Lipoprotein metabolism in human peritoneal cells

life

ELSEVIER

Sciences,Vol.

PII SOO24.3205(96)00138-5

LIPOPROTEIN

METABOLISM

Joy J. Winzerling,

58, No. 19, pp. 1631-1641, 1996 copyright 0 1996 EIrcvier science Inc. Printed in the USA. All rights resend ooz4-3m/sasls.oo t .oo

IN HUMAN PERITONEAL

CELLS

Zeinab E. Jouni and Donald J. McNamara

Department of Nutritional Sciences, and Interdisciplinary Nutritional Sciences Program The University of Arizona Tucson, AZ. 85721 (Received in final form March 11, 1996)

The feasibility of using human cells isolated from peritoneal dialysis effluent as a model for studying lipoprotein and cholesterol metabolism was investigated. Human peritoneal cells degraded low density lipoproteins (LDL) and acetylated LDL (acetylLDL) by saturable, high affinity receptor-mediated processes. Positive correlations of the percentage of macrophage cells with degradation rates of LDL (r=O.742; ~~0.05) and acetyl-LDL (r-0.931; pcO.01) indicated that macrophage cells LDL receptor-mediated significantly contributed to lipoprotein degradation. degradation was calcium dependent, and sensitive to pronase and chloroquine The receptor exhibited specificity for lipoproteins containing treatments. apolipoprotein B (apoB) or apolipoprotein E (apoE). Exposure of cells to LDL for 24 hrs signiticantly down-regulated LDL receptor-mediated degradation. Acetyl-LDL receptor-mediated degradation was calcium independent, inhibited by chloroquine, and was sensitive to pronase and fi.rcoidin treatments. The scavenger receptor exhibited specificity for only acetyl-LDL. These results demonstrate that human peritoneal cells can provide a source of human tissue macrophages suitable for studies of cholesterol and lipoprotein metabolism and offer the opportunity for comparison of metabolic characteristics of in vivo maturated macrophages with available macrophage-like cell lines. Key Words: human peritoneal macrophages, low density lipoprotein receptor, scavenger receptor

The presence of LDL (apo B/E) and modified LDL (scavenger) receptors on the surface of macrophages derived in vitro from blood monocytes has been well established (l-3). Uptake of LDL via the apo B/E receptors is controlled by feedback suppression of receptor synthesis by intracellular cholesterol thereby preventing intraceUu1ar accumulation of cholesteryl esters (4). In contrast, uptake of modified lipoproteins via scavenger receptors is not responsive to intracellular cholesterol concentration and can result in massive accumulation of cholesteryl esters and formation of foam cells (1,4). Foam cells, macrophages loaded with lipid, are a consistent feature of mature atherosclerotic lesions (5,6), and studies have shown that macrophage cells isolated from human aortic fatty streaks possess both types of receptors (6). Address correspondence and reprints requests to: Zeinab E. Jouni, Ph.D., Department of Biochemistry, Biosciences West # 440; University of Arizona, Tucson, Arizona 85721; Tel. (520) 621-1772; Fax: (520) 621-9288; [email protected]

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Studies using in vivo derived tissue macrophages could confirm previous work using cells derived in vitro, as well as provide new insights into foam cell formation. Few such studies exist because acquisition of tissue cells requires invasive procedures. Macrophages represent the primary line of host defenses in the peritoneal cavity (7). Several reports have documented the presence of tissue macrophages in the peritoneal fluid of patients on Chronic Ambulatory Peritoneal Dialysis (CAPD) (8,9,10). These studies indicate that human peritoneal macrophages originate from blood monocytes, exhibit phagocytosiq possess IgG F, and complement receptor activity, and demonstrate other known macrophage characteristics (8,9). We investigated the possibility of using human peritoneal cells as a potential model for studying lipoprotein and cholesterol metabolism. The results of the present study demonstrate that human peritoneal cells possess native LDL receptors, as well as un-regulated scavenger receptors. Materials and Metho& Materi&: Materials for these studies were purchased from Flow Labs, McLean, VA (RPMIl640, glutamine,

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Human Peritoneal Macropbages

1633

and D-ion of Labeled LjpoproteinS: Cellular lipoprotein degradation rates were assessed using freshly isolated peritoneal cells. Cells (lO?ml) were incubated at 3’PC for 4 hr in microfirge tubes containing lipoprotein-depleted medium (LPDM) (RPMI# 1640, 50 mM CaCl,, and 20% human A/B LPDS), and the designated labeled lipoprotein (10 @ml) with or without excess (20-25fold) unlabeled lipoprotein. The incubations were terminated by addition of 0.5 ml of 50% (w/v) trichloroacetic acid (TCA). The tubes were microfiged and 0.25 ml of 10% (w/v) silver nitrate was added to precipitate free lz51(11). TCA soluble radioactivity was determined using a LKB 1272 Clinigamma Counter (Piiand). Tubes containing only lZ51-labeled lipoprotein provided measurement of total degradation rates. Radioactivity measured from tubes containing both labeled lipoprotein and excess unlabeled lipoprotein assessed rates of non-receptor (non-specific) degradation. Receptormediated degradation was calculated as the difference between total and non-specific degradation values. Lipoprotein degradation rates are presented as ng lipoprotein degraded/mg cell protein-4 hr or as a percentage of defined control values. Incubations in the absence of cells served as background.

utake

Cell Identification: Cells were stained by the methods of Kaplow (13) using myeloperoxidase and counter-stained according to Yam et al. (14) using non-specific esterase. Cell populations were determined by counting >300 cells from each stain preparation. Values obtained using these techniques were comparable to those obtained by an outside clinical laboratory. Other Assays: Pronase sensitivity of lipoprotein degradation by peritoneal cells was determined by pre-treating the cells with 3 &ml pronase for 20 minutes prior to conducting LDL and acetyl-LDL degradation assays (15). Lysosomal degradation of lipoproteins was evaluated by pre-incubating the cells in the presence of 50 mM chloroquine (45 min) followed by analysis of degradation rates for assays conducted in the presence of 50 mM chloroquine (15). Protein concentrations were determined by the method of Markwell et al. (16) and read on a Beckman Du-Two Series Spectrophotometer with BSA as a standard. . atrstrcal Analysis: One-way analysis of variance was used to assess differences in all measured parameters (17). Data from individual patients are reported as the mean + S.D. for assays conducted in triplicate. Data for all patients are reported as the mean f. SEM.

Cell Recoveud Population A&&: The average total peritoneal cell population for several isolations from six donors were 10.4 x lo6 + 3.7 cells with a concentration of 0.056 + 0.21 x lb cells/100 mls (TABLE I). Peritoneal cell populations consisted primarily of three different cell types namely: a) red blood cells (RBC) that possess no receptor-mediated degradation of native or modified LDL, b) polymorphonuclear cells (PMC) that exhibit LDL receptors, but no acetyl-LDL receptors, and c) mononuclear cells (MNC) that have high levels of both LDL and acetyl-LDL receptors. Percentages of cell types varied widely among patients; however, MNC were the predominate cell types for donors 1,3 and 6. The majority of studies presented here were completed using cells from these three donors. Some donors were not available for the entire course of study.

L by I: LDL and acetyl-LDL degradation rates of freshly isolated peritoneal cells obtained from different donors are presented in TABLE II. Although both native and modified LDL degradation rates varied widely among and within donors, peritoneal cells obtained from all donors exhibited receptor-mediated degradation for both lipoproteins. Mean degradation rates for acetyl-LDL exceeded that of LDL by about 5-fold.

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TABLE I Cell Populations

Present in the Dialysis Effluent of Individual Patients Cell Populations

(%)

n

REK

1

3

41.1 f 7.8

2

1

71.2

Cl.0

4

20.7 f 10.9

34.8 f 19.9

4

2

94.0195.0

1.o/o.o

5.OlS.O

5

1

78.3

Cl.0

20.6

Donor

3

DM

+

6

+

8

Mean f SEM

8.6+

6

PMC

1.8

58.5 f 13.2

1.8 f

6.7*

MNC 1.7

56.6 f 9.7 27.6 44.6*

1.8

85.0 + 2.7

6.6& 4.8

34.9 * 11.0

Cells were isolated from the peritoneal effluent from the first dialysis following an overnight dwell as described in Methods. Analysis was conducted by counts of >300 cells from both Wright-Giemsa and acid esterase/myeloperoxidase stains. All isolations represent donors free Ii-om infection and on dialysis >3 weeks. RFlC= red blood cells, PMN= polymorphonuclear cells, MNGmononuclear cells, DM = diabetes mellitus, and n = number of isolations. Data are presented as mean + S.D. or values of single or duplicate isolations. TABLE II Receptor-mediated

Degradation

of Acetyl-LDL and LDL bv Human Peritoneal Cells

Receptor-Mediated Donor 1

Acetyl-LDL 439.2 f 200.8 (4)

9.1

Degradation

(ng/mg-4 hr) LDL 46.4 f

15.7 (3)

2

116.0 f

13.8 (3)

not tested

3

22.6 f

7.9 (4)

24.2 (1)

6

1037.8 f 748.3 (6)

160.9 f 113.8(6)

Mean f SEM

403.9 f 194.8 (4)

77.2 f 42.4 (3)

Cells were isolated as described in Methods and incubated at 37°C for 4 hrs in medium containing 20% human AA lipoprotein-depleted serum (v/v) and 10 ug protein/ml of the designated r2Upoprotein in the presence or absence of a 2O(LDL) or 25(Acetyl-LDL)-fold excess of the corresponding unlabeled lipoprotein. Receptormediated degradation was assessed as described in the Methods section. Assays were conducted in triplicate on isolates from donors on dialysis for >3 weeks and free from peritonitis. Data are presented as mean *SD. for the number of analyses shown in parenthesis.

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Human Peritoneal Maerophages

A

0

AcLDL

LDL

1400 1200

1635

1

1000 800 600 400 200 0

_-

-200 0

20

40

% Mononuclear

60

80

100

Cells

Fig. 1 Peritoneal cells were isolated from donors as described in Methods. Dashed line represents receptor-mediated degradation of lz51- LDL and solid line represents receptor-mediated degradation of 1251-acetyl-LDL. Cell percentage was analyzed by counts of > 300 cells from both Wright-Giemsa and acid esterase/myelopeioxidase stains.

Peritoneal cells obtained from donor # 3 exhibited virtually equal degradation rates for LDL and acetyl-LDL. This observation can be attributed to the fact that PMC. which contributed to native, but not modified LDL degradation, accounted for 35% of total cell population relative to 1% - 7% for other donors. Correlation between percentage of MNC (>90% macrophages) and receptor activities for LDL and acetyl-LDL are depicted in Fig. 1. A positive correlation was found between the percentage of MNC and receptor-mediated degradation rates of both LDL (r =0.742, pcO.05) and acetyl-LDL (l=O.930, p
i ) _torg: Peritoneal cells obtained from donor # 6 (PMC = 6.1%, MNC = 60.9%; >90% macrophages) exhibited a saturable receptor-mediated degradation of LDL (Fig. 2, left panel). Analysis of saturation curve indicated that the apparent ligand saturation was about 20 ug protein/ml. Receptor-mediated degradation of acetyl-LDL was saturable at about 50 ug protein/ml (Fig. 2, right panel). When expressed as double reciprocal plots (insets), both LDL and acetyl-LDL degradation curves generated a straight line indicating that a single receptor accounted for the observed lipoprotein degradation rates.

Human Peritoneal Macrophages

1636

5

10

15

20

25

Vol. 58, No. 19, 1996

0

20

40

60

60

100

1*51-Acetyl-LDL (pg protein/ml)

lz51-LDL (wg protein/ml)

Fig. 2 Peritoneal cells were isolated as described in Methods, and incubated

at 37°C for 4 hrs in medium containing 20% human A/B LPDS (v/v) and the designated concentrations of “*I-LDL or ‘251-acetyl-LDL in the presence or absence of a 20-fold excess of unlabeled LDL or 25fold excess of acetyl-LDL. Cells were obtained from donor 6 # (PMC =6.1%, MNC = 60.9%) for LDL degradation studies or from donor # 3 (PMC = 20.7%, MNC = 5 1%) for acetyl-LDL degradation studies. Data were transformed to double-reciprocal plots (inset). Error bars indicate S.D. for triplicate assays. The effect of unlabeled CDL and acetyl-LDL on total degradation rates of I25I-LDL (donor # 6; PMC = 6.1%, MNC = 60.9%) and “‘I-acetyl-LDL (donor # 3; PMC = 62.9, MNC = 3 1.S) are presented in Fig. 3. Cells from donor #3 were used despite relatively high %PMC because PMC do not contribute to acetyl-LDL degradation values. Unlabeled LDL (200 ug/ml) reduced total 12’I-LDL degradation to 8% relative to cells incubated in the absence of unlabeled LDL (control), demonstrating that 92% of the total degradation was receptor-mediated (let? panel). Similarly, unlabeled acetyl-LDL reduced ‘251-acetyl-LDL degradation to 40%, indicating that 60% of the total acetyl-LDL degradation was receptor-mediated (right panel).

To further characterize the specificity of native and modified LDL receptors, competition studies were done using several different lipoproteins (Fig. 4). For both experiments, peritoneal cells from donor # 6 (JbfNC > 60%) were used. At a 20-fold excess of unlabeled competitor lipoproteins, lZ51-LDL degradation was significantly (P
Human PeritonealMaerophages

Vol.58, No. 19,1996

0’ 0

.

= 100

.

*

’

200

300

.

1637

’

400

500

Acetyl-LDL (pg protein/ml)

LDL (vg protein/ml) Fig. 3

Peritoneal cells were isolated 6om donor # 6 (PMC = 6.1%, MNC = 60.9%) for LDL degradation studies and from donor # 3 (PMC = 62.9%, MNC = 3 1.8%) for acetylLDL degradation studies. All cells were incubated at 37°C for 4 hrs in medium containing 20% human A/B LPDS (v/v), 1251-labeled LDL (10 pg protein/ml) (left panel) or ‘251-acetyl-LDL (10 pg protein/ml) (right panel) and the designated concentration of the corresponding unlabeled lipoprotein. Error bars indicate S.D. from triplicate assays.

120, 100

AOLM

-

A....

60 60 40

-

:

mL

Fig. 4 Peritoneal cells from donor # 6 (MNC > 86%) were incubated at 37°C for 4 hrs in medium containing 20% human A/B LPDS (v/v), 10 pg protein/ml 12?-LDL (solid bars) or ‘2SI-acetyl-LDL (AcLDL, hatched bars) and 20-fold excess of LDL, acetylLDL, VLDL, HDL. Degradation of the labeled lipoproteins in the absence of unlabeled competitor served as control (100%). Data are presented as percent control; error bars indicate the SD. from assays done in triplicate. *Significantly different from control value at p
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Fig. 5

Peritoneal cells obtained from donor I# 6 (MNC > 86%) were incubated for 4 hrs at 37°C in medium containing 20% (v/v) human A/B LPDS plus 10 ug protein/ml of ‘*‘ILDL in the presence or absence of 20-fold excess LDL (solid bars) or 1251-acetyl-LDL (hatched bars) in the presence or absence of 25-fold excess of acetyl-LDL. Receptormediated degradation was calculated as described in Methods. Degradation rates were determined in the presence of 2 mM calcium chloride (control) or calcium-free media containing EDTA (ImM), and in the presence of tkcoidin (50 @ml), pronase (3 pg/ml), or chloroquine (50 PM). Values represent mean + S.D. of three assays. * Significantly different from control values p <0.002.

Receotors Characterization: To tiuther characterize native and scavenger LDL receptor regulation, peritoneal cells from donor # 6 (MNC > 86%) were incubated with either “‘1-LDL or ‘251-acetyl-LDL under various conditions: a) in the presence of calcium (control), b) in the absence of calcium and presence of EDTA, c) in the presence of fkoidii a scavenger receptor competitor, d) in the presence of chloroquine, an inhibitor of lysosomal enzyme activity, or e) in the presence of pronase (Fig. 5). Receptor-mediated degradation of LDL was significantly reduced (97%) in the absence of calcium and the presence of EDTA compared to control value (P
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Human Peritoneal Macrophages

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is in agreement with findings by others (819). Human peritoneal cells do not survive in the presence of the dialysate, and thus viable cells present in the effluent are drawn from the surrounding tissues during the dialysis process (18). This would suggest that macrophage cells present in dialysis effluent represent human peritoneal macrophages maturated in the peritoneal tissues. Due to the limited number of cells, isolation and analysis of lipoprotein metabolism for each cell type was not feasible. Receptor-Mediated Dw -of Freshly isolated human peritoneal cells exhibited a specific, saturable LDL receptor. The LDL receptor was specific for apoB and apoE containing lipoproteins. LDL degradation was calcium dependent, sensitive to pronase, and required lysosomal activity. Pre-incubation of peritoneal cells in the presence of LDL for 24 hrs resulted in a significant (3-fold) decrease in the LDL degradation rate (data not shown). Thus, freshly isolated peritoneal cells possess active LDL receptors that can be down-regulated by the presence of cholesterolcontaining lipoproteins. Although peritoneal cells are characterized as heterogenous, the macrophages present contributed significantly to receptor-mediated LDL degradation. This hypothesis is supported by the following observations: first, degradation studies using cell populations that contained greater than 85% MNC, with greater than 90% macrophage cells (donor # 6) resulted in the largest degradation rates of LDL. Second, the percentages of mononuclear peritoneal cells per assay exhibited a positive correlation with rates of LDL degradation (t= 0.742; P
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we were unable to acquire sufficient numbers of purified cells to conduct studies on each cell type. We are evaluating flow cytometry as a method for cell separation and analysis of human peritoneal macrophage cells. The Effect of Activation on Receptors Activities: It has been documented that peritoneal macrophages in the dialysis effluent of un-infected donors are activated (7,26,27). The importance of activation to the use of peritoneal macrophage cells as a model for studies of lipoprotein metabolism as it relates to heart disease is unknown, However, other investigators have shown that macrophage and lymphocyte cells from arterial plaque are activated (2829). In addition, advanced human atherosclerotic plaque is characterized by granulomatous foci, advential infiltration by lymphocytes and monocytic recruitment (5). In summary, our results indicate that human peritoneal cells could be a possible model to study lipoprotein and cholesterol metabolism. In addition, these cells could serve as a potential source of in vivo maturated macrophages that could be used to provide insight into foam cell formation.

Acknowledgments The authors gratefully acknowledge the contributions of Fang Shi, Ph.D., William Kleese, Ph.D., Darrel E. Gall, Ph.D., Ms. Gladys Benavente, R.N., and the patients and staff of Desert Dialysis Supported by funds from the University of Arizona Agricultural Center, Tucson, Arizona. Experiment Station.

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R.G.D. STEEL, AND J.H.TORRIE, Principles of Procedures for Statistkv&l&@&with . . Reference to the Blol~cal Sciemxs McGraw-Hill, New York, 106-l 14 (1960).

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C.S. GOLDSTEIN, J.S. BOMALASKI, R.B. ZURIER, E.G. NEILSON, AND S.D. DOUGLAS, Kidney Intematl. 2 733-740 (1984). Y. MADDOX, M. FOEGI-I, B. ZELIGS, M. ZMUDU J. BELLANTI, AND P. RAMWELL, Stand. J. Immunol. fi 23-29 (1984). A.K. SOUTAR, AND B.L. KNIGHT, Biochem. J. 204 549-556 (1982). J.L. GOLDSTEIN, Y.K. HO, S.K. BASU, AND M.S. BROWN, PROC. Natl. Acad. Sci. USA 26 333-337 (1979). R.W. MAHLEY, T.L. INNERARITY, K.H. WEISGRABER, AND Y. OH. SUK, J Clin. Invest.& 743-750 (1979). M.G. TRABER. AND H.J. KAYDEN. Proc. Natl. Acad. Sci. USA 77 5466-5470 (1980). D.W. BILHEIMER Y.K. HO, M.S. BROWN. R.G.W. ANDERSON, AND J.L. GOLDSTEIN. J. Clin. invest. 61678-696 (1978). A.M. FOLGELMAN, M.E. HABERLAND. J. SEAGER,M. HOKOM, AND P.A. EDWARDS, J. LipidsRes. 22 1131-1141 (1981). G.J. DAUGHERTY, AND W.H. MCBRIDE, J. Clin. Lab. Immunol. 14 l-l 1 (1984). P.K. PETERSON, E. GAIZIANO, H.J. SUH, M. DEVALON, L. PETERSON, AND W.F. KEANE, Infect. Immun. 49212-218 (1985). G.K. HANSSON, L. JONASSON, P.S. SEIFERT, AND S. STEME, Arteriosclerosis 9 567578 (1989). J.M. MUNRO, J.D. VAN-DERWALT, C.S. MUNRO, J.A. CHALMERS, AND E.L. COX, Hum. Pathol.18 375-380 (1987).

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