Enhancement of Mouse Macrophage and Human ... - CiteSeerX

Journal

of Leukocyte

Biology

42:498-503

(1987)

1 a,25 Dihydroxyvitamin D3 and Mononuclear Phagocytes: Enhancement of Mouse Macrophage and Human Monocyte Hydrogen Peroxide Production Without Alteration of Tumor Cytolysis W. Larry Division

Gluck

of Hematology/Oncology, Duke University

and

J. Brice

Weinberg

Department of Medicine, Medical Centers, Durham,

Veterans Administration North Carolina

and

1 a,25 dihydroxyvitamin D3 (1,25 D3) is known to interact with hematopoietic cells. The purpose of this study was to determine the effect of 1,25 D3 on hydrogen peroxide (H202) production and tumor cell killing by mouse peritoneal macrophages and human blood monocytes.

Enhanced

monocyte

and

macrophage

phorbol

mynstate

stimulated H202 production was observed at concentrations of 0.13 to and and was maximal at 1.3 nM. At concentrations of 100 U/mi, gamma y) alone had a similar effect but, in combination with 1,25 D3, there was effect. At concentrations ranging from 0.13 to 130 nM, 1,25 D3 failed to

acetate (PMA)130 nM 1,25 D3

interferon (IFNno cooperative augment tumor

cell lysis by macrophages from peptone-injected normal or bacillus Calmette-Guerin (BCG)-infected mice, or by blood monocytes from normal humans. Our results indicate that 1,25 D3 can activate the monocyte and macrophage for H202 secretion without concomitant activation for tumor cell killing. Key words:

1,25 dihydroxyvitamin macrophage

D3, hydrogen

INTRODUCTION la,25 dihydroxyvitamin D3 (1,25 D3) is a steroid hormone that principally regulates calcium homeostasis by specifically binding to cytosolic receptors in bone, kidney, and intestine. Recently, several unconventional target cells have been identified, and an expanded spectrum of biological activity has unfolded to possibly include cellular differentiation [1,14,16,36], immunoregulation [30], alteration of tumor growth kinetics [10], and modulation of mononuclear cell function [5,6,8-11]. The significance of a receptor for 1,25 D3 on mononuclear cells remains largely unknown. In 1983, BarShavit et al noted the ability of 1,25 D3 to induce macrophage multinucleation by causing cellular fusion [5]. Abe et al, studied the effects of 1,25 D3 on murine alveolar macrophages in vitro and found that incubation with ng/ml concentrations of 1,25 D3 fostered multinucleation, increased glucose consumption and Fc receptor expression, and enhanced cytolysis of B16a mouse melanoma cells [2]. These effects were not seen with inflammatory peritoneal macrophages. Recently, 1,25 D3 has been shown to activate human blood monocytes for secretion of H202, to increase adherence to plastic, and to

© 1987 Alan

R. Liss,

Inc.

peroxide,

cytotoxicity,

monocyte,

increase the activities of certain lysosomal enzymes [7,23,24]. This study was designed to study the effect of 1,25 D3 on H202 production and tumor cell killing by murine peritoneal macrophages and human monocytes. In this report, we show that 1,25 D3 enhances phorbol myristate acetate (PMA)-induced H202 production by peritoneal macrophages from mice injected with sterile peptone or infected with BCG, and by nonnal human blood monocytes. We observe no additive or synergistic effects of 1,25 D3 and IFN-’y on H202 production. Secondly, we demonstrate that 1,25 D3 has no effect on tumor cell killing by mouse peritoneal macrophages and human blood monocytes. MATERIALS Mice

AND

METHODS

and Experimental

Ten-to-fifteen

week

Charles

River

Breeding

Received

January

30,

1987;

Subjects old

female

Lab accepted

C3H/HeN

mice

from

(N. Wilmington,

MA)

were

May

14, 1987.

Reprint requests: J. Brice Weinberg, Hematology/Oncology VA Medical Center, Durham, NC 27705.

1510,

1,25 D3 Activation

‘C

I

C

2

T

T

of Monocytes

and Macrophages

499

I.C

C

6-

a,

0. 0.

E N

E

4-

N

0

E C

0

T

=

2-

N

0 E

C

C 1,25

D3(nM)

IFN-y(U/ml)

0 0

0.13

23

130

0

0

0

0

100

1,25D3(nM) IFN-y(U/mi)

0 0

0.13

1.3

13

130

0

0

0

0

0 100

Fig. 1. Hydrogen peroxide production by macrophages from peptone-injected normal mice (a) or BCG-infected mice (b). The cells were incubated 3 days with the designated concentrations of 1,25 D3 or iFPI-’y, and then peroxide generation in response to 200 nM PMA was determined. These are data from one experiment, representative of 7 experiments done. The bars represent the mean + 1 SEM.

used. Human volunteers were all healthy donors on no medications who donated their blood after giving informed consent according to a protocol approved by the V.A. and Duke University Clinical Investigation Committees. Plateletpheresis residue bags from healthy donors were obtained from the American Red Cross (Charlotte, NC). Preparation Macrophages

and Culture

of Monocytes

and

Mice. BCG infection was established 4-8 weeks before harvest by the intraperitoneal injection of 1 x 106 CFU of BCG of the Phipps Strain. One milliliter of sterile 10% peptone (Difco Laboratories, Detroit, MI) was given intraperitoneally 3 days before harvest of peritoneal cells. The cells (3.5 x 10) in 0.1 ml of medium were added to the 6-mm diameter chambers of microtiter plates (Falcon No. 3040). The cells were allowed to adhere for 90 mm at 37#{176}C in humidified 95% air/5% CO2, and then nonadherent cells were removed by washing two times with phosphate-buffered saline (PBS) with suction through a sterile Pasteur pipette. Intact monolayers were verified by phase-contrast microscopy. Human. Peripheral blood and cells from plateletpheresis residue bags were anticoagulated with acid citrate dextrose (ACD) (3.8% sodium citrate) and the cells were first separated on Ficoll-sodium diatrizoate [28,37]. The interphase layer consisting of lymphocytes and monocytes was then centrifuged at 60g to separate contaminating platelets. The cells were then further separated by use of a discontinuous Percoll gradient as described be-

fore [37]. The purity of monocytes than 85%. After the adherence monocyte population constituted cells.

so isolated step (see over 95%

was greater above), the of the total

Cell Lines The human epithelioid carcinoma cell line Hela and murine tumorigenic fibrosarcoma line 3T 12 were from the American Type Culture Collection (Rockville, MD). All cell lines were maintained by serial passage two-tothree times per week in a 10% heat-inactivated fetal bovine serum (FBS). Drugs Recombinant IFN-’y was a gift from Biogen Research Corporation (Cambridge, MA) and Genentech, Inc. (San Francisco, CA). The la, 25-dihydroxyvitamin D3 was from Hoffman-LaRoche (Nutley, NJ) and was stored in sealed ampules in argon until use. As determined by Limulus amebocyte lysate assay [32], the amounts of IFN-y and 1,25 D3 used in these assays had less than 10 pg/mi lipopolysaccharide (LPS). Supplies Dulbecco’s modified Eagle’s medium (catalog no. 4301600, Grand Island Biological Co., Grand Island, NY) was formulated with endotoxin (LPS)-free water and supplemented with 100 U of penicillin/mi, 100 g streptomycin/ml, 20 mM Hepes, and 1 mg dextrose/mi. Sterilization was by filtration and only medium with < 200 pg/mi LPS as determined by the Limulus amebocyte

Gluck

500

and Weinberg

lysate assay was used [32]. LPS-free fetal and adult bovine serum were obtained from Sterile Systems, Inc. (Logan, UT). Human serum was isolated from subjects at the time of monocyte isolation. Ficoll-sodium diatrizoate, specific gravity 1.077 (Histopaque1077) was from Sigma Chemical Chemical Co., St. Louis, Missouri. Polyvinylpyrrolidone-coated silica (Percoll) was purchased from Pharmacia Fine Chemicals, Division of Pharmacia, Inc. (Piscataway, NJ). Tritiated thymidine (2 Ci/mmol) was from New England Nuclear Corp. (Boston, MA). Hydrogen

Peroxide

Assay

Adherent monocyte/macrophage monolayers were incubated in the indicated additives for 36 h. Human monocytes were cultured in 10% unheated autologous or pooled serum, and mouse macrophages in 10% heat-inactivated adult bovine serum. The production of H202 in response to 200 nM PMA was determined using horseradish peroxidase and phenol red and a microtiter plate reader as described previously [33]. Two hundred nM PMA was used since this dose produces maximal H202 production by mononuclear phagocytes. Protein determinations were done on triplicate lysates (0.05% Triton X-100) by the method of Lowry et al [15]. All samples were done in triplicate and expressed as nanomoles H202/mg protein/hr. Cell-Mediated Monocyte lished

Cytolysis and

Assay

macrophage

as outlined

above.

monolayers Target

tumor

were cells

(3T12

estabfor

‘C

C a, 0 0. 0’

E N

0 I

N

0

E C 1,25D5(nM) IFN-y(U/ml)0

0

0.13

1.3

13

0

0

0

130 0 13 0 100100

Fig. 2. Hydrogen peroxide production by human monocytes isolated from blood drawn into ACD and processed immediately (“fresh”), or monocytes isolated from piateletpheresis residue bags (“pheresis”). The monocytes were incubated 3 days with the designated concentrations of 1,25 D3 or IFN-’y, and then peroxide generation in response to 200 nM PMA was determined. These data are from one experiment, representative of 8 experiments done with “fresh” monocytes and 5 experiments done with “pheresis” monocytes. The bars represent the mean + 1 SEM.

murine experiments and HeLa for human experiments) were prelabeled with [3H]-thymidine as described before [33]. Target cells were added with or without additives as indicated. All experiments using murine macrophages were done in 10% heat-inactivated adult bovine serum, and those using human monocytes were done in 10% unheated autologous serum or 10% pooled, unheated human serum for cells from apheresis bags. Experiments involving 2-h pretreatment only with 1,25 D3 were completed in serum-free medium with the monolayers washed twice with sterile PBS before the final addition of target cells with or without additives. All assays were completed in 10% serum, as noted. After incubation for 60 h, the cell-free supernatants were aspirated and counted individually in Aquasol scintillation fluid [33]. Cell lysates were produced by vigorous mixing of intact monolayers with 0.2% sodium dodecylsulfate (SDS). All samples were done in triplicate. Calculation of percent cytolysis was done as described before [33].

RESULTS Hydrogen Peroxide Macrophages

Production

by Monocytes

and

Murme. Inflammatory macrophages harvested from normal mice demonstrated enhanced H202 production in response to PMA after incubation with 1,25 D3 (Fig. 1A). Monolayers incubated with 0.13 nM 1,25 D3 demonstrated a 5-fold increase in H2O2 production compared to control. Incubation with 13.00 nM and 130.00 nM 1,25 D3 resulted in similar levels of augmentation at 12-fold control. IFN-’y enhanced H202 production comparable to the higher doses of 1,25 D3. In these assays, the combination of IFN--y and 1.3 nM D3 was consistently toxic to the cells with loss of adherence, crenation of cells and disruption of the monolayers. BCG macrophages also demonstrated enhanced H202 production in the presence of 1,25 D3 although there was less evidence of a dose:response effect with the doses of 1,25 D3 used. The BCG macrophages produced 10-fold more H2O2 than did the inflammatory macrophages (Fig. ib). Human. Freshly harvested monocytes from normal donors and monocytes isolated from plateletpheresis residue bags demonstrated a dose-related increase in PMA-stimulated H202 production with increasing concentrations of 1,25 D3 (Fig. 2). IFN-y (100 U/mi) alone had a similar effect on H202 production. The combination of IFN--y and 1,25 D3 was less than additive for freshly harvested cells. Monocytes isolated from plateletpheresis residue bags showed a comparable enhancement by 1,25 D3, but appeared to respond less well to IFN-y.

1,25 D3 Activation TABLE Normal

1. Lysis of 3T12 or BCG-infected

Tumor Mice

Cells

by P eritoneal

Macrop

of Monocytes hages

From

Additivesa IFN-y

(U/mI)

A B C D E F 0

1,25

LPS

(ng/ml)

0 100 0 0 100

13.0

100 0

0

13.0

10

cytolysis’

Normaic

BCGd

0

0

16.2

± 1.3

11.6

± 1.1

0

0

12.3

± 0.7

36.5

± 0.7

0 13.0

10 0

15.1 18.3

± 1.2 ± 1.2

80.3 12.8

± 5.8 ± 0.4

0

12.1

±

1.3

36.0

± 2.0

10

22.4

± 0.9

43.3

± 2.1

17.3

± 0.3

70.5

± 2.8

aprevent throughout the 3-day culture. bMn ± SEM of triplicate samples. cStatistical comparison (t test): A vs. B, P < 0.05;

A vs. C, p > 0.1;

A vs.

501

Peptone-Injected

Percent

D3 (nM)

and Macrophages

D,

p >

0.1;

A vs.

E,

0.1; Bvs. E, p > 0.4; Cvs. 0, p > 0.5. dStatistical comparisons (t test): A vs. B, p < 0.0005; A vs. C, p < 0.0005; A vs. D, p > 0.1; A vs E, p < 0.0005; A vs. F, p < 0.0005; A vs. 0, p < 0.0005; Bvs. E, p > 0.4; C vs. 0, p > 0.1. p < 0.05;

TABLE

2. Lysis

of Hela

Avs.

Tumor

F, p < 0.01;

Avs.

Ce lis by Human

G,

p >

Monocytes

Additivesa

Cytolysis’’

0 0.l3nMI,25D3 13.00 riM 1,25 D3 130.00 nM 1,25 D3

24.6 ± 2.9 20.9±3.1 21.4 ± 2.0 20.4 ± 3.8

100 13.00 100

U/mI

LFN-’y

nM 1,25 /m1

1)3

+

19.0

± 0.5

17.3

±

was present as a 4-h preincubation step or throughout the assay (data not shown). Human. Baseline human monocyte-mediated tumor cell lysis averaged 24.6 ± 2.9% over seven consecutive experiments. The addition of increasing concentrations of 1,25 D3, IFN--y alone or IFN--y in combination with 1,25 D3 failed to augment cytolysis (Table 2).

3.7

IFN-

D3 or IFN-’y were present bMn ± SEM of seven separate cAll values were not statistically

throughout the 3-day experiments. significantly different

determined

(p

a125

by the Student

Tumor Cell Killing Macrophages

t

test

>

DISCUSSION

coincubation. than control

as

0.05).

by Monocytes

and

Murine. Macrophages from peptone-injected mice demonstrated a baseline cytolysis of 7-15%. The addition of 1.3, 13.0, or 130 nM 1,25 D3 produced no significant change. Table 1 shows results from an experiment, representative of six comparable experiments done. IFN-’y alone caused a slight decrease in cytolysis, while LPS alone produced no change. The addition of 1,25 D3 to these additives produced no further change in cytolysis. The combination IFN-y and LPS caused a slight, but significant increase in cytolysis. Similar patterns and magnitudes of cytolysis were generated whether 1,25 D3 was present in a 4-h preincubation period only or continuously throughout the assay (data not shown). Incubation of macrophages from BCG-infected mice with 1,25 D3 also failed to enhance cytolysis, while IFN‘y, LPS, and IFN-y-/LPS together enhanced cytolysis (Table 1). The effect of 1,25 D3 again was not additive to that of IFN-’y or LPS and the combination of all three was no better than IFN-y or LPS alone. The same pattern of cytolysis was consistently produced whether 1,25 D3

1,25 D3 is known to dramatically affect bone and mineral metabolism [22], and recently it has also been shown to alter the function of hematopoietic cells. Various hematopoietic cells have specific receptors for 1,25 D3 [251. Abe et al observed suppression of proliferation and induction of differentiation of murine myeloid leukemia cell lines in the presence of 1,25 D3 [1]. Nanomolar concentrations of 1,25 D3 caused differentiation of the M- 1 cells to cells with a macrophage morphology, an increased content of lysozyme, and enhanced phagocytic activity. Likewise, the human promyelocytic leukemia HL-60 cells differentiate into macrophage-like cells in the presence of 1,25 D3 [16,36]. Receptors for 1,25 D3 have been identified on a wide variety of human cancer cell lines and fresh surgical specimens. Incubation of these cells with supraphysiologic concentrations of 1,25 D3 has produced variable responses but generally results in suppression of proliferation [10]. 1,25 D3 has been shown to cause both fusion and “activation” of mouse alveolar macrophages as demonstrated by enhanced glucose consumption, Fc receptor expression, and tumor cytotoxicity [2]. Peritoneal macrophages from mice injected with thioglycollate in the same setting demonstrated only a modest increase in glucose consumption with no enhancement of fusion or cytolysis in the presence of 1,25 D3 [2]. Our work has failed to demonstrate enhancement of mouse alveolar

502

Gluck

and Weinberg

macrophage-mediated tumor cytolysis by 1,25 D3 (data not shown). Compartmental differences in the activation of macrophages by immunological stimuli have previously been described [261; however, the discordant response to 1,25 D3 observed by Abe et al remains to be clarified. Several recent studies have expanded the number of known mononuclear phagocytic functions which are altered by 1,25 D3 to include enhancement of secretion of H202 by human monocytes [7] and increased activity of selected lysosomal enzymes [24]. Cohen et al provided indirect evidence that the enhancement in H202 production by human monocytes is a receptor-mediated process [71. Several other compounds have been identified which can activate one or more functions of monocytes and macrophages, at least some of which are receptor-mediated [9]. IFN-y is a potent enhancer of mononuclear phagocyte H202 secretion in vitro for both murine and human cells [17,18], and Nathan, et al have reported augmented secretion of H202 by monocytes harvested from cancer patients receiving IFN--y in vivo [20]. Our results indicate that 1,25 D3 can augment PMAinduced H202 production by peritoneal macrophages from peptone-injected or BCG-infected mice and by normal human blood monocytes. Our data confirms the work of Cohen et al [7] demonstrating that both 1,25 D3 and IFN-’y enhance the competence of human monocytes for H2O2 secretion, without additive enhancement when used together. Significantly, we show that 1,25 D3 fails to augment the capacity of mouse peritoneal macrophages or human blood monocytes to kill tumor cells despite the enhancement of H202 producing capacity. Hydrogen peroxide is produced by reactions involving NADPH oxidase and superoxide dismutase. The mechanism by which 1,25 D3 enhances the cells’ capacity for H202 generation is unknown. Several groups have reported alterations in the Km of NADPH oxidase in response to activation of macrophages by in vitro treatment with LPS or in vivo activation by Corynebacterium parvum [6,27,311. The number and affinity of the receptors for phorbol diesters in macrophages with varying capacities for H202 secretion are all comparable [6,35]. The effects of 1,25 D3 on the several disparate functions (e.g., H202 production, lysosomal enzyme content, and Fc receptor expression) suggest that the effects may be mediated through a general system of signal transduction. Mononuclear phagocyte activation for tumor cytolysis is a complex process involving “priming” of the cells with lymphokine (viz., IFN-’y), and “triggering” the cells to kill with an agent such as LPS [3,12,32]. The precise mechanism(s) by which mononuclear phagocytes kill tumor cells is not known. In general, the killing does require close effector:target contact, and macrophage: tumor cell binding appears to be a necessary (but not

sufficient) requirement for the killing [3,12]. Several mechanisms for tumor cell death have been proposed. These include inhibition of tumor cell mitochondrial respiration [II] mediated by a loss of tumor cell iron [13], tumor cytotoxicity caused by the actions of macrophagederived proteases or tumor necrosis factor [4], or tumor cell damage by macrophage-secreted reactive oxygen species [4,21]. In PMA-stimulated and antibody-dependent macrophage-mediated tumor cell killing, H202 appears to mediate the killing [4,19,21]. However, in PMAand antibody-independent systems, oxygen species including H202 appear not be mediators of the killing [8,29,33,34]. This corresponds with our findings here that demonstrate no correlation between the mononuclear phagocyte H202 secretion capacity and tumor cell killing in cells treated with 1,25 D3 in vitro.

ACKNOWLEDGMENTS This work was supported in part by support from NIH Training Grant 5 T32 HLO 7057-09 (WLG), the Veterans Administration, the James Swiger Hematology Research Fund, and NIH Grant CA 11265 and POl-A123308 (JBW).

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D3 Activation

of Monocytes

and Macrophages

503

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