Clearance of Neutrophil-derived Myeloperoxidase by the Macrophage Mannose Receptor Virginia L. Shepherd and John R. Hoidal Veterans Administration Medical Center and the Departments of Medicine and Biochemistry, University of Tennessee, Memphis, Tennessee

Uptake of neutrophil-derived myeloperoxidase by the macrophage mannose receptor was studied. Rat bone marrow-derived macrophages internalized 75 % of [IZ5I]myeloperoxidase through a mannose-specific process. Uptake via the mannose receptor is highly sensitive to treatment with oxidants. Treatment of rat macrophages with I mM HzO z for 30 min resulted in a 94 % reduction in uptake of myeloperoxidase. By Percoll gradient fractionation studies, 38 % of internalized myeloperoxidase was delivered to the lysosomal compartment during a 15-min chase period, similar to findings for delivery of other ligands for this receptor. Once in the lysosome, the myeloperoxidase remained enzymatically active for several hours, with 50% activity remaining at 8 h. Finally, myeloperoxidase-containing macrophages had an increased capacity to down-regulate their own mannose receptors or receptors on neighboring macrophages, possibly through the myeloperoxidase-mediated production of oxidized halogens. Thus, the macrophage mannose receptor plays a potentially physiologic role in regulating extracellular myeloperoxidase levels. The receptor-mediated uptake may either arm the macrophage to contribute to oxidant-mediated tissue damage or may function to clear extracellular myeloperoxidase during the resolution phase of the inflammatory process.

Macrophages have on their cell surface a receptor that clears glycoproteins from the extracellular space through a mannose-specific process (1). The glycoprotein ligands bind to these cell surface mannose receptors through a Ca+z-dependent process, are internalized via the receptor, and are ultimately transported to lysosomes. It has been postulated that this receptor plays an important role in the process of inflammation by clearance of extracellular enzymes that might contribute to the inflammatory state (2). Expression of mannose receptors is highly regulated by agents intimately involved in regulation of inflammation: neutrophil-derived oxidants rapidly down-regulate surface expression of mannose receptors (3); macrophage-activating agents such as endotoxin and phorbol esters down-regulate total synthesis of receptors (4); and anti-inflammatory drugs such as dexamethasone dramatically increase total mannose receptor synthesis and expression of active surface receptors (2, 5).

Mannosylated proteins that might be candidates for physiologic ligands for this receptor are lysosomal hydrolases (2). Recently, it has been demonstrated that peroxidases such as lactoperoxidase (6) and horseradish peroxidase (HRP) (7) can be internalized by the macrophage mannose receptor. One peroxidase that macrophages might come into contact with in an inflammatory milieu is myeloperoxidase (MPO) from degranulating neutrophils. Schwartz and associates (8) demonstrated that alveolar macrophages from cystic fibrosis patients take up neutrophil.;derived MPO and are capable of producing potentially harmful reactive oxygen species. In the present report, we demonstrate that rat bone marrow-derived macrophages and human monocyte-derived macrophages which express high levels of mannose receptors (2, 9) internalize purified neutrophil MPO through a mannosespecific process and are capable of utilizing the MPO to produce reactive oxygen species.

(Received in original form April 26, 1989 and in revised form November 3, 1989)

Materials Yeast mannan, phorbol myristate acetate (PMA), horseradish peroxidase (HRP) , lactoperoxidase, Ficoll-Hypaque, and Percoll were purchased from Sigma Chemical Co. (St. Louis, MO). Chloramine-T was obtained from Eastman Kodak (Rochester, NY), and Na lZ5I from Amersham (Arlington Heights, IL).

Materials and Methods Address correspondence to: Virginia L. Shepherd, Ph.D., VA Medical Center/Research Service, 1310 24th Avenue South, Nashville, TN 37212. Dr. Shepherd's current address: VA Medical Center, Nashville, and the Departments of Medicine and Biochemistry, Vanderbilt University, Nashville, TN 37212. Dr. Hoidal's current address: VA Medical Center, Salt Lake City, and the Department of Medicine, University of Utah, Salt Lake City, UT 84132. Abbreviations: horseradish peroxidase, HRP; myeloperoxidase, MPO. Am. J. Respir. Cell Mol. BioI. Vol. 2. pp. 335-340, 1990

Ligands Mannose-bovine serum albumin (mannose-BSA) containing

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approximately 30 mol of mannose/mol of protein was purchased from E-Y Laboratories (San Mateo, CA). I3-Glucuronidase was purified from rat preputial glands as previously described (10). I3-Glucuronidase was iodinated using a chloramine-T procedure (11). Purification of MPO from Human Neutrophils MPO was provided by Dr. Beulah Gray, Department of Microbiology, University of Minnesota. It was purified from an extract of neutrophil granules using Matrex Gel Orange A chromatography followed by cation exchange chromatography on Bio Rex 70 as previously described (12). The purity and molecular weight were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by staining with Coomassie Blue R-250 overnight and destaining. Assay of MPO Activity MPO activity was measured by the quantitation of the oxidized product produced from o-dianisidine. The following reagents were mixed in a 3-ml quartz cuvette: o-dianisidine, 0.45 mg in 300 p.l methanol; H z02, 300 p.l of a 0.0025 % solution in potassium phosphate/saline buffer, pH 6.0; 2.3 ml of potassium phosphate/saline buffer; 100 p.l ofthe MPOcontaining sample. The reaction was initiated by the addition of HzO z. The resulting absorbance at 460 nm was measured at lO-s interavals for 4 min, and the MPO concentration calculated from a standard curve using known quantities of purified neutrophil MPo. Iodination of MPO Neutrophil MPO was labeled by reaction with Na lz5I as described for-the iodination of lactoperoxidase (13). lodogen (Pierce Chemical Company) was dissolved in chloroform at 1 mg/m!. A lO-p.l aliquot of this solution was added to a glass tube and air-dried. To this tube was added the following: 10 p.l (5 p.g) of MPO; 90 p.l of 0.1 M potassium phosphate buffer, pH 7.4; and 5 p.l (0.5 mCi) of Na ,z5I (Amersham). The reaction was allowed to proceed for 10 min at room temperature. The reaction mixture was fractionated on a 5-ml column of Sephadex G-25, and fractions at the void volume containing the labeled MPO were combined. The resulting specific activity was approximately 5 X lQ6 cpm/ J1-g protein. Assay of Cell-associated HRP Activity Cells were solubilized in 0.1 % Triton, and HRP activity measured as described by Rabinovitch and colleagues (7). Aliquots of cell extracts (25 p.l) were added to 0.9 ml of phenol red solution (5 mg/50 ml PBS). H Z0 2 (5 p.l) was added to a final concentration of 50 p.M. The mixture was incubated at room temperature in the dark for 10 min. The reaction was stopped by addition of 10 p.l of 1 N NaOH and the absorbance of 610 nm was measured. Preparation of Macrophages (1) Rat bone marrow-derived macrophages. Macrophages were prepared as previously described (2). Briefly, bone marrow cells were flushed from rat femurs with phosphate-buffered saline (PBS). Cells were collected by centrifugation and resuspended in Dulbecco's modified medium (DME) containing 10% L-cell-conditioned me-

dium and 10% fetal bovine serum. Cells were plated in 150mm plastic tissue culture dishes for 4 to 5 days, then removed from the plates with cold 5 mM EDTA and gentle pipetting. The cells were reseeded into 24-well plates or used directly in suspension in binding medium (Hanks' balanced salt solution [HBSS] containing 1% BSA). (2) Human monocyte-derived macrophages. Monocytes were isolated from buffy coats as described previously (9). Briefly, the buffy coat was diluted 1:1 with 1 mM EDTA in PBS, pH 7.4, layered over Ficoll-Hypaque, and centrifuged at 2,200 rpm for 20 min at ISO C. The mononuclear layer was removed, and the cells were collected by centrifugation. The cells were resuspended in RPMI containing 10% autologous serum and were cultured for 6 to 8 days. Uptake of [Iz5I]MPO by Rat Bone Marrow-derived Macrophages [ 'Z51]MPO (1 x 105 cpm) was added to macrophages (5 X 105 cells/well) in a 24-well tissue culture plate in a total volume of 400 p.l in binding medium. After incubation at 37° C for 60 min, the cells were washed twice and then solubilized in 0.1 % Triton X-100. Uptake was quantitated by gamma-counting. Nonspecific uptake was determined by addition of excess mannan to companion wells. Inhibition of Uptake of [Iz5Ill3-g1ucuronidase by Other Mannose Receptor Ligands Rat macrophages in 24-well dishes (5 x 105 cells/well) were incubated with 10 p.g oflabeled l3-g1ucuronidase in 400 p.l of binding medium. Mannose-BSA, HRP, lactoperoxidase, or MPO (0.3 to 30 p.g/ml) was added to the medium, and uptake allowed to proceed at 37° C for 60 min. Control wells received only ['Z51]I3-g1ucuronidase. Results are expressed as the percentage of control uptake in the presence of increasing concentrations of inhibitor. Percoll Gradient Fractionation of Rat Bone Marrow Macrophages Containing Endocytosed MPO and I3-Glucuronidase Macrophages (2 X 107 cells/ml) were incubated with MPO (3.5 U) and l3-glucuronidase (20 p.g/ml at 5 X 105 cpm/ p.g) in a total volume of 3 ml in binding medium for 30 min at 37° C. The cells were pelleted, non-internalized ligands removed, and the cells resuspended in 3 ml of binding medium and incubated for an additional 15 min at 37° C. The cells - were collected and then fractionated into plasma membrane and lysosomal fractions on Percoll gradients as described by Wileman and coworkers (13). Briefly, cells were homogenized in 3 ml of 3 mM imidazole buffer, pH 7.4, containing 0.25 M sucrose using a Dounce homogenizer with a tightfitting pestle. Nuclei and unbroken cells were removed by centrifugation, and a crude membrane fraction was collected by centrifugation at 15,000 X g for 10 min. Membranes were resuspended in imidazole/sucrose and fractionated on a 1.07g/ml self-forming Percoll gradient. I3-Hexosaminidase, a lysosomal marker, was found in dense fractions, and the plasma membrane marker alkaline phosphodiesterase was found at the top of the gradient (13). Distribution of (l z51]13glucuronidase was quantitated by gamma-counting, and distribution of MPO was quantitated by measurement of the enzymatic activity as described above.

Shepherd and Hoidal: Myeloperoxidase Clearance by the Macrophage Mannose Receptor

TABLE 1

TABLE 2

Specific uptake of FZ5/jMPO by the macrophage mannose receptor*

Effect of 1 mM HzO z on MPO uptake by the macrophage mannose receptor* . Uptake

[IZ5I]MPO [IZ5I]MPO + mannan (1 mg/ml)

12,550 ± 469 3,078 ± 488

* Rat bone marrow macrophages were seeded into 24-well tissue culture dishes at 5 X 10' cells/well. [IZ'I1MPO (5 x 10' cpm/O.I p.g protein) was added to triplicate wells in HBSS containing 1 % BSA. Companion wells received labeled MPO plus I mg/ml mannan. Cells plus ligand were incubated for 60 min at 37° C, solubilized in 0.1 % Triton, and the amount of cell-associated [ 125 I1MPO quantitated by gamma-counting. Results are expressed as the average of triplicate determinations ± SD and are representative of 2 separate experiments.

Results Uptake of MPO by the Mannose Receptor on Rat Bone Marrow-derived Macrophages Table 1 shows the specificity of uptake of [IZ5IJ-labeled MPO by rat macrophages. Approximately 75 % of the uptake can be blocked by mannan (1 mg/ml), in close agreement with the specificity of this receptor for well-characterized ligands such as mannose-BSA (2), {3-glucuronidase (11), and HRP (7). These results demonstrate that MPO is taken up by rat macrophages through a mannose-specific process. Similar results were obtained with human monocyte-derived macrophages. In further support that the mannose receptor is responsible for MPO uptake, Figure 1 compares the ability of MPO to inhibit uptake of J3-glucuronidase with other peroxidases and mannose-BSA. Mannose-BSA reduced uptake to 50% of control levels at an approximately lO-fold lower concentration than the peroxidases, in agreement with the known affinity for this ligand. MPO was similar to HRP and lactoperoxidase in its inhibitory capacity, suggesting that these three peroxidases bind with similar affinities.

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Figure 1. Inhibition of uptake of [!Z5I]t3-glucuronidase by other mannose receptor ligands. Rat macrophages were seeded into 24well culture dishes at 5 X 10 5cells/well. [IZ5I]t3-glucuronidase was added at 10 ILg/400 ILl (5 X 10' cpm/ILg) in HBSS containing 1% BSA. Cells were incubated with labeled enzyme alone, or plus mannose-BSA (0.3 to 30 ILg/ml), or lactoperoxidase, HRP, or MPO (1 to 30 ILg/ml). After incubation at 37° C for 60 min, cells were solubilized with 250 ILl 0.1 % Triton X-IOO, and cell-associated counts determined by gamma-counting. Each point is the average of triplicate determinations and is representative of 2 experiments.

Ligand

MPO {1-Glucuronidase

-H,O,

+H 20 2

Percentage of Control

17.0 mo 9,289 cpm

1.0 mo 1,132 cpm

6 12

* Rat macrophages were treated with medium containing I mM H,O, or medium alone at 37° C for 30 min. The cells were then washed, and assay medium (HBSS containing 1% BSA) was added. MPO and [ 125 Ill3-glucuronidase were added, and specific uptake was determined. Results are expressed as specific cell-associated counts for l3-glucuronidase and specific cell-associated units of activity for MPO in cells pretreated with or without H2 0,. The data are averages of triplicate determinations and are representative of 2 separate experiments. Effect of HzO z Treatment of Rat Macrophages on Uptake of MPO and {3-Glucuronidase Our laboratory has demonstrated that the activity of the mannose receptor on rat bone marrow-derived macrophages is rapidly down-regulated by treatment with 1 mM HzO z (3). This effect appears to be specific for this receptor, since uptake via another endocytic receptor, the mannose phosphate receptor, and phagocytic uptake were not affected, nor were the cells injured by this treatment. Table 2 shows the effect of oxidant treatment of rat macrophages on MPO uptake. Untreated control macrophages specifically internalized 17 U MPO via the mannose receptor. Pretreatment of macrophages with 1 mM HzO z for 30 min resulted in a decrease in MPO uptake to 6 % of control levels. Similar results were found for uptake of {3-glucuronidase as previously reported (3). These results suggest that MPO is taken up by an oxidant-sensitive receptor and, together with the data in Table 1 and Figure 1, further support the hypothesis that the mannose receptor is responsible for this uptake. Receptor-mediated Endocytosis of MPO followed by Delivery to the Lysosomal Compartment By kinetic analysis, it is known that ligand binds to the mannose receptor at the surface and is rapidly internalized into an endosomal, acidic compartment. Within this compartment, the soluble ligand and membrane-bound receptor separate, and ligand is routed to lysosomes, while receptors recycle to the membrane for further rounds of uptake (14). Wileman and coworkers (13) reported that lysosomal, endosomal, and plasma membrane fractions could be separated on Percoll gradients and that the time of transport of ligand from the surface to the lysosome could be determined. Using this technique, we showed that MPO does not remain on the surface of the macrophage but is transported via the mannose receptor to the lysosome, with kinetics similar to delivery of {3-glucuronidase.Figure 2 shows the results of Percoll fractionation of cells loaded with both [IZ5IJ{3-glucuronidase and MPO for 30 min, followed by a chase period of 15 min. Under these conditions, 38 % of both ligands are detected in the lysosome, in agreement with the findings of Wileman and coworkers (13). Stability of Endocytosed MPO in Macrophage Lysosomes We have shown that [i25IJ{3-glucuronidase is taken up by bone marrow macrophages and delivered to lysosomes,

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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 21990

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