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In vitro assay of spontaneous cytotoxicity by human monocytes and macrophages against tumor cells
Journal oflmmunological Methods, 37 (1980) 233--247 © Elsevier/North-Holland Biomedical Press
233
IN VITRO ASSAY OF SPONTANEOUS CYTOTOXICITY BY HUMAN MONOCYTES AND MACROPHAGES AGAINST TUMOR CELLS
BIJAY MUKHERJI 1 Tufts University School o f Medicine and New England Medical Center Hospital, Boston, MA 02111, U.S.A. (Received 1 April 1980, accepted 28 May 1980) The [3H] proline microcytotoxicity technique has been adopted to examine the spontaneous cytotoxic property of human circulating monocytes (M) derived from the peripheral blood and resident monocytes/macrophages (Me) derived from effusion fluids. A leukocyte fraction is obtained by centrifugation in Ficoll-Hypaque (F-H) gradient. M are then isolated as adherent cells following incubation of the leukocyte fraction in plastic flasks containing 50% fetal calf serum (FCS) in culture medium. M~ are isolated from effusion fluids after first separating the Fc receptor bearing cells (as Fc receptor-7S EA resettes) in F-H gradient and then enriching them as adherent cells. The above techniques yield a M/M~b population of some 90--98% purity. The M]M~ non-selectively lyse tumor cells and allogeneic normal fibroblasts in the 48 h [3H]proline assay. The fibroblasts, however, exhibit considerable resistance to lysis, particularly at lower effector to target ratios. While appreciable levels of cytotoxicity are observed with resident M6 at 6 h, followed by a further increase in the level of cytotoxicity at 2 4 - - 4 8 h, circulating M exhibit a consistent level of cytotoxicity only at 24--48 h. When circulating M and resident M~ from the same donor are examined concurrently for cytotoxicity, resident M6 are consistently found to show significantly higher levels of cytotoxicity when compared to circulating M. This may reflect a true functional heterogeneity within this lineage of effector cells or some degree of in vivo activation of resident M~b in malignant and non-malignant effusion fluids. Further studies of spontaneous cytotoxicity by M]M~ through CMC assay techniques, such as the [3H ]proline microtoxicity technique, will be useful in the examination of the role of M/M6 in cell mediated immunity and immunosurveillance against cancer. INTRODUCTION
T h e cells o f m o n o c y t e ( M ) / m a c r o p h a g e (M~b) l i n e a g e c o n s t i t u t e a n i m p o r t a n t class o f e f f e c t o r cells i n cell m e d i a t e d i m m u n i t y a g a i n s t v a r i o u s m i c r o b i a l i n f e c t i o n s a n d p o s s i b l y a g a i n s t n e o p l a s t i c d i s o r d e r s . M / M e are v e r s a t i l e e f f e c t o r cells ( N e l s o n , 1 9 7 6 ) . T h e i r e f f e c t o r f u n c t i o n s are d i v e r s e , i n c l u d i n g a I Present address: University of Connecticut Health Center, Farmington, CT 06032, U.S.A. Abbreviations used: M = monocytes; M~ = macrophages; M-CMC = monocyte-macrophage mediated microcytotoxicity; CMI = cell mediated immunity; SRBC = sheep erythrocytes; F-H = Ficoll-Hypaque; FCS = fetal calf serum; 7S-EA = sheep erythrocytes sensitized with 7S anti-sheep erythrocytes IgG; 19S-EAC = sheep erythrocytes sensitized with 19S antisheep antibody and complement.
234 phagocytic ability as well as an apparent selective tumoricidal property (Nelson, 1976; Hibbs, 1976). While phagocytosis is an important property of M/M0, selective c y t o t o x i c i t y against cancer cells appears to be another important functional property of these effector cells, at least, in animal systems (Hibbs, 1976). Lately, several studies have emerged to indicate a spontaneous c y t o t o x i c capacity of rodent as well as human M/Me (Holterman et al., 1974; Keller, 1978; Mantovani, 1979a, b). Although spontaneous cytotoxicity of human circulating macrophages is well documented, the specificity of such c y t o t o x i c i t y has not yet been clearly established. In addition, it appears that M/MO can be 'activated' by various types of manipulation and such activated M/M¢ may play an important role in tumor immunity and adjuvant immunotherapy. On the scale of evolutionary development of the immune system, the emergence of M/Me lineage possibly antedates that of the lymphoid system. If a first line defense against cancer exists, M/M0 may play an important role in such an immune surveillance scheme. Further, macrophages are known to home at diverse sites, and natural c y t o t o x i c i t y of M/M0 from diverse anatomical sites has been studied in rodents (Keller, 1978). No coordinated examination of c y t o t o x i c capability of circulating and resident human macrophages has, however, been conducted. This study, therefore, has been undertaken as a broader examination of in vitro c y t o t o x i c properties of M/M~b in a human system; and this manuscript describes some characteristic feature of in vitro cytotoxicity and potential functional diversities of the effector cells of M/M¢ lineage. MATERIALS AND METHODS
Effector cells Peripheral venous blood and effusion fluids (pleural and ascites) are collected in heparinized syringes or bottles (20 units/ml for peripheral blood and 10,000 units/1 of effusion fluid). Healthy donors or cancer patients are bled for 2 0 - - 1 2 0 ml of peripheral blood for monocytes. Effusion fluids are usually obtained in large amounts from patients (with neoplastic and nonneoplastic diseases) who undergo diagnostic or therapeutic thoracentesis or paracentesis. All neoplastic effusions examined in this study were cytologically positive for t u m o r cells.
Target cells Several human malignant melanoma cell lines (RoLe-M, JoCa-M, A1Co-M) and the corresponding a u t o c h t h o n o u s fibroblast lines (RoLe-F, JoCa-F, A1Co-F) established earlier in our laboratory, have been used. The melanoma line RoLe-M and the fibroblast line RoLe-F are used as the reference cell lines in most experiments. A human leiomyosarcoma line SK-LMS-1 (established by, and a gift of, Germaine Trempe of Sloan Kettering Cancer Center,
235 New York) has also been used in this study as another target cell line. The melanoma cell lines and the sarcoma line SK-LMS-1 are transplantable as xenografts according to a technique previously described (Mukherji et al., 1974). The fibroblast line RoLe-F is contact inhibited.
Isolation o f M/M¢ from peripheral blood ieukocytes (PBL) and effusion fluids The basic property of adherence to plastic surfaces by M/M¢ is utilized to isolate these cells from PBL and effusion fluid. In addition, the presence of Fc receptors on M¢ is utilized to separate them as Fc receptor rosettes from other contaminating non-Fc receptor bearing cells in effusion fluids.
Isolation o f M from PBL A leukocyte fraction is first isolated by the technique of B o y u m (1968). The interface band containing the l y m p h o c y t e s and monocytes is gently aspirated, washed once in Hanks Balanced Salt Solution (HBSS) and resuspended in Hams F-10 medium containing 50% fetal calf serum (FCS). Approximately 3 ml of the cell suspension (at 1--2 X 10 ~ cells per ml) in F-10 medium with 50% FCS is incubated in T-30 plastic flasks for 1 h at 37°C in 5% CO2 in air. Following incubation, the non-adherent cells are removed by 6 vigorous rinsings in HBSS. The adherent cells are harvested mechanically by a rubber policeman and are referred to as M to denote them as circulating monocytes.
Isolation o f M/Md from effusion fluids as 7S-EA rosettes Cell pellets are first obtained by centrifugation of 250 ml of effusion fluid at 175 X g for 10 min in conical plastic centrifuge tubes. Most of the supernatant fluid is discarded leaving approximately 2 ml of the fluid at the bottom. The cell pellet is subsequently resuspended, aspirated and pooled. The pooled cell pellet is then washed in HBSS three times and resuspended in Hams F-10 medium. The non-Fc receptor bearing cells (T l y m p h o c y t e s and t u m o r cells) are removed by isolating the Fc receptor bearing cells as 7S-EA rosettes and recovering the 7S-EA rosettes in the pellet after centrifugation in Ficoll-Hypaque (F-H) gradient. To form 7S-EA rosettes, sheep erythrocytes (SRBC) are first thoroughly washed in HBSS three times. The washed SRBC are then sensitized with 7S anti-SRBC IgG (Cordis Laboratory, Miami, FL 33137) at a standard dilution of 1:100 at 37°C for 45 min (the standard dilution of anti-SRBC IgG is determined from pilot experiments). A 5% suspension of the sensitized SRBC is made (containing approximately 1 X 109 cells per ml) in FCS which has previously been absorbed with an equal volume of packed SRBC. Five ml of effusion fluid cell suspension (1 X 107 per ml) is mixed with 5 ml of sensitized SRBC suspension and incubated at 37°C for 45 rain. After incubation
236 t h e cell s u s p e n s i o n is g e n t l y p o u r e d o n t o 10 ml o f F i c o l l - H y p a q u e s o l u t i o n a n d t h e g r a d i e n t is c e n t r i f u g e d at 4 0 0 × g at r o o m t e m p e r a t u r e f o r 30 m i n . T h e pellet is c a r e f u l l y a s p i r a t e d a n d t h e Fc r e c e p t o r bearing e f f e c t o r cells are r e c o v e r e d free o f S R B C a f t e r i n c u b a t i o n w i t h T r i s - b u f f e r e d 0.83% NH4C1, p H 7.2, f o r 10 m i n at r o o m t e m p e r a t u r e as d e s c r i b e d b y B o y l e ( 1 9 6 8 ) . T h e cells are t h e n w a s h e d t w i c e w i t h 50 ml o f H B S S a n d are r e s u s p e n d e d in H a m s F - 1 0 m e d i u m . F o r f u r t h e r p u r i f i c a t i o n o f t h e M/MO f r o m c o n t a m i n a t ing Fc r e c e p t o r bearing B l y m p h o c y t e s , t h e cells r e c o v e r e d a f t e r NH4C1 t r e a t m e n t are i n c u b a t e d f o r 1 h in plastic f l a s k s in F - 1 0 m e d i u m c o n t a i n i n g 50% f e t a l calf s e r u m . T h e n o n a d h e r e n t cells are r e m o v e d b y 6 rinsings in H B S S as b e f o r e , a n d a d h e r e n t cells are h a r v e s t e d m e c h a n i c a l l y . T h e s e cells are h e n c e f o r t h r e f e r r e d t o as M~b t o d e n o t e t h e m as r e s i d e n t m o n o c y t e s / m a c r o p h a g e s . T h e M~b are w a s h e d o n c e m o r e in H B S S a n d r e s u s p e n d e d in F - 1 0 m e d i u m a l o n e or in F - 1 0 m e d i u m s u p p l e m e n t e d w i t h FCS a n d antib i o t i c s f o r m i c r o c y t o t o x i c i t y e x p e r i m e n t s d e s c r i b e d later. T h e y i e l d o f M f r o m P B L varies b e t w e e n 3 a n d 7% o f t h e F-H cell p r e p a r a t i o n , while t h e yield o f M¢ f r o m e f f u s i o n fluids varies b e t w e e n 22 a n d 45%. T h e p u r i t y o f r e s i d e n t M e a c h i e v e d in o u r s t u d y r e a c h e s 9 0 - - 9 5 % w h e n p u r i t y is j u d g e d b y t h e criteria listed in T a b l e 1 (non-specific esterase positive cells s h o w i n g positive r e c e p t o r s f o r 7S-EA a n d 19S-EAC, p h a g o c y t o s i s , a b s e n c e o f r e c e p t o r s f o r n e u r a m i n i d a s e t r e a t e d S R B C a n d bearing no surface i m m u n o g l o b u l i n ) . J u d g e d b y t h e s a m e criteria, t h e p u r i t y o f M~ in t h e effusion fluid cells, isolated b y t h e a d h e r e n c e step o n l y , d r o p s t o 44% a l t h o u g h a v a r i e t y o f t u m o r cells stain p o s i t i v e l y f o r n o n - s p e c i f i c esterase r e a c t i o n . T h e final viability o f t h e a d h e r e n t cells a f t e r scraping ranges f r o m 70 t o 85%.
Iden tifica tion o f M/M¢ T h e M/M~b are i d e n t i f i e d a n d c h a r a c t e r i z e d b y n o n - s p e c i f i c esterase stain as p e r K o s k i et al. ( 1 9 7 6 ) , b y t h e p r e s e n c e o f Fc r e c e p t o r s ( 7 S - E A r o s e t t e TABLE 1 Characteristics of the effector cell. Composite data from 15 donors are shown.
% Positive +- S.E. Blood monocytes
Effusion fluid macrophages
Non-specific esterase stain
7S EA rosettes 19S EAC rosettes Phagocytosis Neuraminidase E-RFC Surface immunoglobulin
-------
98 94 93 63 1.2 1.9
+ 1.5 +- 1.1 -+ 3.3 + 3.0a + 0.1 + 0.02
a Difference is significant at P ~ 0.005 (Student's t-test).
95 93 94 89 0.6 2.3
-+ 2.1 +- 1.8 + 2.6 + 4.7 a + 0.02 + 0.03
237 assay) and complement receptors (19S-EAC rosette assay) as per Bianco (1976), absence of receptors for SRBC (spontaneous) and surface immunoglobulin, and by phagocytosis of heat killed yeast. Natural killer l y m p h o c y t e s (NK cells) are identified as rosettes with neuraminidase treated SRBC (neuraminidase-ERFC) using the methods of West et al. (1977). A simplified yeast ingestion technique is adopted to the phagocytosis assay. Firstly, the yeasts (ordinary baking yeast) are boiled for 1 h and a suspension of yeasts is made in cold HBSS (1 X 107 yeasts per ml). The M/MO are suspended (at 2 X l 0 s per ml) in cold HBSS to prevent clumping. The M/M~b suspension and the yeast suspension are mixed in equal volumes to obtain a M/M¢ to yeast ratio of 1 : 50. The M/M~ yeast suspension is then centrifuged at 175 X g for 5 min and most of the supernatant is discarded by gentle aspiration leaving approximately 0.2 ml of the supernatant fluid. To each tube, thereafter, 0.1 ml of AB+ human serum (heated at 56°C for 45 min) is added. The final reaction mixture is incubated for 37°C for 30 min. The suspension mixture is gently resuspended and following incubation a slide preparation is made with a droplet of the cell suspension. The slides are stained with Wrights-Giemsa stain and are examined for phagocytosis. One hundred effector cells are counted to determine the percentage of phagocytic cells. In vitro M/Md mediated microcytotoxicity (M-CMC) assay The [3H] proline m i c r o c y t o t o x i c i t y assay, originally described by Bean et al. (1973) as an in vitro technique for studying l y m p h o c y t e c y t o t o x i c i t y and extensively used in our laboratory for l y m p h o c y t e microcytotoxicity assay, is adopted for M-CMC assay. The details of the assay have been previously described (Mukherji et al., 1975). Briefly, target cells are prelabelled in a T30 plastic flask in monolayer with 50 pCi of [3H] proline (47.7 Ci/mmole: New England Nuclear, Boston, MA) for 1 8 - - 2 4 h. The labelled target cells are harvested from the flask, resuspended in appropriate medium, and distributed into the wells of linbro 6 mm microcytotoxicity plates (Linbro Chemical Co., New Haven, CT). Usually, 1,000 viable labelled target cells are plated in each well. After the target cells have attached to the well b o t t o m , effector cells suspended in 100 ~l of test medium (Hams F-10) are added to the wells. After initial incubation of effector cells and target cells at 37°C in 5% CO2 for 45 min, 100 pl of F-10 medium with 40% heat inactivated FCS is added to each well to bring the final concentration of FCS to 20% and the incubation is continued for an additional 48 h. Effector cells are added to each well at desired effector to target cell ratios ( 1 2 : 1 - - 1 0 0 : 1 ) . After incubation, the plates are washed, air dried and the b o t t o m s of the wells are punched o u t in scintillation vials. Protosol (0.3 ml) and 0.9 ml of methanol are added to each vial at room temperature 30 min prior to addition of scintillation fluid (4 g of Omnafluor/1 of toluene) (Omnafluor: New England Nuclear, Boston,
238
MA; Toluene: Fisher Chemical Co., Fairlawn, NJ). The vials are kept dark at room temperature for 24 h prior to counting. Usually, each assay is performed in 5--10 replicates. 3H scintillation counting is performed in a Packard Tri-Carb liquid scintillant spectrometer (Packard Instrument Co., Downs Grove, IL). Percentage c y t o t o x i c i t y by a given effector cell is calculated in relation to medium control and then compared for significance by Student's t-test. RESULTS
Characteristics o f the effector cells Table I shows the characteristics of the effector cells isolated as M/M@. It is of interest that, although a very high percentage of cells stain positively for non-specific esterase, reported as a specific stain for M/M@ (Koski et al., 1976), not all of them exhibit phagocytosis. Furthermore, the M@ isolated from neoplastic or non-neoplastic effusions exhibit a significantly higher percentage of phagocytosis (P < 0.005) compared to the M obtained from PBL. The M from PBL and M@ from effusion fluids express receptors for the Fc portion of immunoglobulin IgG and for complement in equal proportion. NK cell and B cell contamination in both preparations are minimal.
In vitro cytotoxicity of M/Md An example of an M-CMC assay is shown in Fig. 1. M from PBL as well as M@ from the effusion fluid exhibit significant levels of cytotoxicity against allogeneic neoplastic as well as non-neoplastic target cells. The fibroblasts
>_ 601
Target
:
R
o
~
Torget: Role-F
g 5ol x
~ 30 u
~ 2o w o.
[
I
12
50
I
I00
I
I
1
12
50
I00
EFFECTOR: TARGETCELLRATO I Fig. 1. S p o n t a n e o u s c y t o t o x i c i t y b y M/M~b against t h e m e l a n o m a cells ( R o L e - M ) a n d t h e a u t o l o g o u s f i b r o b l a s t s ( R o L e - F ) . T h e o p e n circle a n d t h e closed circle r e p r e s e n t r e s i d e n t M~b f r o m ascites a n d c i r c u l a t i n g M r e s p e c t i v e l y f r o m a b r e a s t c a n c e r p a t i e n t . T h e o p e n triangle r e p r e s e n t s c i r c u l a t i n g M f r o m a p a t i e n t w i t h c o l o n c a n c e r a n d t h e closed triangle r e p r e s e n t s t h e s a m e f r o m a n o r m a l d o n o r . C y t o t o x i c i t y e q u a l t o or a b o v e 15% level is significant at P ~< 0 . 0 5 ; o t h e r s are n o t s i g n i f i c a n t . E a c h d a t a p o i n t b e t w e e n t h e r e s i d e n t Mq~ a n d circulating M is significant at P ~< 0.01.
239 TABLE 2 Effect of silica on M/M~b cytotoxicity. Cytotoxicity against target cell RoLe-M Effector cell
Adherent M/M~ a
Non-adherent lymphocyte b
Donor/Diag
Source
Without silica
With silica
Without silica
BM/Normal
PBL
706 + 81 c
818 -+ 76
596 + 40
562 -+ 47
(19) 368 + 23
(6) 769 + 84
(32) 679 -+ 60
(36) 712 -+ 59
RS/Ovarian Ca Ascites and
MD/Normal Medium control
PBL PBL
(58) (12) 638 + 60 854 + 42 (27) (3) 878 + 59
With silica
(23) (19) 508 + 39 534 + 46 (42) (39) 878 + 59
a Circulating M or resident Me (from patient RS) are isolated by methods described in the text. 100 pg of silica (maximum particle size of 5 pm sonicated immediately prior to incubation) is added to flasks contained 1--5 × 106 effector cells (M/M~b as adherent cells, lymphocytes as non-adherent cells) and cultured overnight along with the untreated flasks in 3 ml of F-10 medium containing 10% AB+ human serum and antibiotics. After overnight incubation, the effector cells are harvested (M/M~b mechanically, non-adherent lymphocytes by decanting), washed in HBSS x 3, checked for viability by trypan blue dye exclusion, cell concentration readjusted for viable effector to target cell ratio of 50:1, and added to the microtoxicity plates as described in the text. b Represent nylon wool column passaged non-adherent cells following Ficoll-Hypaque gradient separation. The resultant cells consist of 95--99% lymphocytes. c Mean cpm of 5 samples + S.E.; numbers in parentheses are percentages of cytotoxicity calculated in relation to medium control. e x h i b i t c o n s i d e r a b l e resistance to lysis b y allogeneic M / M ¢ in c o m p a r i s o n t o the m e l a n o m a cells as targets at c o m p a r a b l e e f f e c t o r t o target ratios. T h e r e s i d e n t M~b o b t a i n e d f r o m ascites f r o m a d o n o r having breast c a n c e r e x h i b i t higher c y t o t o x i c i t y (P < 0.01 or better) w h e n c o m p a r e d to circulating M f r o m the same p a t i e n t at all e f f e c t o r t o t a r g e t ratios. Table 2 e x a m i n e s the e f f e c t o f silica (a gift o f J a m e s Murray, S t a n f o r d University Medical S c h o o l , S t a n f o r d , CA) o n c y t o t o x i c i t y e x h i b i t e d b y M/M~. As a c o n t r o l , circulating l y m p h o c y t e s f r o m the same d o n o r s have also been included in the e x p e r i m e n t . T h e l y m p h o c y t e s a n d the circulating M or resident M¢ are t r e a t e d with silica t h e same w a y (for details o f silica t r e a t m e n t see Table 2) prior t o the CMC assay. O n l y the M/M~ t r e a t e d with silica e x h i b i t m a r k e d decrease or c o m p l e t e a b r o g a t i o n o f c y t o t o x i c i t y while s p o n t a n e o u s c y t o t o x i c i t y b y l y m p h o i d cells r e m a i n s u n a f f e c t e d b y silica treatment. T h e kinetics o f M/M~ c y t o t o x i c i t y is s h o w n in Fig. 2. A t 6 h o f i n c u b a t i o n o f e f f e c t o r cell a n d t a r g e t cell, significant levels o f c y t o t o x i c i t y are observed with M o n l y i n c o n s i s t e n t l y . A significant level o f c y t o t o x i c i t y b y M, h o w ever, is c o n s i s t e n t l y o b s e r v e d with 24 h i n c u b a t i o n f o l l o w e d b y a m o d e s t
240 increase in c y t o t o x i c activity at 48 h. T h e M~b, o n the o t h e r hand, show d i f f e r e n t kinetics. Significant levels of c y t o t o x i c i t y are c o n s i s t e n t l y seen at 6 h. T h e c y t o t o x i c activity shows a f u r t h e r significant rise at 24 h, f o l l o w e d b y a m o d e s t rise at 48 h. T h e d i f f e r e n c e in the level o f c y t o t o x i c i t y b y circulating M and r e s i d e n t M~ at the d i f f e r e n t assay p o i n t s and at identical e f f e c t o r t o target ratios s h o w n in Fig. 2 are significant at P < 0 . 0 0 4 or b e t t e r ( S t u d e n t ' s t-test). P o t e n t i a l d i f f e r e n c e in t h e kinetics of M/M~ c y t o t o x i c i t y b e t w e e n n o r m a l d o n o r s and c a n c e r patients, if any, will be e x a m i n e d in a separate m a n u s c r i p t ( u n d e r p r e p a r a t i o n ) . P o t e n t i a l d a y - t o - d a y variation o f M / M e c y t o t o x i c i t y is e x a m i n e d in Fig. 3. Circulating M f r o m t h r e e n o r m a l d o n o r s (bled on 3 c o n s e c u t i v e days) are e x a m i n e d for c y t o t o x i c i t y o n t h r e e c o n s e c u t i v e assays. As can be seen, o n l y m i n o r degrees o f f l u c t u a t i o n in the level o f c y t o t o x i c i t y are seen with t h e circulating M. Statistically significant (P < 0.01) f l u c t u a t i o n is observed with o n e d o n o r in assays on d a y 1 and d a y 3. R e s i d e n t Me f r o m f o u r c a n c e r p a t i e n t s are c o l l e c t e d and e x a m i n e d in several n o n - c o n s e c u t i v e assays. T w o d o n o r s have b e e n t e s t e d twice and t w o d o n o r s have been t e s t e d o n t h r e e occasions. All assays are c o n d u c t e d at identical e f f e c t o r t o target ratios and with target cells at identical passage levels. Significant f l u c t u a t i o n s in the level o f c y t o t o x i c i t y are seen with t h r e e o f these patients. R e s i d e n t M e f r o m t w o p a t i e n t s have b e e n e x a m i n e d within 2 weeks and the o t h e r subjects have been e x a m i n e d o n t h r e e widely separated, n o n - c o n s e c u t i v e days. T h e p o t e n t i a l activating i n f l u e n c e on FCS, which m i g h t c o n t a i n e n d o t o x i n 7060-
60"
5 50-
50"
~ 40-
40. 30-
2O-
20" I0-
I0-
Lenoth of Assoy in Hrs
# Doys Tested
Fig. 2. Kinetics of s p o n t a n e o u s c y t o t o x i c i t y by circulating m o n o c y t e s (open circle) and resident m o n o c y t e s / m a c r o p h a g e s (closed circle). Collective data of circulating m o n o c y t e preparations from 15 donors (10 normal and 5 cancer patients) and resident M~ preparation for 15 donors (12 cancer patients and 3 non-cancer patients) are shown. All M-CMC assays are performed at identical E:T ratio of 50:1. Fig. 3. Day-to-day variations in the spontaneous c y t o t o x i c i t y level of circulating M (solid lines) and resident M~b (interrupted lines). The difference in the cytotoxicity level to circulating M from this donor at points indicated w i t h the letter a is significant at P 0.05 by Student's t-test.
241
TABLE 3 Effect of FCS and human AB+ sera on monocyte cytotoxicity. Monocyte
Residual target cell c p m -+ S.E. a against R o L e - M line
Donor/Diag
FCS # 1 b
GD/Melanoma
1238 + 98 (17) 997 + 108 (33) 1485 +
HC/Normal Medium control
FCS # 2 b
FCS # 3 b
1137 + 121 (23) 870 + 101 (41) 116
AB+
1273 + 87 (14) 1013 + 71 (31) 1485 +
1206 + 73 (19) 1056 + 80 (29) 116
a Mean cpm of 5 samples + S.E.; numbers in parentheses represent percentage cytotoxicity in relation to medium control. b Effector cell isolation and M-CMC assay are performed in medium containing 3 separate batches of FCS. a n d t h e r e f o r e c a u s e M / M ~ a c t i v a t i o n , has b e e n e x a m i n e d t h r o u g h M - C M C e x p e r i m e n t s ( i s o l a t i o n o f e f f e c t o r cells a n d t h e CMC a s s a y ) p e r f o r m e d in t h e i r e n t i r e t y in t h r e e b a t c h e s o f F C S a n d h u m a n AB + s e r u m . While m i n o r d i f f e r e n c e s in c y t o t o x i c level c a n be seen w i t h t h e t h r e e b a t c h e s o f F C S , n o m a j o r f l u c t u a t i o n is seen in t h e levels o f c y t o t o x i c i t y w h e n a s s a y s are p e r f o r m e d w i t h F C S a n d A B + s e r u m ( T a b l e 3).
Functional differences between circulating M and resident Md Table 4 shows a comparative analysis of cytotoxic activity of circulating M and resident Me derived from the same donor and assayed concurrently a g a i n s t t h e m e l a n o m a t a r g e t cell line R o L e - M . T w e l v e o u t o f 18 d o n o r s in t h i s series have m a l i g n a n t d i s e a s e (5 w i t h o v a r i a n c a n c e r , 4 w i t h b r e a s t cancer, 1 with malignant melanoma, and 2 with lung cancer). The nonTABLE
4
Comparison of cytotoxicity between circulating M and resident M~$.
Percentage eytotoxicity against RoLe-M a Nb
Circulating M
18
M e a n -+ S.E.
Range
28 + 3.0
14--43
(<: 0.0001) c
Resident M/M~b
18
57 + 4.6
38--81
a M - C M C assays are performed at effector to target ratio = 50:1. b N u m b e r of donors from w h o m circulating M and resident M/M~b are obtained and assayed for cytotoxicity. c T h e figure in parentheses represents the level of significance in the difference in the percent cytotoxicity.
242 malignant group of donors is comprised of 4 patients with hepatic cirrhosis, one with congestive heart failure, and one with inflammatory pleural effusion. Circulating M and resident M¢ from each patient have been concurrently tested for microcytotoxicity in separate experiments against the reference melanoma target cell line at several identical effector to target ratios. Comparative data at the effector to target ratio with which cytotoxicity is consistently seen is shown in Table 4. Significant difference in the level of c y t o t o x i c i t y between resident MO and circulating M when individually compared is observed in 11 o u t of 12 cancer patients (individual data n o t shown). In all these cases, the resident MO exhibit significantly higher levels of cytotoxicity (P < 0.01 - - < 0.001, in most cases < 0.001). In the non-cancer patient group, significant differences are observed only in three cases and the degree of difference is not as marked as it is in the cancer patient group (P < 0.05-< 0.01). When the percentages of c y t o t o x i c i t y are averaged and compared at identical effector to target ratios, the difference in the level of cytotoxicity by resident Me and circulating M becomes highly significant (Table 4).
Effect of isolation procedure on M/Mql cytotoxicity The effect of the membrane Fc receptor interaction in the isolation procedure on the c y t o t o x i c behavior of M, when examined by exposing the circulating M to SRBC coated with 7S IgG followed by Tris-NH4C1 treatment and assayed for c y t o t o x i c i t y concurrently with untreated M, is found to be non-significant (data not shown). In addition, experiments conducted with circulating M exposed to antibody-coated SRBC and with similarly treated resident Me derived from the same cancer patients in two separate but concurrent CMC assays reveal significantly higher levels of cytotoxicity by the resident M~ compared to the circulating M, whether or not treated with 7S-EA and NH4CI (data not shown).
Specificity of cytotoxicity by M/Md Specificity of cytotoxicity by M/Me has been examined in multiple experiments involving the panel of target cells in various different combinations. Target cell controls in these experiments have included fibroblasts (autochthonous to at least one malignant cell line) as well as non-melanoma t u m o r cells. A detailed examination of specificity of M/M~ cytotoxicity in this system will be presented later (manuscript under preparation). However, it is of interest to note that both circulating M and resident M¢ lyse allogeneic t u m o r cells non-selectively. In addition, allogeneic fibroblasts obtained from the same patients from whom melanoma targets are derived, are also lysed, particularly at higher effector to target ratios. Table 5 demonstrates a series of experiments with M/M~ from 5 donors against 3 sets of target cells (each set consisting of a melanoma line and the a u t o c h t h o n o u s fibroblast lines}. As can be seen, the cytotoxic activity of
243
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,-M
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~
~
~
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v 0
~
~
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~
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~
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244 the allogeneic M/M~b against the melanoma cells are detectable at virtually all effector to target ratios. The fibroblasts are, on the other hand, not readily lysed at the lower ratio, while at higher ratios significant killing against the fibroblasts is detectable. DISCUSSION While the isolation of circulating blood m o n o c y t e s is a relatively easier task by virtue of their adherent property, the isolation of resident M~b from human effusion fluids, particularly malignant effusions, has always been a difficult proposition. The m e t h o d utilized in this study involving isolation of the Fc receptor bearing cells as 7S-EA rosettes followed by enrichment of the macrophages by their adherent property is reasonably effective in removing the contaminating non-lymphomatous malignant cells and the lymphocytes. The lack of uniform phagocytic ability by some of the adherent nonspecific esterase positive cells derived from peripheral blood suggests that all circulating M may not always exhibit phagocytosis in any given assay. The infrequent association of cells showing receptors for neuraminidase treated sheep erythrocytes of cells bearing surface immunoglobulins, and cells exhibiting high frequency of non-specific esterase stain, indicate that the non-phagocytic cells in our preparations do not represent lymphocytic lineage. Since contamination of NK cells or B cells in the effector cell population is exceedingly small, the potential influence of NK cells or adherent B cells in the killing process is, therefore, negligible. The cytotoxic ability of activated guinea pig macrophages has been d o c u m e n t e d by Sharma and Piessens (1978) utilizing the [3H]proline CMC technique. They have used the adhesive property of macrophages as a purification step and shown that activated macrophages exhibit significant degrees of cytotoxicity at 6 h. Spontaneous c y t o t o x i c i t y by M or M~ in mice, rats and humans has also been well d o c u m e n t e d by other investigators (Holterman et al., 1974; Keller, 1978; Mantovani, 1979a, b; Tagliabue et al., 1979). Our studies support and confirm the feasibility of utilizing the [~H] proline CMC technique for in vitro assay for cytotoxicity mediated by these effector cells. The kinetics of human M/M~b cytotoxicity, however, differ. Although at 6 h appreciable degrees of cytotoxicity by circulating M may be detected at times, the consistent activity is observed at 2 4 - - 4 8 h. Tagliabue et al. (1979) have recently described natural cytotoxicity of mouse M and Me. These investigators have noted that the cytotoxic activity of mouse and Me is best seen at 4 8 - - 7 2 h of incubation. In this regard, significant cytotoxicity by human resident M~i at 6 - - 2 4 h of incubation, as seen in our study, is noteworthy. In addition, t w o important characteristics of human M/M~ cytotoxicity emerge from our studies. Firstly, the possibility of existence of considerable functional heterogeneity among the effector cells is evident. In terms of kinetics of c y t o t o x i c i t y and c y t o t o x i c potential, the resident M¢ exhibit
245 significantly higher activity compared to the circulating M. The difference in c y t o t o x i c i t y appears to be more marked with the M¢ derived from neoplastic effusions. The difference does not appear to be due to the different isolation procedures (effects of FCS or Fc receptor interaction). It is tempting to suggest that this difference in the degree of c y t o t o x i c potential of the resident M~b reflects in vivo 'activation' of these effector cells. The higher c y t o t o x i c reactivity of the resident M~b, on the other hand, may reflect true functional heterogeneity inherited as a result of further differentiation along their lineage. It should be pointed out that the degree of difference in cytotoxic potential is not as marked with the benign effusion fluid derived M~. They, nevertheless, exhibit statistically significant higher c y t o t o x i c reactivity compared to the circulating M from the same door. The second important feature that emerges from our study is an observation on the nature and specificity of M/M~ cytotoxicity. It has been noted earlier that activated macrophages recognize t u m o r cells from normal cells and exert cytotoxicity against the t u m o r cells selectively (Hibbs et al., 1972; Holterman et al., 1973; Kaplan et al., 1974; Cleveland et al., 1974; Basic et al., 1974; Piessens et al., 1975; Meltzer et al., 1975, 1976). Our studies indicate that under the experimental conditions e m p l o y e d in our isolation procedure (unstressed in conventional terms) human M/M~ are cytotoxic against t u m o r cells as well as against normal cells. It is possible that some degree of activation of these effector cells may ensue from the purification procedures, but studies indicate that the c y t o t o x i c activity of circulating M may be further enhanced by in vitro activation of these cells with macrophage activation factor (manuscript under preparation). Enhancement of cytotoxicity of human Me against t u m o r cells by human l y m p h o c y t e mediators has been demonstrated by Cameron and Churchill (1979). In addition, whether some degree of activation ensues from our purification procedures or not, the specificity of human M/M~ c y t o t o x i c i t y differs, at least qualitatively, from the specificity of activated macrophages as shown by other investigators in animal systems. In contrast to the selective c y t o t o x i c property of activated mouse macrophages against t u m o r cells or transformed fibroblasts, human M/M¢ appear to show no absolute selectivity for t u m o r cells. Although it is clear that normal fibroblasts tend to exhibit a relative resistance to lysis, significant levels of c y t o t o x i c i t y against allogeneic fibroblasts are routinely seen in our study. It is, however, possible that the same fibroblasts, if transformed by SV-40 or other means, might have shown greater sensitivity to lysis by M/Me. It should, however, be pointed o u t that our relative inability, and that of most other investigators, to obtain and include relevant nonfibroblastic target cells as 'ideal' target cell control in microcytotoxicity assays remains a perennial problem. This obviously limits the scope of assessing specificity of cytotoxicity. Our studies continue and careful examinations of kinetics and further specificities of M/M~ from normal individuals and cancer patients will be presented later. Mean~vhile, in recognition of the role of M/M~ in defense
246 against neoplasia ( L e a r y a n d W h e e l o c k , 1 9 7 4 ) , this s t u d y provides f u r t h e r evidence t h a t h u m a n M/MO are capable o f exhibiting s p o n t a n e o u s c y t o t o x i c i t y against various t u m o r cells a n d the e f f e c t o r cell p o p u l a t i o n usually referred to as M / M e c o m p r i s e s a h e t e r o g e n e o u s p o p u l a t i o n o f cells at least in f u n c t i o n a l terms. F u r t h e r studies designed to e x a m i n e the p o t e n t i a l role o f M/M¢ c y t o t o x i c i t y in t u m o r i m m u n i t y or in i m m u n o s u r v e i l l a n c e will be valuable. ACKNOWLEDGEMENTS This s t u d y was s u p p o r t e d in part b y a grant f r o m Aid to Cancer Research, B o s t o n , MA a n d in p a r t b y Public Health Service G r a n t CA 1 2 9 2 4 f r o m the N a t i o n a l Cancer I n s t i t u t e . We t h a n k Arlene F l o w e r s and L e n o r e R o t h m a n for excellent technical assistance a n d L i n d a R a p a c k i for p r e p a r a t i o n of the manuscript. REFERENCES Basic, I,D., D. Milas, D.J. Girdina and H.R. Withers, 1974, J. Natl. Cancer Inst. 52, 1839. Bean, M.A., H. Pees, G. Rosen and H.F. Oettgen, 1973, Natl. Cancer Inst. Monogr. 37, 41. Bianco, C., 19';6, in: In Vitro Methods in Cell Mediated Tumor Immunity, eds. B.R. Bloom and J.J~. David (Academic Press, New York) p. 407. Boyle, W., 1968, Transplantation 6,761. Boyum, A., 1968, Scand. J. Clin. Lab. Invest. (Suppl. 97) 21, 77. Cameron, D.C. and W.H. Churchill, 1979, J. Clin. Invest. 62,977. Cleveland, R.B., M.S. Meltzer and B. Zbar, 1974, Natl. Cancer Inst. 52, 1887. Hibbs, J.B., L.H. Lambert and J.S. Remington, 1972, Science 177,998. Hibbs, J.B., 1976, in: The Macrophage in Neoplasia, ed. M.A. Fink (Academic Press, New York) p. 83. Holterman, O.A., E. Klein and G.P. Casale, 1973, Cell Immunol. 9,339. Holterman, O.A., I. Djerassi, B.A. Lisafeld, E.G. Ellias, B.W. Papermaster and E. Klein, 1974, Proc. Soc. Exp. Biol. Med. 147,456. Kaplan, A.M., P.S. Monahan and W. Regelson, 1974, J. Natl. Cancer Inst. 52, 1919. Keller, R., 1978, Br. J. Cancer 37,732. Koski, I.R., D.G. Poplack and R.M. Blaese, 1976, in: In Vitro Methods in Cell Mediated Tumor Immunity, eds. B.R. Bloom and J.R. David (Academic Press, New York) p. 359. Leafy, N.H. and E.F. Wheelock, 1974, Adv. Cancer Res. 21,131. Mantovani, A., T.R. Jerrels, J.H. Bear and R.B. Herberman, 1979a, Int. J. Cancer 23, 18. Mantovani, A., A. Tagliabue, J.H. Dean, T.R. Jerrells and R.B. Herberman, 1979b, Int. J. Cancer 23, 28. Meltzer, M.S., R.W. Tucker, K.K. Sanford and E.J. Leonard, 1975, J. Natl. Cancer Inst. 54, 1177. Meltzer, M.S., R.W. Tucker and A.C. Brener, 1976, Cell Immunol. 17, 30. Mukherji, B., A. Flowers, L. Nathanson and D.A. Clark, 1974, Cancer Res. 34, 43. Mukherji, B., D. Vassos, A. Flowers, S. Binder and L. Nathanson, 1975, Cancer Res. 35, 3721. Nelson, D.S., 1976, in: Immunobiology of the Macrophage, ed. D.S. Nelson (Academic Press, New York) p. 617. Piessens, W.F., H.W. Churchill, Jr. and J.R. David, 1975, J. Immunol. 114,293.
247 Sharma, S.D. and W.F. Piessens, 1978, Cell Immunol. 37, 20. Tagliabue, A., A. Mantovani, M. Kilgallen, R.B. Herberman and J.L. McCoy, 1979, J Immunol. 122, 2363. West, W.H., G.B. Cannon, H.D. Kay, G.D. Bonnard and R.B. Herberman, 1977, J. Immunol. 118,355.
233
IN VITRO ASSAY OF SPONTANEOUS CYTOTOXICITY BY HUMAN MONOCYTES AND MACROPHAGES AGAINST TUMOR CELLS
BIJAY MUKHERJI 1 Tufts University School o f Medicine and New England Medical Center Hospital, Boston, MA 02111, U.S.A. (Received 1 April 1980, accepted 28 May 1980) The [3H] proline microcytotoxicity technique has been adopted to examine the spontaneous cytotoxic property of human circulating monocytes (M) derived from the peripheral blood and resident monocytes/macrophages (Me) derived from effusion fluids. A leukocyte fraction is obtained by centrifugation in Ficoll-Hypaque (F-H) gradient. M are then isolated as adherent cells following incubation of the leukocyte fraction in plastic flasks containing 50% fetal calf serum (FCS) in culture medium. M~ are isolated from effusion fluids after first separating the Fc receptor bearing cells (as Fc receptor-7S EA resettes) in F-H gradient and then enriching them as adherent cells. The above techniques yield a M/M~b population of some 90--98% purity. The M]M~ non-selectively lyse tumor cells and allogeneic normal fibroblasts in the 48 h [3H]proline assay. The fibroblasts, however, exhibit considerable resistance to lysis, particularly at lower effector to target ratios. While appreciable levels of cytotoxicity are observed with resident M6 at 6 h, followed by a further increase in the level of cytotoxicity at 2 4 - - 4 8 h, circulating M exhibit a consistent level of cytotoxicity only at 24--48 h. When circulating M and resident M~ from the same donor are examined concurrently for cytotoxicity, resident M6 are consistently found to show significantly higher levels of cytotoxicity when compared to circulating M. This may reflect a true functional heterogeneity within this lineage of effector cells or some degree of in vivo activation of resident M~b in malignant and non-malignant effusion fluids. Further studies of spontaneous cytotoxicity by M]M~ through CMC assay techniques, such as the [3H ]proline microtoxicity technique, will be useful in the examination of the role of M/M6 in cell mediated immunity and immunosurveillance against cancer. INTRODUCTION
T h e cells o f m o n o c y t e ( M ) / m a c r o p h a g e (M~b) l i n e a g e c o n s t i t u t e a n i m p o r t a n t class o f e f f e c t o r cells i n cell m e d i a t e d i m m u n i t y a g a i n s t v a r i o u s m i c r o b i a l i n f e c t i o n s a n d p o s s i b l y a g a i n s t n e o p l a s t i c d i s o r d e r s . M / M e are v e r s a t i l e e f f e c t o r cells ( N e l s o n , 1 9 7 6 ) . T h e i r e f f e c t o r f u n c t i o n s are d i v e r s e , i n c l u d i n g a I Present address: University of Connecticut Health Center, Farmington, CT 06032, U.S.A. Abbreviations used: M = monocytes; M~ = macrophages; M-CMC = monocyte-macrophage mediated microcytotoxicity; CMI = cell mediated immunity; SRBC = sheep erythrocytes; F-H = Ficoll-Hypaque; FCS = fetal calf serum; 7S-EA = sheep erythrocytes sensitized with 7S anti-sheep erythrocytes IgG; 19S-EAC = sheep erythrocytes sensitized with 19S antisheep antibody and complement.
234 phagocytic ability as well as an apparent selective tumoricidal property (Nelson, 1976; Hibbs, 1976). While phagocytosis is an important property of M/M0, selective c y t o t o x i c i t y against cancer cells appears to be another important functional property of these effector cells, at least, in animal systems (Hibbs, 1976). Lately, several studies have emerged to indicate a spontaneous c y t o t o x i c capacity of rodent as well as human M/Me (Holterman et al., 1974; Keller, 1978; Mantovani, 1979a, b). Although spontaneous cytotoxicity of human circulating macrophages is well documented, the specificity of such c y t o t o x i c i t y has not yet been clearly established. In addition, it appears that M/MO can be 'activated' by various types of manipulation and such activated M/M¢ may play an important role in tumor immunity and adjuvant immunotherapy. On the scale of evolutionary development of the immune system, the emergence of M/Me lineage possibly antedates that of the lymphoid system. If a first line defense against cancer exists, M/M0 may play an important role in such an immune surveillance scheme. Further, macrophages are known to home at diverse sites, and natural c y t o t o x i c i t y of M/M0 from diverse anatomical sites has been studied in rodents (Keller, 1978). No coordinated examination of c y t o t o x i c capability of circulating and resident human macrophages has, however, been conducted. This study, therefore, has been undertaken as a broader examination of in vitro c y t o t o x i c properties of M/M~b in a human system; and this manuscript describes some characteristic feature of in vitro cytotoxicity and potential functional diversities of the effector cells of M/M¢ lineage. MATERIALS AND METHODS
Effector cells Peripheral venous blood and effusion fluids (pleural and ascites) are collected in heparinized syringes or bottles (20 units/ml for peripheral blood and 10,000 units/1 of effusion fluid). Healthy donors or cancer patients are bled for 2 0 - - 1 2 0 ml of peripheral blood for monocytes. Effusion fluids are usually obtained in large amounts from patients (with neoplastic and nonneoplastic diseases) who undergo diagnostic or therapeutic thoracentesis or paracentesis. All neoplastic effusions examined in this study were cytologically positive for t u m o r cells.
Target cells Several human malignant melanoma cell lines (RoLe-M, JoCa-M, A1Co-M) and the corresponding a u t o c h t h o n o u s fibroblast lines (RoLe-F, JoCa-F, A1Co-F) established earlier in our laboratory, have been used. The melanoma line RoLe-M and the fibroblast line RoLe-F are used as the reference cell lines in most experiments. A human leiomyosarcoma line SK-LMS-1 (established by, and a gift of, Germaine Trempe of Sloan Kettering Cancer Center,
235 New York) has also been used in this study as another target cell line. The melanoma cell lines and the sarcoma line SK-LMS-1 are transplantable as xenografts according to a technique previously described (Mukherji et al., 1974). The fibroblast line RoLe-F is contact inhibited.
Isolation o f M/M¢ from peripheral blood ieukocytes (PBL) and effusion fluids The basic property of adherence to plastic surfaces by M/M¢ is utilized to isolate these cells from PBL and effusion fluid. In addition, the presence of Fc receptors on M¢ is utilized to separate them as Fc receptor rosettes from other contaminating non-Fc receptor bearing cells in effusion fluids.
Isolation o f M from PBL A leukocyte fraction is first isolated by the technique of B o y u m (1968). The interface band containing the l y m p h o c y t e s and monocytes is gently aspirated, washed once in Hanks Balanced Salt Solution (HBSS) and resuspended in Hams F-10 medium containing 50% fetal calf serum (FCS). Approximately 3 ml of the cell suspension (at 1--2 X 10 ~ cells per ml) in F-10 medium with 50% FCS is incubated in T-30 plastic flasks for 1 h at 37°C in 5% CO2 in air. Following incubation, the non-adherent cells are removed by 6 vigorous rinsings in HBSS. The adherent cells are harvested mechanically by a rubber policeman and are referred to as M to denote them as circulating monocytes.
Isolation o f M/Md from effusion fluids as 7S-EA rosettes Cell pellets are first obtained by centrifugation of 250 ml of effusion fluid at 175 X g for 10 min in conical plastic centrifuge tubes. Most of the supernatant fluid is discarded leaving approximately 2 ml of the fluid at the bottom. The cell pellet is subsequently resuspended, aspirated and pooled. The pooled cell pellet is then washed in HBSS three times and resuspended in Hams F-10 medium. The non-Fc receptor bearing cells (T l y m p h o c y t e s and t u m o r cells) are removed by isolating the Fc receptor bearing cells as 7S-EA rosettes and recovering the 7S-EA rosettes in the pellet after centrifugation in Ficoll-Hypaque (F-H) gradient. To form 7S-EA rosettes, sheep erythrocytes (SRBC) are first thoroughly washed in HBSS three times. The washed SRBC are then sensitized with 7S anti-SRBC IgG (Cordis Laboratory, Miami, FL 33137) at a standard dilution of 1:100 at 37°C for 45 min (the standard dilution of anti-SRBC IgG is determined from pilot experiments). A 5% suspension of the sensitized SRBC is made (containing approximately 1 X 109 cells per ml) in FCS which has previously been absorbed with an equal volume of packed SRBC. Five ml of effusion fluid cell suspension (1 X 107 per ml) is mixed with 5 ml of sensitized SRBC suspension and incubated at 37°C for 45 rain. After incubation
236 t h e cell s u s p e n s i o n is g e n t l y p o u r e d o n t o 10 ml o f F i c o l l - H y p a q u e s o l u t i o n a n d t h e g r a d i e n t is c e n t r i f u g e d at 4 0 0 × g at r o o m t e m p e r a t u r e f o r 30 m i n . T h e pellet is c a r e f u l l y a s p i r a t e d a n d t h e Fc r e c e p t o r bearing e f f e c t o r cells are r e c o v e r e d free o f S R B C a f t e r i n c u b a t i o n w i t h T r i s - b u f f e r e d 0.83% NH4C1, p H 7.2, f o r 10 m i n at r o o m t e m p e r a t u r e as d e s c r i b e d b y B o y l e ( 1 9 6 8 ) . T h e cells are t h e n w a s h e d t w i c e w i t h 50 ml o f H B S S a n d are r e s u s p e n d e d in H a m s F - 1 0 m e d i u m . F o r f u r t h e r p u r i f i c a t i o n o f t h e M/MO f r o m c o n t a m i n a t ing Fc r e c e p t o r bearing B l y m p h o c y t e s , t h e cells r e c o v e r e d a f t e r NH4C1 t r e a t m e n t are i n c u b a t e d f o r 1 h in plastic f l a s k s in F - 1 0 m e d i u m c o n t a i n i n g 50% f e t a l calf s e r u m . T h e n o n a d h e r e n t cells are r e m o v e d b y 6 rinsings in H B S S as b e f o r e , a n d a d h e r e n t cells are h a r v e s t e d m e c h a n i c a l l y . T h e s e cells are h e n c e f o r t h r e f e r r e d t o as M~b t o d e n o t e t h e m as r e s i d e n t m o n o c y t e s / m a c r o p h a g e s . T h e M~b are w a s h e d o n c e m o r e in H B S S a n d r e s u s p e n d e d in F - 1 0 m e d i u m a l o n e or in F - 1 0 m e d i u m s u p p l e m e n t e d w i t h FCS a n d antib i o t i c s f o r m i c r o c y t o t o x i c i t y e x p e r i m e n t s d e s c r i b e d later. T h e y i e l d o f M f r o m P B L varies b e t w e e n 3 a n d 7% o f t h e F-H cell p r e p a r a t i o n , while t h e yield o f M¢ f r o m e f f u s i o n fluids varies b e t w e e n 22 a n d 45%. T h e p u r i t y o f r e s i d e n t M e a c h i e v e d in o u r s t u d y r e a c h e s 9 0 - - 9 5 % w h e n p u r i t y is j u d g e d b y t h e criteria listed in T a b l e 1 (non-specific esterase positive cells s h o w i n g positive r e c e p t o r s f o r 7S-EA a n d 19S-EAC, p h a g o c y t o s i s , a b s e n c e o f r e c e p t o r s f o r n e u r a m i n i d a s e t r e a t e d S R B C a n d bearing no surface i m m u n o g l o b u l i n ) . J u d g e d b y t h e s a m e criteria, t h e p u r i t y o f M~ in t h e effusion fluid cells, isolated b y t h e a d h e r e n c e step o n l y , d r o p s t o 44% a l t h o u g h a v a r i e t y o f t u m o r cells stain p o s i t i v e l y f o r n o n - s p e c i f i c esterase r e a c t i o n . T h e final viability o f t h e a d h e r e n t cells a f t e r scraping ranges f r o m 70 t o 85%.
Iden tifica tion o f M/M¢ T h e M/M~b are i d e n t i f i e d a n d c h a r a c t e r i z e d b y n o n - s p e c i f i c esterase stain as p e r K o s k i et al. ( 1 9 7 6 ) , b y t h e p r e s e n c e o f Fc r e c e p t o r s ( 7 S - E A r o s e t t e TABLE 1 Characteristics of the effector cell. Composite data from 15 donors are shown.
% Positive +- S.E. Blood monocytes
Effusion fluid macrophages
Non-specific esterase stain
7S EA rosettes 19S EAC rosettes Phagocytosis Neuraminidase E-RFC Surface immunoglobulin
-------
98 94 93 63 1.2 1.9
+ 1.5 +- 1.1 -+ 3.3 + 3.0a + 0.1 + 0.02
a Difference is significant at P ~ 0.005 (Student's t-test).
95 93 94 89 0.6 2.3
-+ 2.1 +- 1.8 + 2.6 + 4.7 a + 0.02 + 0.03
237 assay) and complement receptors (19S-EAC rosette assay) as per Bianco (1976), absence of receptors for SRBC (spontaneous) and surface immunoglobulin, and by phagocytosis of heat killed yeast. Natural killer l y m p h o c y t e s (NK cells) are identified as rosettes with neuraminidase treated SRBC (neuraminidase-ERFC) using the methods of West et al. (1977). A simplified yeast ingestion technique is adopted to the phagocytosis assay. Firstly, the yeasts (ordinary baking yeast) are boiled for 1 h and a suspension of yeasts is made in cold HBSS (1 X 107 yeasts per ml). The M/MO are suspended (at 2 X l 0 s per ml) in cold HBSS to prevent clumping. The M/M~b suspension and the yeast suspension are mixed in equal volumes to obtain a M/M¢ to yeast ratio of 1 : 50. The M/M~ yeast suspension is then centrifuged at 175 X g for 5 min and most of the supernatant is discarded by gentle aspiration leaving approximately 0.2 ml of the supernatant fluid. To each tube, thereafter, 0.1 ml of AB+ human serum (heated at 56°C for 45 min) is added. The final reaction mixture is incubated for 37°C for 30 min. The suspension mixture is gently resuspended and following incubation a slide preparation is made with a droplet of the cell suspension. The slides are stained with Wrights-Giemsa stain and are examined for phagocytosis. One hundred effector cells are counted to determine the percentage of phagocytic cells. In vitro M/Md mediated microcytotoxicity (M-CMC) assay The [3H] proline m i c r o c y t o t o x i c i t y assay, originally described by Bean et al. (1973) as an in vitro technique for studying l y m p h o c y t e c y t o t o x i c i t y and extensively used in our laboratory for l y m p h o c y t e microcytotoxicity assay, is adopted for M-CMC assay. The details of the assay have been previously described (Mukherji et al., 1975). Briefly, target cells are prelabelled in a T30 plastic flask in monolayer with 50 pCi of [3H] proline (47.7 Ci/mmole: New England Nuclear, Boston, MA) for 1 8 - - 2 4 h. The labelled target cells are harvested from the flask, resuspended in appropriate medium, and distributed into the wells of linbro 6 mm microcytotoxicity plates (Linbro Chemical Co., New Haven, CT). Usually, 1,000 viable labelled target cells are plated in each well. After the target cells have attached to the well b o t t o m , effector cells suspended in 100 ~l of test medium (Hams F-10) are added to the wells. After initial incubation of effector cells and target cells at 37°C in 5% CO2 for 45 min, 100 pl of F-10 medium with 40% heat inactivated FCS is added to each well to bring the final concentration of FCS to 20% and the incubation is continued for an additional 48 h. Effector cells are added to each well at desired effector to target cell ratios ( 1 2 : 1 - - 1 0 0 : 1 ) . After incubation, the plates are washed, air dried and the b o t t o m s of the wells are punched o u t in scintillation vials. Protosol (0.3 ml) and 0.9 ml of methanol are added to each vial at room temperature 30 min prior to addition of scintillation fluid (4 g of Omnafluor/1 of toluene) (Omnafluor: New England Nuclear, Boston,
238
MA; Toluene: Fisher Chemical Co., Fairlawn, NJ). The vials are kept dark at room temperature for 24 h prior to counting. Usually, each assay is performed in 5--10 replicates. 3H scintillation counting is performed in a Packard Tri-Carb liquid scintillant spectrometer (Packard Instrument Co., Downs Grove, IL). Percentage c y t o t o x i c i t y by a given effector cell is calculated in relation to medium control and then compared for significance by Student's t-test. RESULTS
Characteristics o f the effector cells Table I shows the characteristics of the effector cells isolated as M/M@. It is of interest that, although a very high percentage of cells stain positively for non-specific esterase, reported as a specific stain for M/M@ (Koski et al., 1976), not all of them exhibit phagocytosis. Furthermore, the M@ isolated from neoplastic or non-neoplastic effusions exhibit a significantly higher percentage of phagocytosis (P < 0.005) compared to the M obtained from PBL. The M from PBL and M@ from effusion fluids express receptors for the Fc portion of immunoglobulin IgG and for complement in equal proportion. NK cell and B cell contamination in both preparations are minimal.
In vitro cytotoxicity of M/Md An example of an M-CMC assay is shown in Fig. 1. M from PBL as well as M@ from the effusion fluid exhibit significant levels of cytotoxicity against allogeneic neoplastic as well as non-neoplastic target cells. The fibroblasts
>_ 601
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EFFECTOR: TARGETCELLRATO I Fig. 1. S p o n t a n e o u s c y t o t o x i c i t y b y M/M~b against t h e m e l a n o m a cells ( R o L e - M ) a n d t h e a u t o l o g o u s f i b r o b l a s t s ( R o L e - F ) . T h e o p e n circle a n d t h e closed circle r e p r e s e n t r e s i d e n t M~b f r o m ascites a n d c i r c u l a t i n g M r e s p e c t i v e l y f r o m a b r e a s t c a n c e r p a t i e n t . T h e o p e n triangle r e p r e s e n t s c i r c u l a t i n g M f r o m a p a t i e n t w i t h c o l o n c a n c e r a n d t h e closed triangle r e p r e s e n t s t h e s a m e f r o m a n o r m a l d o n o r . C y t o t o x i c i t y e q u a l t o or a b o v e 15% level is significant at P ~< 0 . 0 5 ; o t h e r s are n o t s i g n i f i c a n t . E a c h d a t a p o i n t b e t w e e n t h e r e s i d e n t Mq~ a n d circulating M is significant at P ~< 0.01.
239 TABLE 2 Effect of silica on M/M~b cytotoxicity. Cytotoxicity against target cell RoLe-M Effector cell
Adherent M/M~ a
Non-adherent lymphocyte b
Donor/Diag
Source
Without silica
With silica
Without silica
BM/Normal
PBL
706 + 81 c
818 -+ 76
596 + 40
562 -+ 47
(19) 368 + 23
(6) 769 + 84
(32) 679 -+ 60
(36) 712 -+ 59
RS/Ovarian Ca Ascites and
MD/Normal Medium control
PBL PBL
(58) (12) 638 + 60 854 + 42 (27) (3) 878 + 59
With silica
(23) (19) 508 + 39 534 + 46 (42) (39) 878 + 59
a Circulating M or resident Me (from patient RS) are isolated by methods described in the text. 100 pg of silica (maximum particle size of 5 pm sonicated immediately prior to incubation) is added to flasks contained 1--5 × 106 effector cells (M/M~b as adherent cells, lymphocytes as non-adherent cells) and cultured overnight along with the untreated flasks in 3 ml of F-10 medium containing 10% AB+ human serum and antibiotics. After overnight incubation, the effector cells are harvested (M/M~b mechanically, non-adherent lymphocytes by decanting), washed in HBSS x 3, checked for viability by trypan blue dye exclusion, cell concentration readjusted for viable effector to target cell ratio of 50:1, and added to the microtoxicity plates as described in the text. b Represent nylon wool column passaged non-adherent cells following Ficoll-Hypaque gradient separation. The resultant cells consist of 95--99% lymphocytes. c Mean cpm of 5 samples + S.E.; numbers in parentheses are percentages of cytotoxicity calculated in relation to medium control. e x h i b i t c o n s i d e r a b l e resistance to lysis b y allogeneic M / M ¢ in c o m p a r i s o n t o the m e l a n o m a cells as targets at c o m p a r a b l e e f f e c t o r t o target ratios. T h e r e s i d e n t M~b o b t a i n e d f r o m ascites f r o m a d o n o r having breast c a n c e r e x h i b i t higher c y t o t o x i c i t y (P < 0.01 or better) w h e n c o m p a r e d to circulating M f r o m the same p a t i e n t at all e f f e c t o r t o t a r g e t ratios. Table 2 e x a m i n e s the e f f e c t o f silica (a gift o f J a m e s Murray, S t a n f o r d University Medical S c h o o l , S t a n f o r d , CA) o n c y t o t o x i c i t y e x h i b i t e d b y M/M~. As a c o n t r o l , circulating l y m p h o c y t e s f r o m the same d o n o r s have also been included in the e x p e r i m e n t . T h e l y m p h o c y t e s a n d the circulating M or resident M¢ are t r e a t e d with silica t h e same w a y (for details o f silica t r e a t m e n t see Table 2) prior t o the CMC assay. O n l y the M/M~ t r e a t e d with silica e x h i b i t m a r k e d decrease or c o m p l e t e a b r o g a t i o n o f c y t o t o x i c i t y while s p o n t a n e o u s c y t o t o x i c i t y b y l y m p h o i d cells r e m a i n s u n a f f e c t e d b y silica treatment. T h e kinetics o f M/M~ c y t o t o x i c i t y is s h o w n in Fig. 2. A t 6 h o f i n c u b a t i o n o f e f f e c t o r cell a n d t a r g e t cell, significant levels o f c y t o t o x i c i t y are observed with M o n l y i n c o n s i s t e n t l y . A significant level o f c y t o t o x i c i t y b y M, h o w ever, is c o n s i s t e n t l y o b s e r v e d with 24 h i n c u b a t i o n f o l l o w e d b y a m o d e s t
240 increase in c y t o t o x i c activity at 48 h. T h e M~b, o n the o t h e r hand, show d i f f e r e n t kinetics. Significant levels of c y t o t o x i c i t y are c o n s i s t e n t l y seen at 6 h. T h e c y t o t o x i c activity shows a f u r t h e r significant rise at 24 h, f o l l o w e d b y a m o d e s t rise at 48 h. T h e d i f f e r e n c e in the level o f c y t o t o x i c i t y b y circulating M and r e s i d e n t M~ at the d i f f e r e n t assay p o i n t s and at identical e f f e c t o r t o target ratios s h o w n in Fig. 2 are significant at P < 0 . 0 0 4 or b e t t e r ( S t u d e n t ' s t-test). P o t e n t i a l d i f f e r e n c e in t h e kinetics of M/M~ c y t o t o x i c i t y b e t w e e n n o r m a l d o n o r s and c a n c e r patients, if any, will be e x a m i n e d in a separate m a n u s c r i p t ( u n d e r p r e p a r a t i o n ) . P o t e n t i a l d a y - t o - d a y variation o f M / M e c y t o t o x i c i t y is e x a m i n e d in Fig. 3. Circulating M f r o m t h r e e n o r m a l d o n o r s (bled on 3 c o n s e c u t i v e days) are e x a m i n e d for c y t o t o x i c i t y o n t h r e e c o n s e c u t i v e assays. As can be seen, o n l y m i n o r degrees o f f l u c t u a t i o n in the level o f c y t o t o x i c i t y are seen with t h e circulating M. Statistically significant (P < 0.01) f l u c t u a t i o n is observed with o n e d o n o r in assays on d a y 1 and d a y 3. R e s i d e n t Me f r o m f o u r c a n c e r p a t i e n t s are c o l l e c t e d and e x a m i n e d in several n o n - c o n s e c u t i v e assays. T w o d o n o r s have b e e n t e s t e d twice and t w o d o n o r s have been t e s t e d o n t h r e e occasions. All assays are c o n d u c t e d at identical e f f e c t o r t o target ratios and with target cells at identical passage levels. Significant f l u c t u a t i o n s in the level o f c y t o t o x i c i t y are seen with t h r e e o f these patients. R e s i d e n t M e f r o m t w o p a t i e n t s have b e e n e x a m i n e d within 2 weeks and the o t h e r subjects have been e x a m i n e d o n t h r e e widely separated, n o n - c o n s e c u t i v e days. T h e p o t e n t i a l activating i n f l u e n c e on FCS, which m i g h t c o n t a i n e n d o t o x i n 7060-
60"
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50"
~ 40-
40. 30-
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Fig. 2. Kinetics of s p o n t a n e o u s c y t o t o x i c i t y by circulating m o n o c y t e s (open circle) and resident m o n o c y t e s / m a c r o p h a g e s (closed circle). Collective data of circulating m o n o c y t e preparations from 15 donors (10 normal and 5 cancer patients) and resident M~ preparation for 15 donors (12 cancer patients and 3 non-cancer patients) are shown. All M-CMC assays are performed at identical E:T ratio of 50:1. Fig. 3. Day-to-day variations in the spontaneous c y t o t o x i c i t y level of circulating M (solid lines) and resident M~b (interrupted lines). The difference in the cytotoxicity level to circulating M from this donor at points indicated w i t h the letter a is significant at P 0.05 by Student's t-test.
241
TABLE 3 Effect of FCS and human AB+ sera on monocyte cytotoxicity. Monocyte
Residual target cell c p m -+ S.E. a against R o L e - M line
Donor/Diag
FCS # 1 b
GD/Melanoma
1238 + 98 (17) 997 + 108 (33) 1485 +
HC/Normal Medium control
FCS # 2 b
FCS # 3 b
1137 + 121 (23) 870 + 101 (41) 116
AB+
1273 + 87 (14) 1013 + 71 (31) 1485 +
1206 + 73 (19) 1056 + 80 (29) 116
a Mean cpm of 5 samples + S.E.; numbers in parentheses represent percentage cytotoxicity in relation to medium control. b Effector cell isolation and M-CMC assay are performed in medium containing 3 separate batches of FCS. a n d t h e r e f o r e c a u s e M / M ~ a c t i v a t i o n , has b e e n e x a m i n e d t h r o u g h M - C M C e x p e r i m e n t s ( i s o l a t i o n o f e f f e c t o r cells a n d t h e CMC a s s a y ) p e r f o r m e d in t h e i r e n t i r e t y in t h r e e b a t c h e s o f F C S a n d h u m a n AB + s e r u m . While m i n o r d i f f e r e n c e s in c y t o t o x i c level c a n be seen w i t h t h e t h r e e b a t c h e s o f F C S , n o m a j o r f l u c t u a t i o n is seen in t h e levels o f c y t o t o x i c i t y w h e n a s s a y s are p e r f o r m e d w i t h F C S a n d A B + s e r u m ( T a b l e 3).
Functional differences between circulating M and resident Md Table 4 shows a comparative analysis of cytotoxic activity of circulating M and resident Me derived from the same donor and assayed concurrently a g a i n s t t h e m e l a n o m a t a r g e t cell line R o L e - M . T w e l v e o u t o f 18 d o n o r s in t h i s series have m a l i g n a n t d i s e a s e (5 w i t h o v a r i a n c a n c e r , 4 w i t h b r e a s t cancer, 1 with malignant melanoma, and 2 with lung cancer). The nonTABLE
4
Comparison of cytotoxicity between circulating M and resident M~$.
Percentage eytotoxicity against RoLe-M a Nb
Circulating M
18
M e a n -+ S.E.
Range
28 + 3.0
14--43
(<: 0.0001) c
Resident M/M~b
18
57 + 4.6
38--81
a M - C M C assays are performed at effector to target ratio = 50:1. b N u m b e r of donors from w h o m circulating M and resident M/M~b are obtained and assayed for cytotoxicity. c T h e figure in parentheses represents the level of significance in the difference in the percent cytotoxicity.
242 malignant group of donors is comprised of 4 patients with hepatic cirrhosis, one with congestive heart failure, and one with inflammatory pleural effusion. Circulating M and resident M¢ from each patient have been concurrently tested for microcytotoxicity in separate experiments against the reference melanoma target cell line at several identical effector to target ratios. Comparative data at the effector to target ratio with which cytotoxicity is consistently seen is shown in Table 4. Significant difference in the level of c y t o t o x i c i t y between resident MO and circulating M when individually compared is observed in 11 o u t of 12 cancer patients (individual data n o t shown). In all these cases, the resident MO exhibit significantly higher levels of cytotoxicity (P < 0.01 - - < 0.001, in most cases < 0.001). In the non-cancer patient group, significant differences are observed only in three cases and the degree of difference is not as marked as it is in the cancer patient group (P < 0.05-< 0.01). When the percentages of c y t o t o x i c i t y are averaged and compared at identical effector to target ratios, the difference in the level of cytotoxicity by resident Me and circulating M becomes highly significant (Table 4).
Effect of isolation procedure on M/Mql cytotoxicity The effect of the membrane Fc receptor interaction in the isolation procedure on the c y t o t o x i c behavior of M, when examined by exposing the circulating M to SRBC coated with 7S IgG followed by Tris-NH4C1 treatment and assayed for c y t o t o x i c i t y concurrently with untreated M, is found to be non-significant (data not shown). In addition, experiments conducted with circulating M exposed to antibody-coated SRBC and with similarly treated resident Me derived from the same cancer patients in two separate but concurrent CMC assays reveal significantly higher levels of cytotoxicity by the resident M~ compared to the circulating M, whether or not treated with 7S-EA and NH4CI (data not shown).
Specificity of cytotoxicity by M/Md Specificity of cytotoxicity by M/Me has been examined in multiple experiments involving the panel of target cells in various different combinations. Target cell controls in these experiments have included fibroblasts (autochthonous to at least one malignant cell line) as well as non-melanoma t u m o r cells. A detailed examination of specificity of M/M~ cytotoxicity in this system will be presented later (manuscript under preparation). However, it is of interest to note that both circulating M and resident M¢ lyse allogeneic t u m o r cells non-selectively. In addition, allogeneic fibroblasts obtained from the same patients from whom melanoma targets are derived, are also lysed, particularly at higher effector to target ratios. Table 5 demonstrates a series of experiments with M/M~ from 5 donors against 3 sets of target cells (each set consisting of a melanoma line and the a u t o c h t h o n o u s fibroblast lines}. As can be seen, the cytotoxic activity of
243
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244 the allogeneic M/M~b against the melanoma cells are detectable at virtually all effector to target ratios. The fibroblasts are, on the other hand, not readily lysed at the lower ratio, while at higher ratios significant killing against the fibroblasts is detectable. DISCUSSION While the isolation of circulating blood m o n o c y t e s is a relatively easier task by virtue of their adherent property, the isolation of resident M~b from human effusion fluids, particularly malignant effusions, has always been a difficult proposition. The m e t h o d utilized in this study involving isolation of the Fc receptor bearing cells as 7S-EA rosettes followed by enrichment of the macrophages by their adherent property is reasonably effective in removing the contaminating non-lymphomatous malignant cells and the lymphocytes. The lack of uniform phagocytic ability by some of the adherent nonspecific esterase positive cells derived from peripheral blood suggests that all circulating M may not always exhibit phagocytosis in any given assay. The infrequent association of cells showing receptors for neuraminidase treated sheep erythrocytes of cells bearing surface immunoglobulins, and cells exhibiting high frequency of non-specific esterase stain, indicate that the non-phagocytic cells in our preparations do not represent lymphocytic lineage. Since contamination of NK cells or B cells in the effector cell population is exceedingly small, the potential influence of NK cells or adherent B cells in the killing process is, therefore, negligible. The cytotoxic ability of activated guinea pig macrophages has been d o c u m e n t e d by Sharma and Piessens (1978) utilizing the [3H]proline CMC technique. They have used the adhesive property of macrophages as a purification step and shown that activated macrophages exhibit significant degrees of cytotoxicity at 6 h. Spontaneous c y t o t o x i c i t y by M or M~ in mice, rats and humans has also been well d o c u m e n t e d by other investigators (Holterman et al., 1974; Keller, 1978; Mantovani, 1979a, b; Tagliabue et al., 1979). Our studies support and confirm the feasibility of utilizing the [~H] proline CMC technique for in vitro assay for cytotoxicity mediated by these effector cells. The kinetics of human M/M~b cytotoxicity, however, differ. Although at 6 h appreciable degrees of cytotoxicity by circulating M may be detected at times, the consistent activity is observed at 2 4 - - 4 8 h. Tagliabue et al. (1979) have recently described natural cytotoxicity of mouse M and Me. These investigators have noted that the cytotoxic activity of mouse and Me is best seen at 4 8 - - 7 2 h of incubation. In this regard, significant cytotoxicity by human resident M~i at 6 - - 2 4 h of incubation, as seen in our study, is noteworthy. In addition, t w o important characteristics of human M/M~ cytotoxicity emerge from our studies. Firstly, the possibility of existence of considerable functional heterogeneity among the effector cells is evident. In terms of kinetics of c y t o t o x i c i t y and c y t o t o x i c potential, the resident M¢ exhibit
245 significantly higher activity compared to the circulating M. The difference in c y t o t o x i c i t y appears to be more marked with the M¢ derived from neoplastic effusions. The difference does not appear to be due to the different isolation procedures (effects of FCS or Fc receptor interaction). It is tempting to suggest that this difference in the degree of c y t o t o x i c potential of the resident M~b reflects in vivo 'activation' of these effector cells. The higher c y t o t o x i c reactivity of the resident M~b, on the other hand, may reflect true functional heterogeneity inherited as a result of further differentiation along their lineage. It should be pointed out that the degree of difference in cytotoxic potential is not as marked with the benign effusion fluid derived M~. They, nevertheless, exhibit statistically significant higher c y t o t o x i c reactivity compared to the circulating M from the same door. The second important feature that emerges from our study is an observation on the nature and specificity of M/M~ cytotoxicity. It has been noted earlier that activated macrophages recognize t u m o r cells from normal cells and exert cytotoxicity against the t u m o r cells selectively (Hibbs et al., 1972; Holterman et al., 1973; Kaplan et al., 1974; Cleveland et al., 1974; Basic et al., 1974; Piessens et al., 1975; Meltzer et al., 1975, 1976). Our studies indicate that under the experimental conditions e m p l o y e d in our isolation procedure (unstressed in conventional terms) human M/M~ are cytotoxic against t u m o r cells as well as against normal cells. It is possible that some degree of activation of these effector cells may ensue from the purification procedures, but studies indicate that the c y t o t o x i c activity of circulating M may be further enhanced by in vitro activation of these cells with macrophage activation factor (manuscript under preparation). Enhancement of cytotoxicity of human Me against t u m o r cells by human l y m p h o c y t e mediators has been demonstrated by Cameron and Churchill (1979). In addition, whether some degree of activation ensues from our purification procedures or not, the specificity of human M/M~ c y t o t o x i c i t y differs, at least qualitatively, from the specificity of activated macrophages as shown by other investigators in animal systems. In contrast to the selective c y t o t o x i c property of activated mouse macrophages against t u m o r cells or transformed fibroblasts, human M/M¢ appear to show no absolute selectivity for t u m o r cells. Although it is clear that normal fibroblasts tend to exhibit a relative resistance to lysis, significant levels of c y t o t o x i c i t y against allogeneic fibroblasts are routinely seen in our study. It is, however, possible that the same fibroblasts, if transformed by SV-40 or other means, might have shown greater sensitivity to lysis by M/Me. It should, however, be pointed o u t that our relative inability, and that of most other investigators, to obtain and include relevant nonfibroblastic target cells as 'ideal' target cell control in microcytotoxicity assays remains a perennial problem. This obviously limits the scope of assessing specificity of cytotoxicity. Our studies continue and careful examinations of kinetics and further specificities of M/M~ from normal individuals and cancer patients will be presented later. Mean~vhile, in recognition of the role of M/M~ in defense
246 against neoplasia ( L e a r y a n d W h e e l o c k , 1 9 7 4 ) , this s t u d y provides f u r t h e r evidence t h a t h u m a n M/MO are capable o f exhibiting s p o n t a n e o u s c y t o t o x i c i t y against various t u m o r cells a n d the e f f e c t o r cell p o p u l a t i o n usually referred to as M / M e c o m p r i s e s a h e t e r o g e n e o u s p o p u l a t i o n o f cells at least in f u n c t i o n a l terms. F u r t h e r studies designed to e x a m i n e the p o t e n t i a l role o f M/M¢ c y t o t o x i c i t y in t u m o r i m m u n i t y or in i m m u n o s u r v e i l l a n c e will be valuable. ACKNOWLEDGEMENTS This s t u d y was s u p p o r t e d in part b y a grant f r o m Aid to Cancer Research, B o s t o n , MA a n d in p a r t b y Public Health Service G r a n t CA 1 2 9 2 4 f r o m the N a t i o n a l Cancer I n s t i t u t e . We t h a n k Arlene F l o w e r s and L e n o r e R o t h m a n for excellent technical assistance a n d L i n d a R a p a c k i for p r e p a r a t i o n of the manuscript. REFERENCES Basic, I,D., D. Milas, D.J. Girdina and H.R. Withers, 1974, J. Natl. Cancer Inst. 52, 1839. Bean, M.A., H. Pees, G. Rosen and H.F. Oettgen, 1973, Natl. Cancer Inst. Monogr. 37, 41. Bianco, C., 19';6, in: In Vitro Methods in Cell Mediated Tumor Immunity, eds. B.R. Bloom and J.J~. David (Academic Press, New York) p. 407. Boyle, W., 1968, Transplantation 6,761. Boyum, A., 1968, Scand. J. Clin. Lab. Invest. (Suppl. 97) 21, 77. Cameron, D.C. and W.H. Churchill, 1979, J. Clin. Invest. 62,977. Cleveland, R.B., M.S. Meltzer and B. Zbar, 1974, Natl. Cancer Inst. 52, 1887. Hibbs, J.B., L.H. Lambert and J.S. Remington, 1972, Science 177,998. Hibbs, J.B., 1976, in: The Macrophage in Neoplasia, ed. M.A. Fink (Academic Press, New York) p. 83. Holterman, O.A., E. Klein and G.P. Casale, 1973, Cell Immunol. 9,339. Holterman, O.A., I. Djerassi, B.A. Lisafeld, E.G. Ellias, B.W. Papermaster and E. Klein, 1974, Proc. Soc. Exp. Biol. Med. 147,456. Kaplan, A.M., P.S. Monahan and W. Regelson, 1974, J. Natl. Cancer Inst. 52, 1919. Keller, R., 1978, Br. J. Cancer 37,732. Koski, I.R., D.G. Poplack and R.M. Blaese, 1976, in: In Vitro Methods in Cell Mediated Tumor Immunity, eds. B.R. Bloom and J.R. David (Academic Press, New York) p. 359. Leafy, N.H. and E.F. Wheelock, 1974, Adv. Cancer Res. 21,131. Mantovani, A., T.R. Jerrels, J.H. Bear and R.B. Herberman, 1979a, Int. J. Cancer 23, 18. Mantovani, A., A. Tagliabue, J.H. Dean, T.R. Jerrells and R.B. Herberman, 1979b, Int. J. Cancer 23, 28. Meltzer, M.S., R.W. Tucker, K.K. Sanford and E.J. Leonard, 1975, J. Natl. Cancer Inst. 54, 1177. Meltzer, M.S., R.W. Tucker and A.C. Brener, 1976, Cell Immunol. 17, 30. Mukherji, B., A. Flowers, L. Nathanson and D.A. Clark, 1974, Cancer Res. 34, 43. Mukherji, B., D. Vassos, A. Flowers, S. Binder and L. Nathanson, 1975, Cancer Res. 35, 3721. Nelson, D.S., 1976, in: Immunobiology of the Macrophage, ed. D.S. Nelson (Academic Press, New York) p. 617. Piessens, W.F., H.W. Churchill, Jr. and J.R. David, 1975, J. Immunol. 114,293.
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