Bone Marrow-derived Mononuclear Phagocytes Autoregulate ...

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Jul 14, 1988 - tions of CSF-1-dependent bone marrow-derived mac- rophages ..... Adherent ptr wall. "IGH. (1.511106 ccllr/ml). 4.2 x IOs. Time (hours). FIG. 2.
THEJOURNALOF BIOLOGICAL CHEMISTRY

Vol. 264,No. 10, Issue of April 5, pp. 5370-5377, 1989 Printed in U.S.A.

0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

Bone Marrow-derived Mononuclear Phagocytes Autoregulate Mannose Receptor Expression* (Received for publication, July 14,1988)

Denis R. ClohisyS, Jean C. Chappel, and StevenL. Teitelbaume From the Department of Pathology and Laboratory Medicine, Jewish Hospital a t Washington University Medical Center, St. Louis, Missouri 63110 and ShrinersHospital for Crippled Children (St. Louis Unit), St. Louis, Missouri 63131

This study extends our previous observation that The mannose receptor is a 175-kDa plasma membrane surface mannose receptor expression by pure popula- component, which in the marrow resides exclusively in cells tions of CSF-1-dependent bone marrow-derived mac- of the monocyte/macrophage family (1).The protein isknown rophagesincreaseswithtime (Clohisy, D. R., Bar- to be involved in recognition and endocytosis of particles and Shavit, Z., Chappel, J. C., and Teitelbaum, s. L. (1987) other substances displaying terminal mannose residues. Thus, J.Biol. Chem. 262,16922-15929). We presently find, it is likely that themannose receptor is pivotal to an activity however, that the progressive enhancement of lZ6I- which characterizes the macrophage phenotype, namely mannose-bovine serum albumin (‘”I-Man-BSA) bind- phagocytosis (2). ing per cell reflects cell number rather than duration Recently, the mannose receptor has also been found to of culture. In fact,macrophages plated at high density serve as a marker of macrophage differentiation. 1,25-Dihybind &fold more ‘”I-Man-BSA than do their low dendroxyvitamin D, an agent known to promote monocytic difsity counterparts, with no difference in receptor-ligand affinity. Furthermore, cells cultured at high den- ferentiation of leukemic cells (3), also accelerates mannose sity are ultimately subjected to lower levelsof exoge- receptor expression (4). Taken with evidence from others that nously provided macrophagegrowth factor, and fewerthe mannose receptor is differentiation-dependent (5), our are in interphase. By obtaining synchronous popula- findings indicate that thismembrane-residing protein may be tions of quiescent bone marrow macrophages, how- used as a hallmark of macrophage maturation. Furthermore, ever, we demonstrate that neithercell cycling nor at- the apparentassociation of mannose-receptor expression and tendant levels of colony stimulating factor-1 influence macrophage differentiation raises the possibility that both events may be functionally related. Hence, an understanding mannose receptor expression. Our nextseries of experiments established that den- of the means by which developing mononuclear phagocytes sity-related mannose receptor expression reflects re- express this protein may yield important clues into the funmoval, by marrow macrophages, of a “down-regulat- damentals of macrophage differentiation. ing” factor contained in culturemedium. To this end, We found, during our priorstudies, that appearance of the we treated mononuclear phagocytes with either mac- mannose receptor on bone marrow macrophage precursors rophage- or control-conditioned medium and found increases with time in culture (4). While this finding may that, via a fetal calf serum-residing protein(s), only reflect the differentiation-associated properties of the recepcontrol medium is capable of noncompetitively reduc- tor, they also raise the possibility that its expression is reguing lZ51-Man-BSAbinding in a dose-dependent man- lated by extracellular (medium-contained) components which ner. Moreover, reconstituted 20-40% (NH4)zS04-pre- are progressively modified by the developing cell. In fact, we cipitablefractionsderivedfrom either sham-condi- demonstrate herein that a serum-residing protein factor tioned medium or fetal calf serum are capable of down“down-regulates” plasma membrane expression of the manregulating mannose receptorexpression.Alternanose receptor and that bone marrow macrophages progrestively, the same fraction obtained from macrophagesively inactivatethis inhibitory agent,thereby leading to conditioned medium contains no such activity. Finally, initial characterization of the down-regulating factor enhanced binding of mannosylated radioligand. reveals it to be acid-activable and trypsin-sensitive, MATERIALS AND METHODS yet resistant to heating toat least 80 “C, ribonuclease A, or freezing and thawing. We conclude that bone Unless otherwise specified, all chemicals were obtained from marrow macrophages up-regulate expression of their Sigma. Horse and fetal calf sera were purchased from Hazelton own plasma membrane mannose receptor by inactivat- Dutchland Research Products (Denver, PA). Murine L929 cells were ing a noncompetitive, serum-residing inhibitory pro- a gift of H. S. Lin (Washington University Medical School, St. Louis, MO), mannosylated BSA’ (42 molof sugar/mol of protein) was tein(s).

* This work wassupported in part by National Institutes of Health Grant DE05413, a grant from the Shriners Hospital for Crippled Children (St. Louis Unit),and a gift from the Jewish Hospital Auxiliary. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “acluertisement” in accordance with 18U.S.C. Section 1734 solelyto indicate this fact. A NASA Research Associate and Research Fellow of the Orthopaedic Research and Education Foundation. 5 To whom reprint requests should be addressed Dept. of Pathology, Jewish Hospital at Washington University Medical Center, 216 S. Kingshighway, St. Louis, MO 63110.

+

purchased from E-Y Laboratories (San Mateo, CA), and ribonuclease A was from Boehringer Mannheim. Preparation of Stage Z CSF-1-Prepared by modification of method of E. R. Stanley (6). Serum-free conditioned medium from L929 cells was chromatographed by a batch calcium phosphate method. Specifically, 250 ml of calcium phosphate gel (300 ml of 0.4 M Na3P04plus 3 liters of 0.057 M CaC12) were added per liter of L929 conditioned

The abbreviations used are: BSA, bovine serum albumin; lz5IMan-BSA, ‘251-mannosylatedbovine serum albumin; CSF-1, colony stimulating factor-1; MEM, minimum Eagle’s medium; PBS, phosphate-buffered saline: Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.

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Mononuclear Phagocytes Autoregulate Mannose Receptor media and stirred overnight. The supernatantwas recovered and the gel rinsed twice with 3 mM N a p 0 4 (pH 6.5) and once with 94 mM Na3P04. Supernatantswere collected from the high molarity phosphate rinses and dialyzed against deionized water. The Lowry assay was used for protein determination (7). Final specific activities were typically lo6 units/mg protein. Complete Culture Medium-a modification of Eagle'sMedium (MEM) (Sybron, Washington, D. C.) with 500 units/ml Stage I CSF1, 15% fetal calf serum, penicillin (100 units/ml), and streptomycin (100 rg/ml). Marrow Cells-Nonadherent cells were obtained from bone marrow cultures of 9-12-week-old male A/J mice (Jackson Labs, Benton Harbor, MI) and prepared as described previously (4). The ends of freshly harvested femurs were excised, and the cells collected by flushing the medullary cavity with ice-cold a-MEM through a 25gauge needle. The marrow plug was dispersed by several passages through an 18-gauge needle, and the cells were pelleted (800 x g for 7 min a t 4 "C). The pellet was resuspended in ice-cold a-MEM and the number of nucleated cells determined by counting an aliquot of the resuspended cells in 2% acetic acid. Cells (1 X lo6 cells/ml) were then seeded into tissue culture dishes (Falcon Plastics) at a density of 3.4 X lo5 cells/cm2 in the presence of complete medium containing 900 units/ml of Stage I CSF-1. After a 24-h incubation, nonadherent cells were collected and theadherent cells discarded. The nonadherent population was then pelleted (800 X g for 7 min at 4 "C),resuspended in 1ml of Pronase solution (0.02%w/v Pronase, Bgrade, Calbiochem, 1.5 mM EDTA in PBS/107 nucleated cells) and incubated for 15 min at 37 "C. Pronase treatment was stopped by the addition of horse serum (0.2 m1/10 ml Pronase solution), and the suspension layered onto 15 ml of ice-cold horse serum and incubated on ice for 15 min. The cell suspension was then pelleted (1200 X g for 7 min at 4 "C), resuspended in complete medium and nucleated cell number determined. This group of cells, which is 24 h post-bone marrow cell isolation and freshly recovered from Pronase treatment, is designated "marrow cells." When these cells are cultured in the presence of 500 units/ml CSF-1 for 7 days, all colony forming units are positive for the monocyte-specific enzyme, a-napthyl acetate esterase. Adherent Bone Marrow-derived Mononuclear Phagocytes-Marrow cells were cultured at the concentrations indicated in 24-well plates (NUNC) in 2 ml of complete medium/well. After designated periods, adherent cells were used for cell cycle analysis and binding studies. Adherent Cell Counts (6)-Adherent mononuclear phagocytes were washed with cold PBS, detached from the tissue culture plastic after a 5-minincubation at 20 "C in 0.005% Zwittergent (Calbiochem), and counted by hemocytometer. Analysis of DNA Contentper Nuclei-Adherent mononuclear phagocytes in 24-well plates were rinsed three times with PBS and1.0 ml of Krishan's reagent (9). After the cells detached, they were harvested and rinsed twice with fresh Krishan's reagent. The resulting nuclei were placed on ice and DNA content per nucleus determined in a Coulter EPICS V fluorescence-activated cell sorter. PHlThymidine Incorporation Assay-Adherent mononuclear phagocytes were pulsed with 50pCi of [3H]thymidine (ICN) and incubated a t 37 "C. After a 60-min incubation, the cells were rinsed with PBS and incubated (37 "C) for 30 min in 10% trichloroacetic acid. They were then re-rinsed with an ethano1:ether (3:l) solution and extracted for counting in 0.1 N NaOH. Preparation of Control- and Macrophage-conditioned MediumMarrow cells were plated into 24-well plates at 1.5 X lo6 cells/ml and 2 ml/well in complete medium. After 48 h in culture,the macrophageconditioned medium from each well was collected, concentrated 1:lO (Amicon-YM5),and stored at 4 "C. Control-conditioned medium was generated in a similar but cell-free manner. Mannose-Bouine Serum Albumin Iodination (10)-100 pg of mannose-bovine serum albumin (Man-BSA) were mixed with 1 mCi of NalZ5I(Amersham Corp.) and 300 pg of chloramine T in 80 pl of 0.1 M Na3P04 buffer (pH 7.6). The reaction was terminated after 10 min on ice by addition of 190 pl of sodium metabisulfate (2.4 mg/ml) and 190 p1 of potassium iodine (10 mg/ml). The sample was then run on a Sephadex G-50 column (1 X 20 cm) buffered in 10 mM Tris-HCL (pH 7.5). 0.4-mlsamples were collected and active fractions identified by y counting. Protein determination was performed by the Miller method (11) and specific activity was typically 5-8 X IO6 cpm/pg Man-BSA with >95% of total counts trichloroacetic acid-precipitable. Ligand was used within 2 weeks of iodination. lZ51-Man-BSABinding Assay-Binding determinations at 4 "C of lZ51-Man-BSAto adherent bone marrow mononuclear phagocytes involved slight modifications of techniques previously described (10).

5371 Such procedures result in nonspecific binding representing 10-20% of total cell-associated counts. The cells were washed three times (0.4 ml/well/wash) with HHBG (Hank's Balanced Salt Solution, 10 mM Hepes, 10 mM Tris, 0.1% glucose, and 10 mg/ml BSA, pH 7.1) and incubated with 0.2 ml of various concentrations of lZ5I-Man-BSAin HHBG plus 0.2 ml of HHBG & 4 mg/ml mannan (final volume 0.4 ml/well). Equilibrium binding was achieved after a 48-h incubation, and the level of cellassociated ligand determined. After 48 h, the incubation medium was aspirated, and cell layers quickly rinsed six times with Hank's Balanced Salt Solution. Cellswere dissolved in 1.0 N NaOH (0.5 ml/ well) and cell-bound radioactivity of NaOH-solubilized material was measured by y counting. Duplicate values were determined for all binding points. Transformation of binding data to determine dissociation constants and estimate the number of available binding sites was performed by methods of Scatchard (12). Evaluation of the Influence of Conditioned Medium and Fetal Calf Serum on lZ5I-Man-BSABinding-Marrow cells were plated at 0.3 X lo6 cells/ml in 24-well plates (1 ml/well). Twenty-four hours later, various aliquots of either macrophage- or sham (control)-conditioned medium or fetal calf serum were added to appropriate wells. After the indicated additional culture period (12-36 h), cells were placed at 4 "Cand the specific binding of lZ5I-Man-BSAper mg of cell protein determined by incubation with 2 pg/ml "'1-Man-BSA & 2 mg/ml mannan. Cell-associated binding was assessed by the method of Lowry (7) and standardized perunit of protein. CSF-1 Levels-Radioimmunoassay determinations of CSF-1 levels were kindly performed by E. R. Stanley (Albert Einstein Medical Center, Bronx, NY). Ammonium Sulfate Precipitation-All ammonium sulfate samples were precipitated in a percent-to-percent methodology described by Green and Hughes (13). The precipitated samples were dissolved in PBS, which had been diluted 1:lO a t a concentration 10 times that of the original sample. The solutions were dialyzed against a-MEM prior to use. Acid Actiuation-The 20-40% (NH4)2SO4-precipitablefraction of fetal calf serum was redissolved and concentrated 10 times in PBS and dialyzed against a-MEM. The solution was then titrated to pH 2.0 with 1 N HC1 for 5 min at 4 "C, during which time a precipitate formed which was removed by microfugation (12,000 X g for 5 min). The supernatantwas aspirated and left at 4 "C for 1week after which it was dialyzed against a-MEM overnight. Control material was treated identically except for acidification. A suboptimal down-regulating volume (10 pl) of material was tested for its effects on lZ5IMan-BSA binding. RESULTS

Effect of Cell Number on the Binding of lZ5I-Man-BSAby Bone Marrow Mononuclear Phagocytes-We recently reported that lZ5I-Man-BSAbinding by bone marrow mononuclear phagocytes increases with time in culture (4). Since these cells are actively dividing, we explored the possibility that such binding actually relates to cell number and not culture duration. To this end, we plated various concentrations of marrow cells (1.5, 7.5, and 15 X lo6 cells/ml) and after 2 days assessed lZ5I-Man-BSAbinding at 4 "C. As seen in Fig. L4, specific binding of lZ5I-Man-BSAper lo5 cells increases progressively with cell density. These differences are reflected by maximal specific bindings of 0.3,0.8, and 2.6 ng ofradioligand/W cells at cell densities of 5.0, 18.1, and 42.5 x lo4 cells/well, respectively. Accompanying Scatchard plots (Fig. 1B) demonstrate that this progressive enhancement of binding reflects increased available sites per cell, as receptor affinities are similar. Specifically, low, intermediate, and high density cells display approximately 2.5,6.0, and 20 x lo4 receptors/cell, respectively. These data demonstrate that mannose receptor expression is influenced by cell number and not durationof culture. Effect of Cell Density on CSF-1 Levels and Cell CyclingHaving determined that lZ51-Man-BSAbinding per cell increases with cell number, we next sought to identify distinct characteristics of high and low density populations which may alter radioligand binding. Two parameters which we consid-

Mononuclear Phagocytes Autoregulate Mannose Receptor

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TABLEI Characterization of high and low density cell populations Marrow cells were plated at high (1.5 X lo6 cells/ml) or low (0.15 X lo6 cells/ml) numbers. After 48 h, adherent cell number, attendant culture medium levels of CSF-1, and DNA content per nucleus were determined. Morrow Cells Plolcd

Adherent Cell Numbcr ptr wall

"IGH (1.511106 ccllr/ml)

4.2 x IOs

DNA Contat per Nuckus

0.5 n

0.5 1.0 1.5 2C 12SI-MANNOSE BSA ADDED (pg/ml)

J

3.a 12sI-MANNOSE BSA BOUND (ng)/lOs CELLS

FIG. 1. Effect of cell number on 1261-mannoseBSA binding by bone marrow-derived mononuclear phagocytes. Marrow cells were plated at various densities and '261-Man-BSAbinding determined per lo6 cells after 48 h. A , saturation binding isotherms; B , Scatchard analysis of binding data. All samples shown are the means of duplicate determinations. A, 4.2 X lo6 cells/well; 0, 1.8 X lo5 cells/well; 0, 0.5 x lo5 cells/well.

ered likely to change with cell density and are known to influence macrophage physiology, are ambient concentrations of CSF-1 and the proportion of cells in interphase (6, 11).To address this issue, we plated both low (0.15 x lo6 cells/ml) and high (1.5 x 106/ml)numbers of marrow cells in 500 units/ ml CSF-1 and after 2 days measured medium CSF-1 levels and determined cell cycle distribution by flow cytometry. Table I demonstrates that at high cell density 1) ambient levels of CSF-1 are lower (106 versus 344) and 2) fewer cells are in interphase (10% high density versus 30% low density). As '251-Man-BSAbinding increases dramatically with increasing cell density (Fig. l ) , these data raised the possibility that CSF-1 levels and/or cell cycling may dictate lZ5I-Man-BSA binding. Characterizing a Model to Isolate Cycling and Noncycling Populations of Bone Marrow-derived Mononuclear Phagocytes-With the intent of ultimately defining the role of cell cycling in regulating '251-Man-BSAbinding, we adopted previously described techniques designed to yield populations of bone marrow-derived macrophages enriched with quiescent

Time (hours) FIG. 2. Regulation of mononuclear phagocyte mitogenesis. Asynchronously dividing bone marrow macrophages received 50 units/ml CSF-1 for 24 h and were then reexposed ( A )to either a low (50 units/ml) or mitogenic dose (1000 units/ml) of CSF-1. Thymidine incorporation per well wasdetermined a t designated times, and DNA content per nucleus examined by flow cytometry analysis at points A , C, and D (see respective inserts). 0, asynchronous cells; A, 50 units/ ml CSF-1; 0, 1000 units/ml.

(GoGI phase) or cycling (S-phase) cells (14). Such methods are predicated on the finding that cell survival and proliferation are dependent on the macrophage growth factor, CSF-1 (8).To obtain quiescent cells, we determined that 50 units of CSF-l/ml is sufficient to maintain marrow macrophage survival without stimulating mitogenesis (data notshown). More specifically, 24 h after treating asynchronously dividing cells with 50 units of CSF-l/ml, we found a greater than 90% decrease in [3H]thymidine incorporation and more than 90% of the cells in GoGl (i.e. pre-DNA synthetic) phase by flow cytometry analysis (Fig. 2, insert A ) . These data characterize such cells as apopulation of noncycling, bone marrow-derived macrophages. With the capacity to produce quiescent cells in hand, we turned to theisolation of their cycling counterparts. To this end, we treated quiescent cells with increasing quantities of CSF-1 and determined that 1000 units/ml induces maximal mitogenesis (data not shown). Further, as shown in Fig. 2, when quiescent cells (point A ) are exposed to 1000 units of

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Mononuclear Phagocytes Autoregulate Mannose Receptor CSF-1, they exhibit a time-dependentincrease in [3H]thymidine incorporation. Most notably, after addition of the mitogen (Fig. 2, point A), [3H]TdR incorporation, and thus entry into S-phase,commences after 12-16 h with the peak in DNA synthesis occurring at 36 h. Accompanyinginserts of analysis of DNA content per nucleus estimate that after a 36-h exposure to 1000 units/ml CSF-1, 45% of bone marrow macrophages are in S-phase (Fig. 2, insert D) as compared to less than 5% exposed for the same time period to 50 units/ml CSF-1 (Fig. 2, insert C). Comparison of lZ5I-Man-BSABinding byCycling versus NoncyclingMononuclear Phagocytes-This set of experiments was designed to determine if cell cycling influences '251-ManBSA binding. Culture methods described in the previous section were instituted to obtain simultaneously separate populations of noncycling (Fig. 2, point C) and cycling (Fig. 2, point D) cells and their respective capacities to bind 1251Man-BSA are compared in Fig. 3. The datademonstrate that regardless of cell cycling dissociation constants as well as estimated number of available binding sites are similar. Influence of CSF-1 on lZ5I-Man-BSABinding by Bone Marrow Macrophages-We next sought to determine if CSF-1 influences expression of the lZ5I-Man-BSA binding site. Hence, we cultured quiescent cells (Fig. 2, point A ) with either 1000 units of CSF-l/ml, or 50 units of CSF-l/ml, and after8 h (Fig. 2, point B ) assessed their binding of lZ5I-Man-BSA. Fig. 4 demonstratesthat both binding affinity and capacity are nearly indistinguishable regardless of CSF-1 concentration. It should be noted that since the cells in this particular experiment bound relatively high levels of lZ5I-Man-BSA(1 ng/105 cells), we also examined the influence of CSF-1 on those expressing fewer receptors (i.e. low density cultures) and found that in this circumstance CSF-1 also fails to impact on lZ5I-Man-BSAbinding (data not shown). The Influence of Medium-contained Factors on lZ5I-ManBSA Binding-To evaluate the possibility that bone marrow macrophages may alter marrow binding by modifying their environment, we incubated complete culture medium for 48 h in either the presence of bone marrow macrophages (macrophage-conditioned medium) or in their absence (controlconditioned medium) concentrated 10-fold. One hundred p1 of either macrophage- or control-conditioned mediumwas then added to bone marrow macrophage cultures, and after 12, 24, or 36 h, '251-Man-BSAbound per mg cell protein was determined at asaturating dose of the ligand (2 pg/ml). Results displayed in Table I1 document that the specific binding of lZ5I-Man-BSAby cells treated with either control-

0.5 1.o 1251-BSA MannoseBound (ng)/lW Cells

FIG. 3. Effect of cellcycling on "'I-Man BSA binding. Quiescent (point C, Fig. 2) or cycling cells (point D,Fig. 2) were incubated with Iz5I-Man-BSAat 4 "C. Scatchard analysis of saturation binding data is shown. All values are the means of duplicate determinations. 0--0, quiescent cells; 0--0, cycling cells.

0.k 1.o 1251-BSA Mannose Bound(ng)/lOs Cells FIG. 4. Effect of CSF-1 on '261-Man-BSAbinding by quiescent cells. lZ5I-Man-BSAbinding by macrophages was assessed after 24 h of a quiescent CSF-1 dose (0h) (pointA in Fig. 2) or subsequently following an 8-hre-exposure (point B in Fig. 2) to either a quiescent or amitogenic dose of CSF-1. Scatchard analysis of saturation binding data is shown. All values are the means of duplicate determinations. 0--0, t = 0 h; A--A, t = 8 h, quiescent CSF-1 dose; 0--0, t = 8 h, mitogenic CSF-1 dose.

TABLEI1 Effect of control versus macrophage conditioned medium on Iz5I-Man-BSAbinding After 24 h in culture, cells were treated with 1OO-gl aliquots of either control-conditioned or macrophage-conditioned medium. At the times indicated, Iz5I-Man-BSA(2 pg/ml) was added at 4 "C and nanograms bound/mg of cell protein determined. All values shown are the means of auadruulicate determinations. '2SI-Mannose BSA Time after addition

Control-conditioned medium

ng bound/mgprotein

h

12 24 36

Macrophage-conditioned medium

13 f 5 18 f 4 33 f 6

34 f 5 59 f 2 136 f 2

or macrophage-conditioned medium increases in a stepwise fashion with time in culture. More importantly, however, those cells exposed to macrophage-conditioned medium bind more radioligand at each time point than do theirsham medium-treated counterparts. Fig. 5 documents that the enhanced binding capacity of the macrophage-conditioned medium-exposed cells reflects a greater than 2-fold increase in the number of sites per cell with unaltered affinity. These differences in lZ5I-Man-BSAbinding capacity could, however, represent either"up-regulation" of available binding sites by macrophage-conditioned medium or "down-regulation" by control-conditioned medium. To resolve this issue, cells were exposed to increasing aliquots of either form of medium for 36 h. As before, lZ5I-Man-BSAspecific binding was determined at a saturating concentrationof the ligand (2 pg/ml). The results of this experiment demonstrate that macrophages incubated with control-conditioned medium bind progressively less lZ5I-Man-BSA (Fig. 6). Specifically, untreated cells bind 103 f 11 ng of '251-Man-BSA/mgprotein, those exposed to 150 pl of control-conditioned medium, only 23 f 8 ng/mg protein, and macrophages incubated with up to 150-p1 aliquots of macrophage-conditioned medium bind as much radioligand (96 f 15 ng/mg protein) as do virgin cells. Influence of Fetal Calf Serum on lZ5I-Man-BSABindingWe next turned to identifying the inhibitor of lZ5I-Man-BSA binding present in control-conditioned culture medium, which conceivablycould include components of modifiedEagle's Medium, Stage I CSF-1, or fetal calf serum. Data not shown

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Mononuclear Phagocytes Autoregulate Mannose Receptor

I

1251

1

I

0.5 1.o 1.5 IzsI-MannoseBSAAdded (pg/rnl)/lOs Cells

1

1

50

100 Medium Added @I)

150

FIG.6. Effect of increasing volumes of control- or macrophage-conditioned medium on '2'I-Man-BSAbinding. After 24 h in culture, cells were supplemented with increasing volumes of control (0)-or macrophage-conditioned medium (0).Thirty-six hours later cells were incubated with a saturating quantity of '"1Man-BSA (2 pg/ml) at 4 "C and radioligand binding per mg cell protein determined. All values shown represent the means S.E. of quadruplicate determinations

*

-

50-

i3 a -

0.5 1.o 12sI-Mannose BSA Bound (ng)/l05 Cells

FIG.5. Effect of control versus macrophage-conditioned medium on "'I-Man-BSA binding. After 48 h in culture, cells wereexposed to 100 pl of either control-conditioned medium (0--0) or bone marrow-derived macrophage-conditioned medium (0--0). Twenty-four hours later, cells were incubated with lZ6IMan-BSA at 4 "C: A , saturation binding isotherms plotted per lo6 cells; B , Scatchard analysis of binding data. All values shown are the means of duplicate determinations.

demonstrate that the inhibitory factor is maintained within M, 14,000 exclusion dialysis tubing, precluding the possibility that the factor of interest is modified Eagle's Medium. In addition, data presented under "Influence of CSF-1 on lZ5IMan-BSA Binding by Bone Marrow Macrophages," excludes the possibility that CSF-1 or any otherelement of our Stage I CSF-1 preparationis responsible for regulation of lZ5I-ManBSA binding. Consequently, we explored the effect of fetal calf serum on lZ5I-Man-BSAbinding. Thus, marrow cells were cultured for 24 h in complete mediumfollowedby addition of various volumes of 10-fold concentrated fetal calf serum (0-100 pl). After an additional 36 h, lZ5I-Man-BSAbinding was determined at a saturating quantityof the ligand (2 pg/ml). As shown in Fig. 7, lZ5I-Man-BSAbinding by bone marrow macrophages falls with increasing volumes of serum. In fact, whereas nonsupplemented cells bind 43 f 3 ng of '"I-ManBSA/mg of protein, those treatedwith 100 plof concentrated calf serum bind only 27 k 6 ng/mg protein. These observations raised the possibility that fetal calf serum simply competes for available 'T-Man-BSA binding sites and is, in effect, acting as cold ligand. This concern was investigated by simply preincubating bone marrow macro-

e

i 8m

3020-

I

I

I

I

25

50

75

100

FetalCd~mAdded@l)

FIG.7. Effect of fetal calf serum on regulation of "'I-ManBSA binding. After 24 h in culture, cells were supplemented with increasing volumes of 10-fold concentrations of fetal calf serum. Thirty-six hours later cells were incubated with lZ6I-Man-BSA(2 pg/ ml) a t 4 "C and radioligand binding per mg cell protein determined. All values shown represent the means S.E. of quadruplicate determinations.

*

phages at 4 "C in 1 ml of binding buffer (HHBG) plus0-100 pl of fetal calf serum for 6 h, rinsing the wells, and then measuring lZ5I-Man-BSAbinding per mg cell protein by our standard assay. Data shown in Fig. 8 document that fetal calf serum does not compete with lZ5I-Man-BSAfor available binding sites. Taken together, these findings demonstrate that a component of fetal calf serum truly "down-regulates" lZ5I-Man-BSAbinding sites. Influence of Fetal Calf Serum Ammonium Sulfate-precipitable Fractions on lZ5I-Man-BSABinding-Initial characterizations of element responsible for down-regulating mannose receptor expression was undertaken by assessing the capacity of (NH4)zS04-precipitablefractions of fetal calf serum to

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Mononuclear Phagocytes Autoregulate Mannose Receptor

2oor

FIG. 8. Effect of fetal calf serum on the assay for '251-ManBSA binding. Cells wereincubated for 6 hat 4 "C in 1ml of binding buffer (HHBG) plus 0-100 pl of fetal calf serum. The wells werethen rinsed, incubated with lZ6I-Man-BSA(2 pg/ml) at 4 "C and binding per mg cell protein was determined. All values represent the mean k S.E. of quadruplicate determinations.

FIG. 10. Effect of 20-40% (NH&SO,-precipitable fractions of control- and macrophage-conditioned medium on '"I-ManBSA binding. After 24 h in culture, cells were supplemented with from 100p1 of reconstituted 20-40% (NH4)zS04-precipitable fractions control and bone marrow-derived macrophage-conditioned media. Thirty-six hours later, '261-Man-BSA(2 pg/ml) was added at 4 "C and binding per mg cell protein determined. All values shown represent the mean k S.E.of quadruplicate determinations. 8, controlconditioned medium; @ bone marrow-derived macrophage-conditioned medium.

TABLE I11 Effect of trypsin treatment of 2040% (NH4)2S04-precipitableserum fraction on 'z5Z-Man-BSAbinding 100 pl of reconstituted and dialyzed 20-40% (NH4)zS04-precipitable serum fraction, which had been treated with various concentrations of trypsin for 1h a t 37 "C followed by a 4-fold excessof soybean trypsin inhibitor, were added to cells precultured for 48 h. Forty-eight hours later the cells were incubated with a saturating quantityof '"IMan-BSA (2 pg/ml) a t 4 "C and the specific binding per well determined. Negative control represents cultures to which no (NH4)ZS04Precipitable protein was added. 0 0-20 20-40 40-60 60-80 Ammonkrm Sulfate FmdplWe Fradion (%)

FIG. 9. Effect of treatment with reconstituted (NH&S04-

precipitable fractions of fetal calf serum on '251-Man-BSA in culture, cells were supplemented with 100 p1 binding. After 24 h22,770 of reconstituted, (NH4)2S04-precipitable fractions of fetal calf serum (0, 0-20, 20-40, 40-60, or 60-80%). Thirty-six hours later, '"I-Man49,597 added at 4 "C and binding per mg cell protein BSA (2 pg/ml) was64,727 determined. All values shown represent the means S.E. of quadruplicate determinations.

*

regulate lZ51-Man-BSAbinding. Thus, marrow cells incubated in complete medium for 24 h were supplemented with 100 pl of each (NH4)2S04-precipitable fraction (0, 0-20, 20-40, 4060, and 60-80%), and lZ5I-Man-BSAbinding determined after 36 h. Fig. 9 shows that macrophages treated with either the 0-20,40-60, or 60-80% component bind asmuch radioligand as do sham-treated cells (0%). In contrast, however, cells exposed to reconstituted proteins from the 20-40% fraction bind approximately one-half as much lZ5I-Man-BSAas do control cells. Thus, the serum-residing element responsible for down-regulation of lZ6I-Man-BSAbinding sites is located in the protein fraction precipitable by 20-40% (NH4)2S04. Comparison of 20-40% Ammonium Sulfate-precipitable Fractions in Control and Macrophage-conditioned MediumBecause the 20-40% (NH)2S04-precipitable fraction of fetal calf serum appeared to contain the component of culture medium responsible for down-regulating the mannose receptor, we examined the effect of a similar fractionfrom controland macrophage-conditioned medium on expression of the mannose binding site. We found that cells cultured with the 20-40% fraction from control-conditioned medium bind 16 Ifr 4 ng of lZ5I-Man-BSA/mgof protein which those exposed to

'"I-Man-BSA cprn boundlwell

Negative control Trypsin concentration (pg/ml) 0

150 500 1,000 1,500

64,271 17,207 41,647

TABLE IV Effect of ribonuclease A treatment of2040% (NH4)~SO~precipitable serum fraction on '25Z-Man-BSA binding 100 pl of reconstituted and dialyzed 20-40% (NH&S04-precipitable serum fractions, eitheruntreated or exposed to 10 pg/ml of ribonuclease A, a quantity capable of degrading at least 15 pg of total RNA (data not shown), for 1 h a t 37 "C, were added to cells cultured for 48 h. Forty-eight hours later, the cells were incubated with a saturating quantity of "%Man-BSA (2 pg/ml) at 4 "C and specific binding per well determined. Negative control represents culturesto which ribonuclease A but no (NH4)2S04-precipitableprotein was added. '251-Man-BSA cprn bound/well

Negative control 20-40% (NH4)2S04fraction Untreated Ribonuclease A-treated

27,936 3,239 1,765

similarly treated macrophage-conditioned medium bind 28 f 6 ng (p < 0.05) (Fig. 10). These data demonstrate that bone marrow-derived mononuclear phagocytes inactivatea medium-residing protein factor contained inthe 20-40% (NH)2S04-precipitable fraction of fetal calf serum responsible

5376

Mononuclear Phagocytes Autoregulate

Mannose Receptor

TABLEV ume of material was subjected to a pH of 2.0 for 1 week and Effect of freezing and thawing of 20-40% fNH4)2S04-precipitable then dialyzed against a-MEM, however, down-regulating acserum fraction on "'I-Man-BSA binding tivity increased more than 6-fold (Table VII). 100 pl of reconstituted and dialyzed 20-40% (NH4)2S04-precipitable serum fractions, either untreated or frozen and thawed, were DISCUSSION added to cells preculturedfor 48 h. Forty-eight hours later, thecells The mannose receptor is among the most studied of the were incubated with a saturating quantity of I2'I-Man-BSA (2 pg/ml) at 4 "C and specific binding per well determined. Negative control macrophage surface proteins and, based upon its enhanced protein was expression with time in culture, and its induction by 1,25represents cultures to which no (NH4)2S04-precipitable

added.

dihydroxyvitamin D, it appears to be a marker of monocytic differentiation. Furthermore, itsappearance parri passu with cpm bound/well monocytic maturation suggests that the phagocytic capacity control Negative 64,271 of macrophages is indeed a developmentally related event. 20-40% (NHa)&04fractions Thus, with the potential importance of progressive expression 17,207 Untreated of the mannose receptor in mind, we explored the means by Freeze-thaw 19,847 which bone marrow-derived mononuclear phagocytes bind increasing quantities of 1251-Man-BSAwith time. TABLEVI Our first step was to examine the possibility that thetimeEffect of heating 20-40% (NH4)2SO4-precipitable serum fraction on related increase in radioligand binding reflects an event other lZ51-Man-BSAbinding than duration of culture. As the cells are actively dividing, we 100 pl of reconstituted and dialyzed20-40% (NH&S04-precipita- questioned whether the enhanced appearance of the mannose ble serum fractions either untreated or heated to60, 70,80, or 100 "C receptor is a manifestation of increased cell number. To this for 1 h were added to cells precultured for 48 h. Forty-eight hours end, we determined the relative numbers of available '"Ilater the cellswere incubated with a saturating quantity of '261-ManBSA (2 pg/ml) at 4 "C and specific binding per well determined. Man-BSA binding sites at three different cell densities on the NeEative control represents cultures towhich no (NH4)2S04-precipi- same day of culture. If mannose receptor expression is related only to duration of culture, then one would expect each tabie protein was added. macrophage to display similar lZ5I-Man-BSAbinding, regard'=I-Man-BSA less of cell density. We found, however, that after 48 h, cpm bound/well mannose receptor expression per cell mirrors cell number control Negative 26,350 (Fig. 1).Hence, the previously reported phenomenon of in20-40% (NH4)2S04 fractions creased mannose receptor expression with duration (4) apUntreated 3,239 60 "C 3,543 pears to reflect the influence of events sensitive to cell density 70 'C 96 and notnecessarily time. 80 "C 163 Two circumstances which are usually cell density-depend100 "C 21,137 ent and can impact on ligand binding by macrophages are entry into interphase and exposure to themacrophage-specific TABLEVI1 growth factor, CSF-1 (8, 13). We found, in fact, that similar Effect of acidification of (NH4)2S04-precipitable serum fraction on to mannose receptor expression, both the distribution of cells '"I-Man-BSA binding in interphase andthe attendantlevels of CSF-1 vary with cell 10 pl of reconstituted and dialyzed 20-40% (NH4)2S04-precipitable number (Table I). With this information in hand, we develserum fractions, either untreated or maintained at pH 2.0 for 1 week, oped a method for simultaneously altering the cell cycling were added to cells precultured for 48 h. Forty-eight hours later the cells were incubated with a saturating quantity of "'I-Man-BSA (2 patterns of bone marrow macrophages (Fig. 2). Thus, asynpg/ml) at 4 "C and specific binding per well determined. Negative chronously dividing cells were placed in GoGl by exposure to control represents cultures to which no (NH&S04-precipitablepro- low levels of CSF-1 for 24 h. These quiescent and synchronous tein was added. cells were then recultured in either low or mitogenic concen1z61-Man-BSA trations of the macrophage growth factor for an additional 24 h yielding noncycling or cycling populations, respectively. cprn bound/well Utilizing this system, we found mannose receptor expression control Negative 67,423 to be uninfluenced by entry into interphase(Fig. 3) or ambient 20-40% (NH4)2S04 fractions 34,169 Untreated levels of CSF-1 (Fig. 4). Pre-acidified 5,372 Having determined that neither entry into the cell cycle nor exposure to CSF-1 areresponsible for density-dependent expression of the mannose receptor, we examined the possifor down-regulation of '251-Man-BSAbinding sites. Partial Characterization of Mannose Receptor Down-regu- bility that the phenomenon reflects macrophage-mediated luting Factor-Initial characterization of the serum factor modification of the extracellular environment. These experiresponsible for down-regulating the mannose receptor was ments involved exposure of bone marrow mononuclear phagperformed on the 20-40% (NH4),S04-precipitablefraction of ocytes to aliquots of macrophage-conditioned medium which fetal calf serum. The inhibitory activity is trypsin-sensitive led to enhanced lZ5I-Man-BSAbinding when compared to in a dose-dependent manner (Table 111) but ribonuclease A- control-conditioned medium-treated cells (Table 11, Fig. 5). resistant(Table IV). It also resists freezing and thawing We also noted that thisdifferential expression of radioligand (Table V) and temperaturesof at least 80 "C, but is inactivated binding reflects down-regulation of the mannose receptor by cells exposed to control medium (Fig. 6). Furthermore, the by boiling (Table VI). We next turnedto theeffects of acidification on inhibitory down-regulating activity is localized to theprotein fractionof activity. Thus, we exposed macrophages to 10% optimal in- both fetal calf serum and control medium precipitable with hibitory volumes (10 p1) of the 20-40% (NH4)2S04-precipita- 20-40% (NH4)&04(Fig. 9). The fact that the same fraction ble fraction of fetal calf serum resulting in an approximately recovered from macrophage-conditioned medium does not 50% decrease in lZ5I-Man-BSAbinding. When the same vol- inhibit mannose receptor expression indicates that the cells '*'I-Man BSA

Mononuclear Phagocytes Autoregulate Mannose progressively inactivate the down-regulating factor. Failure of pretreatment with fetal calf serum at 4 "C to reduce radioligand binding indicates that our observations do not merely reflect competition for the mannose receptor by a nonradioactive moiety. Finally, we have begun to characterize the down-regulating factor. It is a protease-sensitive, acid-activable moiety with what appears to be exceptionally stable characteristics. For example, the activity resists freezing, thawing, and heating to at least 80 "C. Thus, our experiments document the presence of a serumresiding protein factor capable of down-regulating membrane expression of the mannose receptor and thatmacrophages are capable of inactivating this regulatory protein(s). These findings raise important issues regarding the phagocytic capacity of macrophages and underscore the importance of carefully defining culture conditions in studiesinvolving expression of this surface binding site. Acknowledgments-We gratefully acknowledge the expert secretarial assistance provided by Jane Wodicker and also thank Dr. E. R. Stanley for measuring CSF-1 levels.

Receptor

5377

REFERENCES 1. Wileman, T. E., Lennartz,M. L., and Stahl, P.(1986) Proc. Natl. Acad. Sci. U. S. A. 8 3 , 2501-2505 2. Stahl, P., Schlesinger, P. H., Sigardson, E., Rodman, J. S., and Lee, Y.C. (1980) Cell 1 9 , 207-215 3. Bar-Shavit, Z., Teitelbaum, S. L., Reitsma, P., Hall, A., Pegg, L. E., Trial,J.,andKahn, A. J . (1983) Proc. Natl. Acad. Sci. U. S. A. 80,5907-5911 4. Clohisy, D. R., Bar-Shavit, Z., Chappel, J. C., and Teitelbaum, S. L. (1987) J . Biol. Chem. 2 6 2 , 15922-15929 5. Shepard, V. L., and Stahl, P. D. (1984) in Lysosomes (Dingle, J. T., Dean, R. T., and Sly, W., eds) pp. 83-98, Elsevier Scientific Publishing Co., Amsterdam 6. Stanley, E. R. (1985) Methods Enzymol. 1 1 6 , 564-587 7. Lowrv. 0. H., Rosebroush. N. J.. Farr, A. L.. and Randall. R. J. (195i) J. B&. C k m . i93,265-275 8. Tushinski. R. J.. Oliver. I. T..Guilbert. L. J.. Tvnan. P. W.. Warner,'J. R., and Stanley, E . R. (1982) Cell 28,"71-81 9. Krishan, A. (1975) J . Cell Biol. 6 6 , 188-193 10. Konish, M., Sheperd, V., Holt, G., and Stahl, P. (1983) Methods Enzymol. 9 8 , 301-304 11. Miller, G. L. (1959) Anal. Chem. 3 1 , 964 12. Scatchard, G. (1949) Ann. N. Y. Acad. Sci. 5 1 , 660-672 13. Green, A. A., and Hughes, W. L. (1955) Methods Enzymol. 1,6790 14. Gandour, D. M., and Walker, W. S. (1983) J . Zmmunol. 1 3 0 , 1108-1112 '