cytokine-induced differentiation of cultured nonadherent macrophages

CELLULAR IMMUNOLOGY 144,203-2 16 ( 1992)

Cytokine-Induced Differentiation of Cultured Nonadherent Macrophages’

VAN TSAI, GARY S. FIRESTEIN, WILLIAM AREND,? AND NATHAN J. ZVAIFLER

Division qfRheumatology, University of California, San Diego, La Jolla, California 92103-8417; and tRheumatology Division, Department of Medicine, University of Colorado

Health Science Center, Denver, Colorado 80262

Received December IO, 1991; accepted July I, 1992

Monocytes were isolated from peripheral blood and cultured in vitro for more than 3 weeks in glass chamber slides. Phenotypically and ultrastructurally these nonadherent macrophages (NAM) appear similar to connective tissue resident macrophages. They constitutively secrete a high amount of IL-lra and little or no IL-la or IL-ID. When exposed to GM-CSF, IL-2, or IFN- y for 24 hr, NAM become adherent and undergo dramatic morphological changes. Cytokines treatment primes NAM for increased LPS-mediated TNF production and these GM-CSF- and LPS-treated NAM are cytotoxic to WEHI 164, a TNF-sensitive target. Morphological changes and TNF production are both inhibited by antimetabolites and a variety of antineoplastic drugs. Although morphology inhibition is reversible under certain circumstances, inhibition of TNF synthesis is irreversible. These findings suggest that cytokines might play a role in differentiation and maturation of long-term cultured monocytes. Furthermore, the effects of antimetabolites and antineoplastic drugs on arresting the differentiation processes may significantly impair antitumor fUnCtiOIIS Of macrophages. 0 1992 Academic PESS, IX.

INTRODUCTION

Macrophages are multifunctional cells capable of performing a wide spectrum of immune functions. They are involved in antigen presentation, tumor cytotoxicity, cytokine production, matrix remodeling, and immune surveillance (1, 2). To accom- plish these various functions, they must be able to migrate into tissues and sites of injury and differentiate into specialized cells. Experiments using chromosome markers and cell surface antigens provide strong evidence that monocytes, resident tissue macrophages (i.e., macrophages found in connective tissues without any exogenous stim- ulus), inflammatory macrophages, and highly specialized tissue macrophages (alveolar macrophages, Kupffer cells, osteoclasts, etc.) probably represent different stages in the life history of cells from the same lineage (3-6). The signals that initiate such changes are not well understood, but cytokines such as GM-CSF, IL-2, and IFN-y can up- regulate accessory functions, tumor cytotoxicity, and TNF production in some monocyte/macrophage populations (7, 8). In addition, GM-CSF has been shown to play a role in differentiation and maturation of certain mononuclear cells (5, 7).

’ Supported in part by a grant from the Concern Foundation and NIH Grants AR40525, AR39576, AM39950. AM40 135. and AR40770.

203

000%8749/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

204 TSAI ET AL.

In this report, we show that isolating blood monocytes without using an adherence step and avoiding exposure to xenoantigens, such as fetal calf serum, allows us to maintain them in culture for more than 12 weeks. These long-term cultured nonadherent macrophages (NAM) can respond to cytokines and have morphologic, phenotypic, and functional characteristics usually ascribed to resident tissue macrophages.

MATERIALS AND METHODS

Donors. All blood donors were normal volunteers between the ages of 20 and 50. Regents. Recombinant hGM-CSF (sp act lo* U/mg, LPS content of the concen-

tration stock < 0.025 rig/ml, purity > 95%) and hIFN-y (sp act 2 X lo7 IRU/mg, LPS < 0.048 rig/ml, purity > 98%) were provided by Amgen Biologicals (Thousand Oaks, CA) and were used at 80 and 100 U/ml, respectively. Recombinant hIL-2 (sp act 3 X lo6 U/mg, LPS < 0.012 rig/ml, purity > 95%) was obtained from Cetus Corp. (Emer- yville, CA) and was used at 3-30 U/ml. LPS (Sigma, St. Louis, MO) was used at 10 pg/ml.

Two murine monoclonal antibodies were used for class II major histocompatibility antigen determination. Leu 10, IgG-1 (Becton-Dickinson, Mountain View, CA) is specific for HLA-DQ, and SC2, IgG-2 (kindly provided by Dr Robert Fox, Scripps Clinic and Research Foundation, San Diego, CA) is specific for HLA-DR. Monoclonal antibodies used for CD 14 and CD1 lc were Leu M3 and Leu M5 (both were IgG-2b, Becton-Dickinson), respectively.

Isolation of pheripheral blood monocytes. Blood leukocytes were isolated from 30 to 60 ml heparinized whole blood samples by Ficoll-Hypaque density gradient cen- trifugation (Pharmacia, Piscataway, NJ) followed by three washes with RPM1 1640 (Irvine Scientific, Santa Ana, CA). The leukocytes were resuspended in 3 ml of 10% human pooled AB serum (ABS) (co.06 EU LPS/ml, Pel-Freez, Brown Deer, WI) in RPM1 supplemented with gentamicin, penicillin, streptomycin, and glutamine (complete medium) and layered over a discontinuous Percoll gradient (Pharmacia). The discontinous Percoll gradient was made in a 50-ml Coming polystyrene tube by first underlaying 15 ml of 50.5% Percoll (made with 10% ABS) with 5 ml of 75% Percoll (made with PBS) followed by overlaying 5 ml of 40% Percoll (made with PBS) onto the 15 ml of 50.5% Percoll; 5 ml of 30% Percoll (made with 10% ABS) was placed on top of the 5 ml of 40% Percoll. Finally, 3 ml of cells (up to lo8 cells) was layered onto the 30% Percoll and centrifuged at 4008 for 30 min at room temperature. Monocytes were harvested from the middle part of the 50.5% Percoll. These cells were >95% esterase positive and >90% Leu M3 positive by FACS.

The gradient-purified monocytes were washed three times and resuspended in 10% ABS at l-3 X 105/ml and cultured in 4-chamber Lab-Tek slides (Nunc, Naperville, IL) at 1 ml/chamber at 37°C 5% CO*. These cultures were fed with freshly made complete medium once a week. This is done by carefully removing 50% of the old media without removing any of the cells and replenishing the cultures with an equal volume of new media. These cultures can be maintained for up to 12 weeks.

Phagocytosis of Candida albicans by nonadherent macrophages and superoxide generation. Three-week cultured NAM were incubated with opsonized (40% human AB serum for 30 minutes at 4°C) heat-killed Candida albicans at a cell to yeast ratio of 1 to 10 and nitroblue tetrazolium (NBT) at 1 mg/ml. The mixture was rocked gently for 1 hr at 37°C. An aliquot of the mixture was removed, cytospun onto a glass

DIFFERENTIATION OF NONADHERENT MACROPHAGES 205

slide, and stained with Wright-Giemsa. Phagocytosis was confirmed by the observation of intracellular yeast particles and superoxide generation was determined by the presence or absence of formazan (dark blue) precipitation.

Treatment of S-week cultured macrophages with cytokines and LPS. After 3 weeks, the cultured macrophages were nonadherent and appeared round with short processes. These cells were then exposed to cytokines for 24 hr followed by the addition of LPS. Twenty-four hours after LPS treatment, morphological changes were observed and supernatants collected for the TNF assay.

Morphological changes were recorded by photographing through the Lab-Tek slides with a Nikkon Diaphot TMD inverted microscope with phase-contrast objectives. All photos were taken with Tmax ASA 400 black and white film under the same mag- nification.

Cytotoxicity of NAM on TNF-a-sensitive target cells. Target cells (the TNF-a-sensitive mouse fibrosarcoma tumor cell line-WEHI 164) were radiolabeled with sodium Cr” ( 100 &i/ lo6 cells) for 3 hr in the presence of actinomycin D ( 1 pg/ml). The targets were washed free of unbound Cr” and 1 X 104-labeled target cells were added to 1 X lo5 long-term (3 week) cultured nonadherent macrophages (NAM) in Lab-Tek chambers in a final volume of 1 ml of medium as follows: targets in medium (5% FCS/RPMI) alone for spontaneous release determination; targets in medium containing 1% Triton X-100 for maximum release determination; targets added to untreated NAM; targets added to NAM pretreated with GM-CSF (80 units) or with or without 24 hr of LPS (10 hg) treatment. Following an 18-hr incubation at 37°C 100 ~1 of medium from triplicate chambers was sampled and the radioactivity released determined in a gamma counter. The percent killing which is measured as specific Cr51 release is calculated from the formula 100X (XC-SR/TC-SR) where XC is experimental counts, SR is spontaneous release, and TC is total counts added.

TNF bioassay. TNF was measured in culture supernatants as previously described (9). Briefly, L929 cells were grown to confluency and removed from culture flasks with trypsin-EDTA (GIBCO, Grand Island, NY). Cells were washed and resuspended in the presence of 1 pg/ml of actinomycin D (Sigma) at 6 X lo5 cells/ml. The cells were cultured in 96 flat-bottom plates (Linbro, Flow Lab, Hamden, CT) in a total volume of 100 pi/well. Supernatants were added to the L929 cells in duplicate or triplicate. Serial dilutions of recombinant hTNF-cu (Amgen) standards were included in each plate. The L929 cells were incubated overnight and TNF activity was determined spectrophotometrically after staining with 0.2% crystal violet, 10% formalin, and 0.0 1 phosphate (pH 7.1).

Assays for IL-lra, IL-la, and IL-ID. IL- 1 ra protein was measured in a specific sandwich ELISA using affinity-purified polyclonal rabbit anti-IL- 1 ra antibodies as the primary antibody and a biotinylated IgG fraction from this rabbit anti-serum as the secondary antibody as previously described (10). The ELISA was developed with a peroxidase-streptavidin system and has a sensitivity about 0.2 rig/ml. The rIL-lra protein used as standard in the ELISA was generously provided by Synergen, Inc. (Boulder, CO). IL- 1 a and IL- 1 p proteins were measured in supernatants using ELISA with specific monoclonal antibodies as the primary antibodies and biotinylated IgG rabbit antibodies as the secondary antibodies. The sensitivity of the IL- 1 a! and IL- 10 ELISA was 63 pg/ml.

Treatment of cytokine-primed NAM with drugs. NAM were treated with the following drugs at a 1 PM concentration: doxorubicin, methotrexate, cyclosphosphamide, dexa-

206 TSAI ET AL.

methasone, indomethacin (indo), actinomycin D (Act D) and prostaglandin E, (PGE,). All were purchased from Sigma (St. Louis, MO). These drugs were added simulta- neously with the cytokines to the cultures. Twenty-four hours after the addition of cytokines and drugs, LPS was added to the cultures as above. Twenty-four hours later, the cultures were photographed (see above) and aliquots of supernatant were collected and dialysed (MW cutoff of tubing was 10,000) overnight against RPMI. The supernatants were then sterilized by ultrafiltration and assayed for TNF activities.

Viability of the cells was determined by trypan blue dye exclusion method, and cell number was determined by counting in a hemacytometer.

Phenotyping of NAM by immunofluorescent analysis using flow cytometry and in- direct fluorescent microscopy. Flow cytometry was performed on Ortho Cytofluoro- graph 11s (Ortho Diagnostics, Raritan, NJ), and data were analyzed on an Ortho Di- agnostics Model 2 15 1 computer. Cells were washed twice with cold RPM1 and incubated with 10% ABS on ice for 20 min. Appropriate monoclonal antibodies were added and the cells were incubated again on ice for 45 min. The cells were then washed twice and exposed for 15 min to normal goat serum (5%) before phycoerythrin-con- jugated goat anti-mouse secondary antibody was added and incubated for an additional 30 min. Cells were washed twice with PBS and 5 X lo3 cells were analyzed for each antibody tested. In some experiments, cells were stained on poly-L-lysine (PLL)-coated slides as previously described (11). Three hundred cells were counted using a Zeiss Epifluorescence microscope.

Electron microscopy. Three-week-cultured NAM were adhered onto PLL-coated Nalgene polymethyl pentene (PMP) 100 X 20-mm petri dishes for 20 min at room temperature. The cells were fixed at room temperature in a fresh mixture of 2% glu- teraldehyde (Electron Microscopy Sciences, Fort Washington, PA), RPM1 1640, 20 mM Hepes (GIBCO), and 0.1 M sucrose, pH 7.4, for 1 hr. They were rinsed with PBS and postfixed with 1% osmium tetroxide for 1 hr on ice. Cells on PMP dishes were embedded in EPONS 12 (EMS) for transmission electron microscopy analysis.

RESULTS

Phenotype and Morphology of Long- Term Cultured Nonadherent Macrophages

Peripheral blood monocytes isolated by Percoll density discontinuous gradients were cultured with 10% human AB serum in chamber slides (see Materials and Meth- ods). After 3 weeks, the cultured cells were uniform in size and nonadherent (Fig. 1A). The number of cells in these cultures remained constant, and viability at the third week was >95%. The cells had some characteristics of macrophages in that they showed an increased amount of cytoplasm and vacuoles and possessed lamellipods as seen with transmission electronmicroscopy (Fig. 2). The NAMs had a small nucleus and a large heterogenous cytoplasm. The cytoplasm of NAM was abundant in mitochondria and vacuoles, had few lysosomes and granules, and was devoid of Golgi apparatus.

The cell surface antigens of freshly isolated blood monocytes and macrophages cultured for various periods of time were compared. As shown in Table 1, the freshly isolated monocytes expressed CD1 lc and CD 11 b and displayed varying amounts of the class II MHC antigen DR and DQ (as determined with MAb SC2 and Leu 10, respectively). Conversely, all of the long-term NAM expressed CD I 1 c and expressed high amounts of DR but little DQ and few expressed CD1 1 b. The amount of CD14,


FIG. 1. Long-term cultured macrophages develop unique morphological changes when treated with cytokines. Phase-contrast photomicrographs of 3-week cultured macrophages treated with various cytokines for 24 hr. (A) Untreated cells are nonadherent, round cells with numerous short lamellipods. (B) GM-CSF- treated cells are adherent and exhibit distinct processes that form a complex interconnecting network with other cells. (C) IL-2-treated cells are adherent, exhibit bipolar fibroblast-like morphology, and rarely make contact with each other. (D) IFN-y-treated cells are adherent, exhibit a “fried egg” appearance, and rarely make contact with each other. (400X)

a monocyte/macrophage marker, was stable and was seen on fresh monocytes as well as NAM. Transferrin receptors (CD7 1) were not found on fresh monocytes but were present in large amount on NAM.

Phagocytosis and NBT Reduction by NAM

Co-culture of opsonized yeast (heat-killed Candida albicans) with 3-week-old NAM in the presence of nitroblue tetrazolium results in the rapid binding and ingestion of the yeast particles. As seen in Fig. 3, the intracytoplasmic yeast particles are surrounded by dark precipitation of formazan, the reduction product of NBT. The change in color (yellow NBT to blue formazan) indicates a robust respiratory burst by the NAM in response to phagocytosis.

Capacity of NAM to Kill TNF-a-sensitive Target Cells

Minimal cytotoxicity (C?’ release) was observed when the TNF-a-sensitive murine fibrosarcoma cell line WEHI 164 was co-cultured with 3-week-old NAM in the presence of medium alone (Fig. 4). There was a slight increase in cytotoxicity (from 4 to 9%) in the presence of LPS (10 rig/ml) (P > 0. l), but the combination of LPS plus GM- CSF (80 U/ml) resulted in a marked increase in killing (P = 0.01). Of interest, GM-

TSAI ET AL.

FIG. 2. Transmission electron micrograph of a NAM. Note the abundance of vacuoles and mitochondria in the cytoplasm; Golgi apparatus is absent. x2500.

CSF alone decreased WEHI killing, indicating that both LPS and GM-CSF are required for full expression of the activated state.

Production of IL-lra and IL-l in NAM Cultures

Supematants from I- and 3-week cultured NAM were harvested from LabTek chamber slides at the end of the appropriate week. As shown in Table 2, IL- 1 ra was detected in


TABLE 1

Phenotvpe of Fresh Monocytes and Cultured Macrophages

Fresh Plastic (1 week) Chamber slide (3 weeks)

CD14 (Leu M3) 93 36 95 DR (SC2) 15 98 99 DQ (Leu 10) 4 18 0 CD1 lb (OKMl) 92 85 6 CD1 Ic (Leu M5) 83 98 98 CD7 1 (Transferrin receptor) 5 55 90

Note. This is a representative experiment showing expression of different phenotypic markers on fresh monocytes and cultured macrophages. The percent positive cells was determined by FACS analysis.

supematants from both time points, but the 3-week cultured cells produced significantly more ILlra, averaging 26 &ml/lo5 cells from three experiments. In these same supernatants, there was little or no detectable IL- 1 (Y or IL l/3. Treatment of these same 3-week cultured NAM for 24 hr with GM-CSF (100 U/ml) with and without the addition of LPS ( 10 rig/ml) but had no effect on IL- 1 ra production (data not shown).

NAM Develop Unique Morphology When Treated with Cytokines

When the 3-week-old NAM were treated for 24 hr with various cytokines (GM-CSF 80 U/ml, IL-2 30 U/ml, or EN-7 100 U/ml), they developed distinctive reproducible

FIG. 3. Light photomicrograph showing two NAMs surrounded by yeast particles. The yeast ingested by NAMs are outlined with a dark precipitate of forma-ran, the reduced product of NBT. (250X)

210 TSAI ET AL.

Control

GM-CSF

LPS+GM-CSF

0 IO 20 30 40 50

Cytotoxicity ( R specific release of 5kr)

FIG. 4. Cytotoxicity of NAM using TNF-sensitive WEHI 164 as targets. Effector to target ratio was 10 to 1. Control, NAM was cultured with medium; GM-CSF, NAM was stimulated with GM-CSF (80 U/ml) for 48 hr at the end of 3 weeks; LPS + GM-CSF, NAM were stimulated with GM-CSF (80 U/ml) for 24 hr followed by 24 hr of LPS (10 rig/ml) treatment; LPS, NAM treated with LPS (10 q/ml) for 24 hr after 3 weeks of cultures. The cytotoxicity data were from four experiments. P = -co.01 for LPS + GM-CSF compared to GM-CFS or LPS alone.

morphological changes. As shown in Fig. lB, GM-CSF-treated NAM became adherent and exhibited long processes (up to 100 pm) that formed a complex interconnecting network with neighboring cells. IL-Ztreated macrophages also became adherent, but they developed a bipolar fibroblast-like morphology; these cells rarely made contact with each other (Fig. 1C). EN-y-treated macrophages were also adherent but exhibited a “fiied- egg” appearance and did not make contact with each other (Fig. 1 D). These morphological changes occurred between 18 and 48 hr after the addition of cytokines. The macrophages retained their unique induced morphology for up to 2 weeks in the presence of cytokine. If the cytokines were then replaced with fresh medium, they changed back to their round nonadherent shape in 72 hr. Reexposure to cytokines rapidly induced the unique shape caused by the specific cytokines. Cytokine-induced morphological change was dose-de- pendent. Concentrations of 0.8 U/ml of GM-CSF, 3 U/ml of IL-2, or 50 U/ml of IFN- y were sufficient to initiate morphological changes.

GM-CSF, IL-2, and IFN-7 increase LPS-Mediated TNF Production NAM were primed with GM-CSF (80 U/ml), IL-2 (30 U/ml), or EN-7 (100 U/

ml) as above. The following day, LPS (10 pg/ml) was added to the cytokine-primed

TABLE 2

IL- 1 ra and IL- 1 Secretion by NAM

Age of NAM IL-lra* IL-la” IL-l/3”

Donor 1 3 weeks 22.5 <0.06 <0.06 Donor 2 1 week 3.0 0.15 <0.06

3 weeks 21.4 -co.06 0.14 Donor 3 1 week 12.8 co.06 co.06

3 weeks 27.6 <0.06 <0.06

* ng/ml/lO’ NAM.


macrophages and TNF measured in the culture supernatant that were collected 24 hr later. As shown in Table 3, a small amount of TNF was produced constitutively by NAM. LPS treatment of GM-CSF- and IL-2-primed NAM secreted significantly more TNF. IFN-y priming also increased TNF production, although it did not reach sta- tistical significance until after 48 hr of priming (data not shown). Surprisingly, LPS did not induce significant TNF production in control unprimed NAM. Similarly, cytokine treatment alone did not increase TNF production. The minimum concentrations of cytokine needed to prime long-term cultured macrophages were 0.8 U/ml for GM-CSF, 3 U/ml for IL-2, and 50 U/ml for IFN-y.

Effect of Drugs on Morphological Changes in NAM-Cultured Macrophages The effects of various compounds on cytokine-treated macrophages were examined.

When drugs such as cycloheximide (protein synthesis inhibitor), Act D (RNA synthesis inhibitor), and indomethacin (cycle-oxygenase inhibitor) were added to the cultured NAM at the same time as cytokine, they blocked the development of the characteristic morphological changes. Except for Act D, the drugs did not influence the viability of the NAM as assessed by trypan blue dye exclusion or their phagocytic capability (Table 4). As can be seen in Fig. 5, GM-CSF-treated macrophages failed to change morphology in the presence of any of these drugs and retained the appearance of nonadherent, round cells. In addition, both indomethacin- and PGEi-treated NAM formed large aggregates (Fig. 5). The effect of indomethacin on cytokine-primed macrophages was not reversed by the simultaneous addition of PGE, . The antineoplastic drugs methotrexate, cyclophosphamide, doxorubicin, and dexamethasone also inhibited the induction of morphological changes in cytokine-treated macrophages (see example in Fig. 5). None of them caused the homotypic aggregation seen in indomethacin- and/or PGE,-treated cultures. In order to determine whether the inhibition of morphologic change was reversible, the cultures were washed free of drugs and rested for 1 week before reexposing them to cytokines. Despite the fact that the cells were viable under all conditions, only cyclophosphamide- and dexamethasone-treated macrophages were able to change morphology when cytokines were later added back to the cultures.

l?fect of Drugs on TNF Production by Cytokine-Treated NAM The relationship between the inhibition of morphologic changes by drugs and TNF

production was then examined. NAM were treated with both groups of drugs along

TABLE 3

TNF Production by Cytokine-Primed Macrophages

LPS concentration

Treatment 0 &ml 10 e/ml P values*

Untreated 127 f 14” 200 + 17 0.5 GM-CSF (80 U/ml) 119 * 13 1904 f 35 0.00 1 IL-2 (30 U/ml) 70* 10 1638 k 41 0.006 IFN--y (100 U/ml) 100 -t 12 409 f 23 0.13

“TNF concentration in supernatants of IO5 macrophages expressed as means & SEM pg/ml (n = 12). * Comparing IO pg/ml of LPS to 0 fig/ml LPS.

212 TSAI ET AL.

TABLE 4

Influence of Drugs on NAM

Drugs Viability” Phagocytic function ’

None LPS ( IO &ml) Methotrexate (1 &f) Cyclophosphamide (I p,QZ) Doxorubicin (1 j&f) Dexamethasone (1 PM) Cycloheximide (10 &ml) Indomethacin (1 pLM) Indo + prostaglandin E, Prostaglandin E, (1 PM) Actinomycin D (1 PM)

99% 88% 99% 93% 99% 99% 74% 80% 89% 15% 0%

Yes Yes Yes Yes Yes Yes No Yes Yes Yes No

a Viability determined by trypan blue exclusion. * Ingestion of opsonized I micron latex beads.

with GM-CSF or IL-2 for 24 hr. Subsequent treatment of these cultures with LPS resulted in markedly diminished TNF production compared to control cells treated in the absence of drugs (see Table 5). The effects of the drugs on LPS-stimulated TNF

FIG. 5. Drugs inhibit unique morphological development by GM-CSF-treated long-term cultured macrophages. Phase contrast photomicrographs of long-term cultured macrophages treated with GM-CSF for 24 hr with or without drugs. GM-CSF, cells exposed to GM-CSF; Indo, cells exposed to both GM-CSF and indomethacin; PGE, , cells exposed to GM-CSF, indomethacin, and PGE,; MTX, cells exposed to GM- CSF and methotrexate.


production were irreversible; if the NAM were washed free of drugs and cytokines and cultured with fresh medium for 5 days, there was no increase in TNF secretion in the supernatants of these cultures after reexposure to cytokines and LPS.

DISCUSSION

Macrophages are multifunctional cells that play an important role in host defense. They arise as pro-monocytes in the bone marrow and migrate into the blood as cir- culating monocytes. After a brief period in the circulation they enter the tissues where they live as resident macrophages (12, 13). Although there is abundant information on monocytes derived from the blood and retained in culture for short periods of time, much less is known about their long-lived progeny. In tissue culture fresh blood monocytes rapidly adhere to plastic, spread, and modify their phenotype and functions ( 14). Initially, cytotoxic activity and lymphokine responsiveness are enhanced; over time, the macrophages in culture have decreased surface expression of class II histocompatibility antigens, secretion of reactive oxygen intermediates, and release of monokines (15). In serum-free medium, they ordinarily die after 7- 10 days, although this can sometimes be prevented by the addition of serum or growth factors such as CSF- 1 (M-CSF) ( 16).

The cells described in this paper differ in a number of ways from conventional cultured monocyte/macrophages. They are nonadherent, two to four times larger than freshly derived blood monocytes, and survive in culture for more than 12 weeks. Failure to adhere to the treated glass surface of the chamber slides is most likely significant since several lines of evidence suggest that adherence “primes” monocytes to secondary signals that induce differentiation ( 17). In vitro plastic adherence rapidly induces an increase in the steady state levels of mRNA for IL-l, TNF-(r, CSF-1, and c-fos, while down-regulating the protooncogene c-fms (17, 18). Likewise, adherence to extracellular matrix molecules such as fibronectin or collagen can modulate morphology, polysaccharide synthesis, CSF- 1 induction, and response to stimulators such as PMA ( 19).

TABLE 5

Effect of Drugs on the TNF Production by NAM Treated with Cytokines

Treatment * GM-CSF (80 U/ml) + LPS IL-2 (30 U/ml) + LPS

Medium Doxorubicin (I PM) Methotrexate (I PLM) Cyclophosphamide (1 PM) Dexamethasone (1 PM) Indomethacin (I pLM) Prostaglandin E, (1 &) Indo + prostaglandin E, Actinomycin D (1 &f)

2533 f 46” 67 f 7

317 f 18 258 f I5

70 +13 188 f 4

0 +-0 0 +o 1.5 f 0

1682 k 41” 110 + 20 30* 5

500 + 3 16” 4

200 f 3 lo* 3 Ii 0 ND

a TNF concentration in supernatants of lo5 macrophages expressed as &ml. LPS (10 &ml) was added to cultures 24 hr after cytokines. The mean + SEM of three separate experiments is shown.

b P value 4 0.01 for medium compared to al1 the conditions in both GM-CSF and IL-2 experiments.

214 TSAI ET AL.

Electron micrographs of the non-adherent macrophages demonstrate a number of unusual features. Unlike conventional monocytes, which have a large nucleus and a nucleus to cytoplasm ratio that approaches 1, the NAM have a small nucleus and an abundant cytoplasm containing abundant mitochondria and vacuoles, but there are few lysosomes and no detectable Golgi apparatus. In this regard, the NAM are very similar to the sessile resident macrophages seen in connective tissues as described by Fawcett and Cohn (3,20). Under appropriate stimulations these resident macrophages migrate into sites of inflammation. There they become activated and transformed into inflammatory macrophages with the acquisition of lysosomes, phagocytic vacuoles, and an elaborate Golgi apparatus and a decrease in the number of mitochondria.

The cell surface antigens of NAM also distinguish them from conventional plastic adherent monocytes, which at the end of 3-5 days show large amounts of the adhesion molecules CD 11 b and CD 11 c, transferrin receptor, and HLA-DR and -DQ antigens. In contrast, NAM show increased CD1 lc, no detectable CD1 lb, and although there is bright staining with antibody to DR, the DQ antigen cannot be demonstrated. In contrast, CD14 expression is considerably greater in NAM than in plastic adherent macrophages. Hogg and Sellvendran (21) and Radzun et al. (22) found CD1 1 b and CD1 lc on tissue macrophages and in vitro expression of these antigens increased with time during culture. Surprisingly, although only a small percentage of our long-term cultured macrophages are CD 11 b positive, they are capable of phagocytosing opsonized Candidu albicans and reducing nitrotetrazoleum dye, an indication of a successful respiratory burst (data not shown).

As human blood monocytes differentiate in vitro into macrophages they lose the ability to make IL- 1 fi (23) but constitutively produce IL- 1 ra protein (24). This product can be detected in both cell lysates and supernatants (25). Exposure to GM-CSF throughout the 7 days of culture results in a doubling of the intracellular concentrations of IL- lra, but this increase is not reflected in an increase in the excreted protein. The results with NAM are similar in that significant amounts of IL- 1 ra protein are present in supernatants collected after the first week in culture and this increases further by the third week, although NAM cell numbers remain constant. At the same time IL- 1 a and IL- 1 p are only detected in very small amounts. Failure to detect an increase in secreted IL-lra when GM-CSF and LPS are exposed to the NAM culture for 24 hr does not exclude a rise in intracellular IL- lra protein, but suggests that monocyte differentiation induced by the addition of exogenous growth factor is not a requirement for the switch from IL-l to IL-lra protein production in NAM.

The long-term cultured NAM undergo dramatic morphologic changes when exposed to cytokines. In most reports examining cultured blood monocytes, GM-CSF and IIN-7 induce monocytes to adhere and then over the course of 2 weeks terminally differentiate into giant cells and epithelioid cells ( 14,26). These cytokine-treated blood monocytes have low cytolytic capacity, reduced generation of reactive oxygen, and a decrease antigen presenting ability that correlates with a decrease in HLA-DR expression (14, 15, 27). The morphology of IFN-y-treated NAM is reminiscent of findings by Kaplan and Gaudernack studying mononuclear cells cultured on glass ( 14). After 3 weeks of culture they found the RN-y-treated monocytes progressed to epithelioid- like cells and multi-nucleated giant cells. This differentiation did not occur in any of the NAM cultures over the course of 12 weeks.

Macrophages are known to play an intimate role in tumor immunology via a number of possible mechanisms, including tumor antigen presentation to T cells, Fc-mediated


phagocytosis, and direct cellular cytotoxicity (28-30). The latter process is likely mediated in part through the elaboration of TNF-a. Obviously, if therapy directed against neoplastic disease has an effect on macrophage function it could have a significant impact on the ability of the immune system to recognize and eliminate malignant cells. Using the NAM, we have demonstrated that this is a real possibility because antineoplastic agents with a broad range of specificities and mechanisms of action interfered with the ability of cytokines to alter NAM morphology and effectively blocked cytokine-primed TNF secretion.

Although all of the agents tested completely blocked cytokine-mediated morphologic changes, there were some individual differences noted in the potency of TNF inhibition. The effects of doxorubicin and dexamethasone were the most pronounced, while cyclophosphamide was the least. There was also a possible difference noted in the sus- ceptibility of NAM to IL-2 or GM-CSF methotrexate-treated cultures, with much greater inhibition observed in the latter. Exposure to the drugs appeared to disable NAM permanently with regard to TNF production since the cells did not recover even after the drug was removed. In contrast, the effect of morphology was temporary for cyclophosphamide and dexamethasone; NAM treated with these drugs recovered this ability within a week after culturing in fresh medium.

The mechanism of these effects on NAM function and phenotype is not clear, as each drug interferes at different sites with cell growth or differentiation. For instance, methotrexate is an anti-metabolite that competitively inhibits dihydrofolate reductase, cyclophosphamide is an alkylating agent, and doxorubicin intercollates into double- stranded DNA. Dexamethasone has a variety of functions that result from its interaction with the glucocorticoid receptor and subsequent interference with proto-oncogenes. It is unlikely that the primary mechanism of NAM inhibition is mediated through an effect on DNA synthesis, since these cells divide very slowly and are clearly viable despite exposure to the drugs. Protein synthesis and gene transcription are required, as evidenced by the effects of cycloheximide and actinomycin D. The action of indomethacin is difficult to explain; although this is a prostaglandin synthesis inhibitor, concomitant addition of exogenous prostaglandin E to cultures did not reverse this effect.

The precise nature of many of the signals responsible for the induction of monocyte differentiation in vitro is not known. Dougherty and McBride suggested the monocyte differentiation is a process with variable outcomes that are readily modulated by interaction with other cells, extracellular matrix proteins, and various soluble mediators (3 1). Two features, however, appear to be important. First is the presence of human serum, since other serum sources support only limited maturation (32, 33). The responsible factors in human serum are not known. Second is the evidence that adherence per se is a critical step, making the monocyte more responsive to secondary signals responsible for differentiation. As noted above, the particular substrates (i.e., plastic, glass, extracellular matrix) seem to dictate the outcome. The NAM in these studies are cultured in glass chamber slides treated with a proprietary method. The use of untreated glass petri dishes resulted in a mixed population of adherent and nonadherent cells. The NAM in chamber slides appear to be intermediate between the fresh blood monocyte and tissue macrophages found in inflammatory lesions. Clearly they are not terminally differentiated, since they can be invoked to display reversible, unique morphologic features with cytokines. Thus they appear more like the resident cells in

216 TSAI ET AL.

various tissues that are the forerunner of alveolar macrophages, Kupffer cells, and osteoclasts.

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