4, 8, 12, 24, 48, and 72 h post-LPS administration. Whole blood was analyzed for surface CD163 or centrifuged at 450 g for 12 min to generate plasma. Plasma was stored at -70°C for subsequent assays.

Soluble CD163 ELISA
Soluble CD163 was detected in the plasma of LPS-infused volunteers using a previously described ELISA protocol [17 ]. Capture mAb Mac 2-158 was obtained from Maine Biotechnology Services, and biotinylated mAb RM3/1 for detection was obtained from Bachem Bioscience Inc. (King of Prussia, PA). A standard curve was generated for each plate based on the titration of a reference plasma standard previously determined to have 18.5 ± 0.19 µg/mL CD163.

Flow cytometry on LPS infusion subjects
Fluorescently labeled mAb [FITC-IgG1 control or FITC-anti-CD163 (Maine Biotechnology Services), allophycocyanin-CD14, PerCP-anti-CD45 (Becton Dickinson, San Jose, CA)] were used to triple-stain whole blood samples. The monocyte population, as determined by CD45 plus side-scatter gating [24 ] and CD14 positivity [25 ], was analyzed first for nonspecific antibody binding (IgG1 control), and this value was subtracted from the CD163 fluorescence intensity. For most samples, 5000 monocytes were examined per measurement. Samples with fewer than 1000 monocytes were excluded from analysis. Identical settings for all samples were used, and analysis was performed using a Becton Dickinson FACSCalibur flow cytometer calibrated daily for forward-scatter, side-scatter, and all fluorescence parameters using Rainbow beads (RCP-30-5) from Spherotech Inc. (Libertyville, IL) and Calibrite beads from Becton Dickinson. Day-to-day variation was less than 5%.

Statistical analysis
SYSTAT 9 for Windows (SPSS Science, Chicago, IL) was used to perform a two-way, repeated-measures ANOVA with post-hoc analysis using the Bonferonni correction. A two-sided P value of less than 0.05 was taken as indicative of statistical significance.


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RESULTS
 
Increased plasma CD163 following intravenous LPS administration
To model the in vivo inflammatory response to infection, eight healthy volunteers were administered a 4 ng/kg bolus infusion of LPS. Plasma samples were obtained at time points ranging from baseline to 72 h post-LPS administration and analyzed by ELISA for soluble CD163. The average baseline plasma CD163 concentration of 8.59 ± 2.8 µg/mL (mean±SD) increased significantly by 1 h following endotoxin administration (P<0.05) and peaked by 1 or 2 h to an average of 35.21 ± 9.6 µg/mL (Fig. 1a ). The range of increase from baseline to peak varied from two- to sixfold among the eight subjects. The kinetics of rise in plasma CD163 were similar to those of TNF-{alpha}, which increased from undetectable levels at baseline to a 2-h average value of 1157 ± 634 pg/mL for the five subjects in which it was measured (unpublished results). As each subject served as their own control, plasma CD163 was monitored on control days (bolus saline infusion) as well as LPS infusion days. Average plasma CD163 values were not statistically different between baseline and 1-h post-saline infusion for the six subjects tested on control days. The average control-day plasma CD163 value was 9.60 ± 3.1 µg/mL and 10.03 ± 4.7 µg/mL at baseline and 1-h post-saline infusion, respectively (Fig. 1b) .



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  		Figure 1. Endotoxemia increases plasma CD163. Plasma CD163 levels in (a) human volunteers (n=8) i.v. administered 4 ng/kg E. coli LPS at time 0 with blood drawn at the indicated time points; (b) the same human volunteers (n=6) i.v.-administered PBS at time 0 with blood drawn at the indicated time points.

LPS decreases surface CD163 expression similar to PMA in vitro
Previous reports have indicated that LPS decreases CD163 expression in vitro when cultured for 24 h [13 ]. We now show that LPS is acting at much earlier time points to decrease receptor expression levels. Droste et al. [23 ] have published that PMA activation induces a rapid decrease in CD163 expression as a result of receptor shedding. To further examine the acute effects of LPS on CD163 expression, we performed a time course comparing the effects of PMA and LPS. Following isolation, PBMC were cultured for 24 h in complete medium. Subsequently, cells were treated with medium alone, PMA, or LPS for 15 min to 4 h. Untreated cells had baseline CD163 mean fluorescence intensity (MFI) of 83 ± 6 (mean±SD) for Donor 1 and 75 ± 4 for Donor 2. By 2 h of treatment, LPS and PMA decreased expression to an average for both donors of 56% and 20% of control, respectively (P<0.05; Fig. 2 ). Average CD163 expression by LPS versus PMA-treated monocytes was different at 2 h for both donors (Fig. 2 ; P<0.05). Figure 2 is representative of 10 out of 10 experiments in which LPS decreased CD163 expression by 2 h but consistently to a lesser extent than PMA treatment.



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  		Figure 2. LPS and PMA decrease monocyte CD163. Human mononuclear cells from healthy donors cultured for 24 h with medium alone followed by complete medium, PMA (10-8 M), or LPS (5 ng/mL) treatment for a 4-h time course. Cells were stained for CD163 expression with mouse IgG1 mAb Mac 2-158 (Maine Biotechnology Services) followed by goat anti-mouse F(ab')2 FITC secondary antibody (Caltag). Data shown are representative of four comparable experiments, and values are mean ± SD of triplicates. *, P < 0.05.

LPS causes shedding of monocyte CD163 in vitro
Recognizing that PMA rapidly stimulates shedding of surface CD163 [23 ], we investigated whether shedding might be the cause of the decreased receptor expression levels following acute exposure to LPS (Fig. 2) . Figure 3 shows the effects of a 2-h treatment with LPS or PMA on CD163 expression by monocytes previously cultured for 24 h with medium alone or medium supplemented with Dex (to up-regulate CD163 expression; ref. [10 ]). Flow cytometric analysis of surface expression and assay for soluble CD163 by ELISA demonstrated that LPS, like PMA, decreased cell surface expression (Fig. 3a) and simultaneously increased culture supernatant CD163 concentrations (Fig. 3b) for control monocytes and those with glucocorticoid-induced, high CD163 expression (P<0.05).

TAPI-0 inhibits monocyte CD163 shedding by LPS
LPS is known to activate metalloproteinases in human monocytes and induce shedding of a variety of surface receptors and precursor proteins such as TNF-{alpha} [26 ], macrophage-colony stimulating factor receptor [27 ], and IL-1 receptor type II [28 ]. Therefore, we examined the effects of an inhibitor of metalloproteinases on LPS-induced shedding of CD163. Figure 4 shows that the average baseline MFI for CD163 expression on control monocytes from two donors was 137 ± 8 and 113 ± 8 after 24-h culture (Fig. 4a) . Following pretreatment with Dex, monocyte CD163 expression increased to an average MFI of 480 ± 48 and 477 ± 21 (Fig. 4b) . LPS treatment for 1 h reduced CD163 levels to an MFI of 75 ± 3 and 65 ± 2 for control cells and 169 ± 3 and 164 ± 5 for cells pretreated with Dex. Preincubation with 10 µM TAPI-0 completely prevented the LPS-induced reduction in surface CD163 expression of control and Dex-treated cells. TAPI-0 (10 µM) alone had no effect on CD163 expression nor did its presence with or without LPS affect another monocyte surface receptor, CD64 (unpublished results). Figure 4 is representative of five out of five experiments in which 10 µM TAPI inhibited the LPS-induced decrease in monocyte surface CD163 expression.



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  		Figure 4. LPS-inducible shedding of monocyte CD163 is mediated by metalloproteinase activity. Human mononuclear from healthy donors cultured for 24 h with (a) medium alone or (b) Dex (200 nM) for 48 h, followed by LPS (1 ng/mL) treatment in the presence of none or 10 µM TAPI-0. Cells were stained for CD163 expression with mouse IgG1 mAb Mac 2-158 (Maine Biotechnology Services) followed by goat anti-mouse F(ab')2 FITC secondary antibody (Caltag). Data shown are representative of five comparable experiments, and values are mean ± SD of triplicates. *, P < 0.05 compared with medium alone.

Monocyte surface CD163 is increased 24 h following intravenous LPS administration
We monitored monocyte surface CD163 expression following in vivo LPS administration using a three-color, whole-blood staining protocol and flow cytometric gating on side-scatter plus positive staining for CD45 and CD14. Cell surface expression of CD163 was significantly increased over baseline at 24 h following LPS infusion, at which time monocyte numbers were within the normal range (Fig. 5 ). CD163 MFI averaged 18 ± 14 for the eight subjects at baseline and 42 ± 24 at 24-h post-LPS infusion (P<0.05). An attempt was made to also monitor loss of CD163 at earlier time points, but the monocytopenia that occurs following LPS infusion [9 ] precluded definitive evaluation of surface expression by monocytes during the first 4-h post-infusion.



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  		Figure 5. A 24-h increase in monocyte surface CD163. Monocyte surface CD163 expression was measured in whole blood obtained from human volunteers at baseline and 24 h post-LPS administration. Values are mean ± SD of triplicates. *, P < 0.05 compared with baseline CD163 values.


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DISCUSSION
 
A new and important finding of this study is the rapid rise in plasma CD163 during experimental endotoxemia (Fig. 1a) , identifying CD163 as an early acute-phase reactant. There was a highly significant increase in plasma CD163 for all eight subjects infused with LPS. The magnitude of increase varied, with peak plasma CD163 values ranging from two- to sixfold over baseline. When plasma CD163 was measured in the same subjects following saline infusion, there was no significant change compared with baseline (preinfusion) time points (Fig. 1b) . Our baseline values are in close agreement with those reported by Moller et al. [18 ], and our post-LPS values are comparable with those reported to be associated with infections [18 ]. Our finding that plasma CD163 levels peaked at 2 h and began to decline by 4 h following LPS infusion suggests that elevated levels may be indicative of an ongoing, inflammatory process. The onset kinetics for increased plasma CD163 in response to LPS were very reproducible and followed approximately the same time course as the rise in TNF-{alpha}, one of the earliest known responses to endotoxin [9 , 29 ]. TNF-{alpha} and CD163 peaked by the 2-h time point, whereas elevations in plasma IL-6 and IL-10 occurred later (unpublished results), consistent with other reports [9 , 30 ]. As purified, soluble CD163 has been reported to inhibit human T-lymphocyte activation [19 ], the significant increase in plasma CD163 following LPS challenge suggests that CD163 shedding could contribute to down-regulation of inflammatory responses.

A second, new finding is that LPS induces shedding of monocyte surface CD163. Although LPS has previously been shown to reduce surface CD163 expression, this was attributed to inhibition of mRNA levels [13 ]. We show here that surface CD163 decreases within 15 min following the addition of LPS to isolated monocytes (Fig. 2) . As it is unlikely that effects on mRNA caused such a rapid change in surface expression, and the studies of Droste et al. [23 ] showed that PMA caused protease-dependent release of surface CD163, we investigated whether shedding could also play a role in the increased plasma levels noted above. Using flow cytometry in conjunction with the CD163 ELISA [17 ], we now show that like PMA, the LPS-induced decrease in surface CD163 is coincident with increased, soluble CD163 in supernatants of cultured monocytes (Fig. 3) . Dex-treated monocytes, which have higher surface CD163, were equally susceptible to LPS-induced shedding. Following LPS or PMA treatment, supernatants from Dex-treated monocytes contained more soluble CD163 than those from non-Dex-treated cells. However, although PMA decreased surface CD163 expression more than LPS (Fig. 3a) , we did not measure more soluble CD163 in supernatants of cells treated with PMA as compared with LPS (Fig. 3b) . This suggests that PMA may activate proteases, which in addition to inducing shedding, also degrade soluble CD163, resulting in loss of the epitope that one or both of our anti-CD163 antibodies recognize. Alternatively, PMA may be more potent than LPS for the activation of another mechanism that would lead to decreased surface expression, such as receptor internalization [31 ].

A third new finding is that a metalloenzyme is responsible for LPS-induced shedding. Many soluble proteins are generated by post-translational cleavage of membrane-bound molecules. Some examples of these include TNF-{alpha} [32 , 33 ], L-selectin [34 ], and fractalkine [35 ]. One group of metalloenzymes responsible for shedding membrane proteins is the "a disintegrin and metalloproteinase" (ADAM) family. ADAMs are multidomain, transmembrane proteins that have been implicated in cell migration [36 , 37 ] and tissue remodeling [38 ]. Our in vivo endotoxemia experiments indicated that soluble CD163 was detectable at the same time as TNF-{alpha}, and it is well-established that TNF-{alpha} release from monocytes is dependent on TNF-{alpha}-converting enzyme (TACE)/ADAM 17 [32 , 33 , 39 , 40 ]. Therefore, we hypothesized that a metalloproteinase, possibly TACE, was also responsible for cleavage of CD163. To investigate this, we tested the effect of the hydroxamic acid-based compound TAPI-0, an inhibitor of TACE and matrix metalloproteinases [40 , 41 ], for its effect on LPS-induced CD163 shedding. As our results in Figure 4 indicate, TAPI-0 completely prevented CD163 shedding induced by LPS in untreated and Dex-treated human monocytes. As we have not determined which specific protease is responsible for cleavage of CD163, we cannot rule out the possibility that other classes of proteases may induce shedding of CD163 following differential activation. We report here the new finding that the metalloproteinase class of enzymes plays a major role in shedding of CD163 following LPS treatment.

Lastly, we show that after a delay, CD163 expression was consistently increased in vivo on circulating monocytes in all endotoxemia subjects at the 24-h time point (Fig. 5) . Although the mechanism remains to be definitively characterized, this increased surface CD163 is likely a result of endotoxemia-associated increases in cortisol, IL-6, and IL-10, known inducers of CD163 synthesis [11 , 13 ]. Blood levels of all three mediators are increased following endotoxin administration [9 ]. We observed average increases in surface CD163 of 2.1-fold from baseline to 24 h in the LPS infusion study (Fig. 5) . Thus, although the pool of circulating monocytes has likely changed, our studies show clearly that human monocyte expression of CD163 is elevated 24 h following exposure to LPS in vivo. As the flow cytometric histogram of CD163 expression showed a unimodal distribution (unpublished results), this increase in CD163 expression occurred in the entire monocyte pool, not just in a subpopulation.

In summary, our results show striking inflammation-associated changes in cell surface and plasma CD163. It will now be important to further elucidate the specific mechanisms that account for these changes, as well as the roles that cell surface and plasma CD163 play in the wide range of inflammatory states in which cortisol, IL-6, and IL-10 are elevated.


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ACKNOWLEDGEMENTS
 
This work was supported by the Englert Cell Analysis Laboratory, a shared resource of the Norris Cotton Cancer Center, which is supported by the Rippel Foundation and an NCI Cancer Center Grant (CA23108). These studies were funded by the Hitchcock Foundation and National Institutes of Health (DA09162, AI40686). The authors thank Dr. James Leiter for his assistance with the statistical analysis. Additionally, advice and suggestions by Drs. Allan Munck and Jack Bodwell, and the support of Timothy Sulahian, Katie Goonan, John Myers, and the surgical and nursing staff at Dartmouth-Hitchcock Medical Center are sincerely appreciated.

Received December 6, 2001; revised June 3, 2002; accepted July 10, 2002.


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