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Properties of a murine monocytic tumor cell line J-774 in vitro
Printed in Sweden Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/78/l 151~0053$02.00/O
Experimental
PROPERTIES
Cell Research 115 (1978) 53-61
OF A MURINE CELL
LINE
I. Morphology G. KAPLAN
MONOCYTIC
TUMOR
J-774 IN VITRO and Endocytosis and B. M@RLAND
institute of Medical Biology, University of Tromsti, Tromsr, Norway
SUMMARY A murine monocytic tumor cell line J-774 was maintained in culture in the presence or absence of endotoxin, in an attempt to induce differentiation similar to that found in activated peritoneal macrophages. The morphology and Fc and Cs receptor functions of attachment and ingestion were compared in the treated and untreated cultures. J-774 cells maintained in culture for 72 h seemed to resemble endotoxin-activated macrophages rather than normal peritoneal macrophages. A striking amount of ruffhng was observed on the surface of the cells cultured for 34 days both in the presence and in the absence of endotoxin. As compared to the peritoneal macrophage where particles attached to the C, receptors are not ingested unless the cells are activated, J-744 cells attached and ingested particles via the C, receptor even without stimulation. The presence of endotoxin in the culture medium of these cells gave rise to more efficient phagocytosis but did not effect the temperature sensitivity of the phagocytic receptors. Both in treated and untreated cultures attachment to the Fc receptor was less dependent on the temperature than that of the C3 receptor while ingestion was more sensitive in the Fc receptor as compared with the C, receptor.
Normal mouse mononuclear phagocytes ture conditions was compared with that of have been shown to differentiate during in- murine peritoneal macrophages. Endotoxin has been shown to effectively flammation as a result of stimulation by factors available in the extravascular compart- inhibit the proliferation of the J-774 cells in ment at the inflammatory site [ 11. A similar vitro [7]. It was of interest to establish differentiation of murine peritoneal macro- whether endotoxin at concentrations which phages can be obtained both in vivo and in are cytostatic but not cytotoxic could induce in these cells any differentiated propvitro by exposure of the cells to endotoxin [2], and other chemical compounds. This erties similar to those found in the actidifferentiation is expressed by changes in vated peritoneal macrophages. Phase contrast and scanning electron mimorphology, phagocytic capacity, cytotoxicity of the cells, and increase in their croscopy (SEM) were carried out on the cells. The Fc and C, receptor functions of lysosomal enzyme activity [l-5]. A murine reticulum cell sarcoma (J-774) attachment and internalization of opsonized has been described, which in ascites form particles were investigated. has the macrophage properties of adherMATERIALS AND METHODS ence, morphology, receptors for immunoglobulins and complement, and antibody- Mouse cell lines dependent lysis of target cells [6]. The J-774 tumor was obtained from Dr P. Ralph from The J-774 cells’ response to in vitro cul- the Sloan Kettering Institute for Cancer Research, Exp Ceil Res I15 (1978)
54
Kaplan and M&and
New York. The ascites tumor was passaged intraperitoneally in BALB/c mice (0.2-0.4 ml). Ascites cells with diameter about 30 urn were reckoned as tumor cells [6]. They constituted about 90% of the cellular elements in the J-774ascites, the remaining 5-10% were smaller cells with a diameter about lo-pm. Experiments were performed with ascites J-774 A cells, showing greater than 90% viability by trypan blue exclusion.
Macrophage culture conditions Mouse oeritoneal macrouhages were obtained from outbred*NMRI mice. Hat!ves&g and culture methods were essentially those of Cohn & Benson [l]. Cells were cultured in MEM Earle (G&co, Grand Island, N.Y.) with 20% heat-inactivated fetal calf serum (FCS) (Gibco), 100 W/ml of penicillin, and 100 wg/ ml streptomycin, The cells were seeded in Linbro plates (Linbro them. Co., New Haven) with glass coverslips (14 mm 0), in 5% CO* in air, at 37°C. After 2 h the cultures were washed and new medium added. Cells were seeded to give 2.5~ 105cells/culture.
J-774 culture conditions Harvesting and culture conditions were the same as above. Cells were seeded to give 2.5~ 105cells/culture.
Sensitivity to endotoxin assay Lyophilized endotoxin (Escherichin coli 026 : B6, Boivin preparation, Difco Laboratories, Detroit, Mich.) was suspended in sterile saline and stored frozen at a concentration of 1 n&ml. Different concentrations of endotoxin were added to 6 and 24 h normal macrophage and J-774 cell cultures in MEM Earle 20% FCS [8]. The cultures were subsequently incubated for 3 days, and the extent of cell death in each culture was recorded. A cell which was rounded up, detached from the surface or showed signs of disintegration was recorded as dead.
and used within a week. Rabbit anti-sheep red cell immunoalobulin M (IeM) and rabbit anti-sheen red cell imm\noglobulin G(IgG) were obtained from Cordis Lab., Miami, Fla. A 5% solution of SICr-labelled E [9] was incubated with 5 pg/ml IgG or 50 pg/ml IgM antibodies for 15 min at 37°C in serum-free MEM Hepes (Gibco) giving E-IgG and E-IgM, respectively. The E-IeM were incubated with a 1 : 20 dilution of C, deficienrfresh mouse serum from AKR mice in Verol r&buffered glucose with Ca2+, Mg*+ and 0.1% gelatin, for 30 min at 37°C giving E-IgMC. The cells were washed and suspended in MEM Hepes.
Phagocytosis of erythrocytes For attachment of E-IgG and E-IgMC to macrophages, 2.5 x lo5 phagocytes and 0.2 ml of 0.05 % red cells were incubated in MEM Hepes for varying lengths of time. Controls containina E and E-IaM did not attach to the macrophages, and-sheep red cells used for absorbing the AKR serum were not attached or internalized. Some of the cultures were washed and kept to calculate the attachment, while others were washed and further incubated for internalization of the attached particles. At the end of the incubation period a 0.14 M solution of NH,Cl was added to lyse attached, noninternalized red cells. The percentage of phagocytes that attached or internalized red cells was calculated. At least 300 cells were scored in each culture. In other cases the attachment and internalization were estimated by counting the S’Cr content of the cultures (Packard, autogamma scintillation spectrometer).
Phase contrast microscopy After washing, the cultures were fixed in 2% &araldehyde in 0.1 M cacodylate buffer @H 7.3) with 0.1 M sucrose. Coverslips were washed and mounted on a drop of Aqua-mount (Edward Gurr Ltd., London). The slides were sealed and examined with a Zeiss phase contrast photomicroscope. Kodak Panatomic-X black and white film was used for photography.
Autoradiography
Scanning electron microscopy (SEM)
An overnight pulse (12 h) of 1 /.&i/ml [3H]thymidine (thymidine(methyl)-3H), Institutt for Atomenergi, Kjeller, Norway) was added to cultures. The cells were washed well and fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer with 0.1 M sucrose (pH 7.3). The coverslips were dipped in a 1 : 3 dilution in water, emulsion in gel form (Ilford Ltd) and dried. The slides were developed after 1 week with D-19 developer (Kodak) and fixed-with Ranid fixer (HVDXI fixer, Ilford). The cells were examined in a phase contrast and light microscope and 300 cells were scored for rH]thymidine incorporation. A cell with 10 grains or more over the nucleus was counted as positive.
The cultures were fixed as described above. The cells were dehydrated in alcohol, transferred to amyl acetate, and critical point dried (Hitachi CPI, Tokyo), in carbon dioxide. The specimens were coated with gold (Polaron SEM Coating Unit E SOOO) and examined with a high resolution Hitachi SEM (HHS/;?R) at 20 kV and a tilt angle of 20”. Pictures were taken on Ilford FP4 film.
Opsonization of sheep red blood cells Sheep red blood cells (E) stored in Alsever’s solution were obtained from National Institute of Health, Oslo, Exp Cell Res~ll5 (1978)
RESULTS Toxic effect of endotoxin on cells in culture In order to establish the toxic effect endotoxin had on the J-774 cells, 24 h old cultures were incubated with varying concen-
Murine monocytic tumor cell line J-774. I a
Table 1. Cytotoxic effect of endotoxin on macrophages and J-774 cells in vitro
55
300
Results expressed as extent of viability after 72 h of incubation with the agent in MEM Earle with 20% FCS; + + +, all cells viable; + + , more than 2/3 of the cells viable; + -, less than half of the cells viable; - , all cells dead
260 220 1 180.
Endotoxin cont. (&ml) Cells
0.1
0.5
1
10
30
140.
Macrophages J-774
+++ +++
+++ +++
+++ ++
+++ +-
++ -
100
y
oi-?---1
0
trations of the agent for 3 days. The cultures were maintained in medium with serum. Damaged J-774 cells resuspend from the surface very easily and the cultures could not be washed or disturbed before the killing effect was evaluated. These two factors made it necessary to use direct morphological methods for the evaluation of cell death. Endotoxin was found to kill a large number of J-774 cells, at concentrations which do not damage normal macrophages (table 1). At concentrations below 1 pglml most of the cells were undamaged. Attempts to activate these cells with endotoxin were therefore carried out at low concentrations (la. 1 pg/ml). Eflect of in vitro cultivation on cell number
As tumor cells have higher and irregular amounts of both DNA and protein/cell than normal macrophages, these parameters were considered inaccurate for the estimation of cell numbers. We have rather used the number of cells per culture, calculated from the cell counts of 10 random microscopic fields. Fig. 1 shows the results for J-774 and normal macrophage cultures under the different conditions. The J-774 cultures maintained in culture
6
24
48
72
b
300 I 260.
I
/”
220iao-
,4o-c 100 1
i 04 0
I 6
24
48
, 72
Fig. 1. Abscissa: time (hours); ordinate: no. of cells/ field. Average cell members per microscopic field calculated from counts of 10 random fields, of in vitro cultures of (a) normal macrophages and (b) J-774 cells. All results varied by about +lO% of counts. (a) n , No endotoxin; 0, 0.1 &nl endotoxm; 0, 1 &nl endotoxin; 0, 10 &ml endotoxin; (b) A, no endotoxin; A, 0.5 &ml endotoxin.
for 72 h in the presence of serum showed a rise in cell number. The presence of 0.5 &ml endotoxin caused a net cell loss. The normal macrophages showed a decrease in cell number after 72 h cultivation. This decrease was independent of the culture conditions used. The differences in cell numbers in the J774 cultures with and without endotoxin Exp Cell Res 115 (1978)
56
Kaplan and M&and
Table 2. Percent of cells incorporating
[3H)-
thymidine At 24 or 72 h, a 12 h pulse was given to the cultures. Results expressed as percent of cells with grains fS.E.M. Time in culture (hours)
Incubation with endotoxin (0.5 dml)
J-774 24 J-774 24 J-774 72 J-774 72 J-774 72
None 6-24 h None 6-72 h 6-48h
% of cells incorporating thymidine 12+2 3L4 2 39+2
were found to be a result of two effects. A drop in the net number of cells was observed during the first 24 h, and an inhibition of DNA synthesis was apparent as long as endotoxin was present with the cells (table 2). The inhibition of thymidine incorporation by endotoxin was completely reversed by the removal of the agent from the 2. SEM micrographs of J-774 cells. The cells medium. Normal macrophages do not in- Fig. were cultured for (a) 6 h in MEM Earle 20% FCS; corporate thymidine in the presence or ab- (b) 72 h in MEM Earle 20% with 0.5 fig/ml endotoxin. x27.50. sence of endotoxin. Effect of in vitro cultivation cell morphology
on
After adherence to the glass the J-774 cells started to spread out very quickly. By 6 h in culture in medium with FCS, many of the cells had aquired an elongated bipolar shape and had flattened out on the glass. The surface of the cells had a very irregular shape. Many ridges and villi were observed, much longer and more pronounced than those found in normal macrophages (fig. 2a). Some ruffling was observed. With time in culture the cells flattened and spread much more but never lost the numerous ridges and villi. Endotoxin treatment of the cells in culture produced larger and even more spread out cells. The amount of ruffling Exp Cell Res 115 (1978)
went up and appeared to be the same at 0.1 pg/ml endotoxin as at 0.5 or 1 pg/ml (fig. 2b). The density of the cultures had some effect on the morphology. When the cells were seeded at a low density they were bigger, more spread out and with more ruffling on their surface. Dense cultures gave more rounded cells with more villi and ridges. Phagocytosis
Attachment to the Fc receptor of J-774 was found to be very effkient in all cultures tested, as can be seen from table 3 A. The percent of cells which internalized the attached particles went up from 6 to 24 h in
Murine monocytic tumor cell line J-774. I
57
Table 3. Phagocytosis mediated by the Fc and C3 receptors of (A) J-774 cells; and (B) mouse peritoneal macrophages Opsonized particles (0.1 ml of 0.5%) were added to 0.25~ IV cells for 1 h at 4°C for attachment and 1.5 h at 37°C for internalization. The results are expressed as percent of cells that attached or internalized particles (calculated by counting 300 cells/culture in the phase microscope) + S.E.M. J-774
CS
Fc
A
Culture conditions
6h 24 h 4 days 4d-ays
MEM + 20 % FCS MEM + 20 % FCS MEM + 20 % FCS MEM+20%FCS+ 0.1 pg/ml endotoxin 4 days MEM + 20 % FCS + 0.5 /.&nl endotoxin. 4 days MEM + 20 % FCS + 1 pg/ml endotoxin 4 days MEM + 20 % FCS + 10 pg/ml endotoxin
Attachment, % of cells
Intemalization, % of cells
Attachment Internalization ~ ~ % of Average no. % of Average no. cells particles cells particles
91f5 98 95+4
59f9 87+4 82+3
9Of6 97+3 95fl
9.5k2.7 11.7f1.7 9.5+ 1.0
40+ 12 47+12 64f7
3.2kO.6 3.3f0.4 3.7f0.3
99
89fl
98
>20
67+7
4.4k0.6
99
9Of2
98
>20
65f5
6.3f0.5
99
89+ 1
98
>20
56f 15
6.0f0.7
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
Macrophages Fc B 6h 24h 4 days 4 days
Culture conditions
MEM + 20 % FCS MEM + 20 % FCS MEM + 20% FCS MEM + 20 % FCS + 0.1 &ml endotoxin 4 days MEM+ZO% FCS + 0.5 pg/ml endotoxin 4 days MEM + 20% FCS + 1 &ml endotoxin 4 days MEM + 20 % FCS + 10 pg/ml endotoxin
G
Attachment, % of cells
Internalization, % of cells
Attachment, % of cells
Internalization, % of cells
N.T. 97kO.4 98f0.3
sN;+T;,3 9520:4
N.T. 97+0.9 98kO.3
N.T. 20+ 1.7 22k1.9
98kO.4
97f0.3
98kO.3
42k3.0
N.T.
N.T.
N.T.
N.T.
99f0.3
99
99
51+20
99
99f0.3
99
80f2.1
culture from 59 to 87 % respectively and remained at the same level after 4 days regardless of the presence of endotoxin in the medium. In the case of the C3 receptor a different pattern was found. The percentage of cells which attached particles was very high in all cultures, but the average number of parti-
cles attached to each cell was dependent on the culture conditions. The presence of endotoxin in the medium caused a significant rise in the efficiency of attachment, while the time in culture had very little effect (table 3 A). Endotoxin-treated and nontreated cells not only attached but also internalized particles by the Cbreceptor (fig. Exp CeNRes 115(1978)
58
Kaplan and M&and a
Fc Attachment
b
C3 Attachment
Fc Internalization
d
C3 Internalization
60
b
0
30
60
'90
120
Fig. 3. Abscissa: time (min); or&are: %. (a) Attachment 25°C and (b) internalization 37°C of l , E-IgG and A, E-IgMC by J-774 cells maintained in culture for 3 days in the presence of 0.5 pg/ml endotoxin. Results expressed as percent of maximum Wr internalized + S.D.
Fig. 4. Ordinate: %. The effect of temperature on attachment and internalization of E-I& (Fc) and E-IgMC (Cd by J-774 cells maintained in culture 3 days with 0.5 pg/ml endotoxin. Results expressed as percent of attachment or internalization at 37°C + SD.
Sa). The percent of cells which showed internalization of the attached particles went up with time while the average number of internalized particles seemed to be independent of the time in culture but dependent on the concentration of endotoxin in the medium. Normal peritoneal macrophages were found to attach opsonized particles at about the same efficiency under all culture conditions, both in the Fc and in the C3 (table 3B). Unstimulated macrophages gave no significant C3 mediated internalization. The 20 and 22 % of cells that did internalize particles (table 3B) only internalize an average of l-2 particles/cell, which was less than 5 % of the attached particles. A triggering of efficient internalization of the parti-
cles was observed in the presence of endotoxin. Medium supplemented with FCS and 10 pg/ml gave macrophage cultures where 80% of the cells were found to internalize the opsonized particles attached to the C3 receptor (45% of total 51Cr-labelled particles [2]). This was therefore the concentration of endotoxin we used routinely for macrophage activation [2].
Exp
Cell
Res
115 (1978)
The function of the Fc and the C3 receptors All observations were made on 3 day old J-774 cells cultured in the presence or absence of endotoxin. Essentially the same results were obtained regardless of the presence of endotoxin in the medium (unless otherwise mentioned). The results shown
Murine monocytic tumor cell line J-774. I
59
Fig. 5. Phase and SEM micrographs of phagocytosis by J-774 cells maintained in culture for 3 days. (a) Phase contrast micrograph of E-IgMC internalized via the C, receator of unstimulated 72 h old J-774 cells in culkre; 6) SEM of E-IgMC (E) being internalized by a J-774 cell (M), via the C3 receptors (30 min at 25°C). The particles are sinking down into the cell cytoplasm. A large amount of ruffling is observed
adjacent to the site of ingestion, but not necessarily in contact with the particle being ingested @rows); (c) SEM of E-IgG internalized via the Fc receptors, The particles are being internalized by a creeping up of the phagocyte membrane over them (30 min at 37°C). Close contact between the membrane and the particles is observed (arrows). (a) x665; (b) x 11000; (c) x9000.
here are for 3 day old cells cultured in the presence of 0.5 pg/ml endotoxin. When the kinetics of attachment and internalization by the Fc and the C3 receptors were looked at an obvious difference between the two receptors is observed. Attachment to the Fc receptor at all temperatures is very fast. At room temperature a high percentage of the particles attach within the first 30 min (fig. 3~) and thereafter a levelling out of the rate of attachment is observed. The limiting factor seems to be the surface area of the cells. Attachment to the C, receptor at 25°C is linear with time and at about 60 min a large percent of the attachment has taken place. The sedimentation rate of the particles is
not a rate limiting factor as most of the particles have sedimented by 10-15 min. Attachment levels out when the entire cell surface is covered by particles. This is not the case with cells cultured without endotoxin, where attachment seems to be less efficient (table 3A). Internalization of the attached particles at 37°C follows very similar kinetics. As can be seen from fig. 3b, by 30 min internalization mediated by the Fc receptor is close to maximal. In the case of the C, receptor it takes over 60 min for maximal intemalization to take place. The number of attached particles is not the rate limiting factor here as only about l&20% of the attached particles are internalized by the cells. Exp Cell Res 115 (1978)
60
Kaplan and Msrland
As mentioned before attachment to the Fc receptor is very efficient at all temperatures and is not affected much by a drop in temperature from 37 to 4°C (fig. 4a). On the other hand attachment to the C, receptor goes down significantly as the temperature goes down (fig. 4b). Internalization of the attached particles is different. Here the Fc receptor shows a high sensitivity to a drop in temperature from 37 to 25°C (fig. 4c) while the C3 receptor is not much affected (fig. 4d). Internalization of particles attached to the C, receptor at 25°C is almost as effective as at 37°C. The morphology of attachment and internalization Morphological observations on the attachment of opsonized particles to the Fc and the C3 receptors shows random attachment over the cell surface of the cell. Particles attached to the Fc receptor cover the entire surface of cultures maintained with or without endotoxin. As the cultures in the presence of endotoxin were less dense and therefore the cells were larger the attachment per cell was higher here. Attachment to the C3 receptor is random too. In cells cultured with endotoxin most of the cell surface is covered, less. so in cells cultured without endotoxin. A difference is observed at the extreme periphery of the cells and on long thin cell extension where attachment to the Fc receptor is much more common than that to the C3 receptor. This is the case with internalization of the particles too. The mode of internalization of particles attached to the two types of receptors is different and resembles that reported for Kupffer cells [lo] and activated macrophages [ 111.In the case of the Fc the membrane can be seen to creep up over the Exp Cell Res I IS (1978)
particles (fig. 5b). Particles attached to the C3 receptor sink directly into the cell cytoplasm, even at 25°C (fig. 5 c). DISCUSSION Attempts to activate the J-774 cells in vitro with endotoxin gave rise to some quantitative rather than qualitative changes. Untreated cells seemed to resemble endotoxin activated macrophages rather than normal peritoneal macrophages. A striking amount of ruffling was observed on the surface of the cells cultured for 34 days in the presence or the absence of endotoxin. Ruffling has been found to be a morphological indication of macrophage activation [l-2]. The presence of endotoxin gave a somewhat larger amount of ruffling. This could be as a result of the cytostatic effect on the cells which stopped the density of the cultures from going up with time, and gave larger, more spread out cells. The phagocytic properties of the cells showed some resemblance to those of activated macrophages too. Unstimulated J774 cells internalized particles attached to the C3 receptor. This is not the case with normal macrophages where internalization mediated by the C3 receptor is dependent on cell activation [12]. In the presence of endotoxin attachment and internalization were more efficient but no difference in the temperature sensitivity and kinetics was found whether endotoxin was present in the incubation medium or not. The J-774 system was therefore considered to be useful for a comparative study of the Fc and the C3 receptor functions. Both attachment and internalization mediated by the two receptor types could be studied under normal unstimulated culture conditions. Differences between the two phagocytic systems could not be attributed to the effect endo-
Murine monocytic tumor cell line J-774. I
toxin had on the cells but rather seen as an inherent difference. Griffin et al. [13] reported on a requirement for circumferential attachment of particle-bound ligands to the receptors on the macrophage plasma membrane. Michl et al. [1415] showed that the two immunological receptors are inhibited by 2-deoxyglucose in a similar manner. However, other evidence has been accumulating in favour of a different role and function for the two receptors. The Fc and C3 receptors,of mouse macrophages have been shown to be not only separate molecules with a distinct specificity [l&18] but to function independently of one another during attachment and internalization of opsonized particles [ 19201. We have previously presented observations on the morphology of phagocytosis by the two receptors that point to a different mode of internalization of the attached particles in the two systems [ 111.The observations in this paper provide more evidence in favour of a difference in the function of the two receptors. The difference in sensitivity to temperature during attachment and even more so, during internalization could indicate a different dependence on the fluidity of the plasma membrane in the ingestion phase mediated by the two receptors. Internalization via the Fc receptor drops proportionally to the drop in temperature. In the case of the C3 receptor ingestion is not affected much by lowering the temperature to 25°C or even 20°C. A sudden drop in activity was observed at temperatures of 17-20°C (preliminary results, unpublished). The differences in the mode of internalization by the two types of receptors were the same as those reported for activated macrophages [ll] and Kupffer cells [lo]. Ingestion mediated by the Fc receptors in-
61
volved creeping up of the cell membrane over the attached particles in circumferential attachment. The C&-mediatedingestion seemed to involve a sinking down of the particles directly into the cell cytoplasm. Creeping up of the phagocyte membrane over the attached particle did not seem to be essential for ingestion. We are deeply indebted to Professor Rolf Seljelid for critical reading and stimulating discussion of the manuscript. We are gratelul to Dr P. Ralph for making this work possible by. providing us with the tumor _ ceil line. This investigation was supported in part by a grant from Landsforeningen mot Kreft, Norway.
REFERENCES 1.
2. 3. 4. 5. 6. 7. 8. 9.
Cohn, Z A &Benson, B, J exp med 121(1%5) 153. M&and, B t Kaplan, G, Exp cell res 108 (1977) 279. Cohn, Z A, Hirsch, J G & Fedorko, M E, J exp med 123(1966) 747. Gordon, S, Unkeless, J C & Cohn, Z A, J exp med 140 (1974) 995. Alexander, P & Evans, R, Nature new biol 232 (1971) 76. Ralph, P, Richard, J & Cohn, M, J immunol 114 (1975) 898. Ralph, P & Nakoinz, I, Cancer res 37 (1977) 546. Wahl, L M, Wahl, S M, Mergenhagen, S E & Martin, G R, Proc natlacad sci US 71(1974) 3598. E71som, H L SelJehd, R, J exp med 137 (1973)
10. Mu&he-Kaas, A, Kaplan, G & Seljelid, R, Exp cell res 103 (1976) 201. 11. Kaplan, G, Stand j immunol6 (1977). In press. 12. Bianco, C, GriIEn, F M & Silverstein, S C, J exp med 141(1975) 1278. 13. GrilBn, F M, Griflin, J A, Leider, J E & Silverstein, SC, J exp med 142 (1975) 1263. 14. Michl, J, Ohlbaum, D J & Silverstein, S C, J exp med 144 (1976) 1465. - J exp med 144 (1976) 1484. :2: Lay, W H & Nussenzweig, V, J exp med 128 (1968) 991. 17. Holland, P, Holland, N H & Cohn, Z A, J exp med 135 (1972) 458. 18. Mantovani, B, Rabinovitch, M & Nussenzweig, V, J exp med 135(1972) 780. 19. Griffin, F M & Silverstein, S C, J exp med 139 (1974) 323. 20. Griffin, F M, Bianco, C & Silverstein, S C, J exp med 141(1975) 1269. Received June 29, 1977 Revised version received November 24, 1977 Accepted February 24, 1978 Exp Cell Res 115 (1978)
Experimental
PROPERTIES
Cell Research 115 (1978) 53-61
OF A MURINE CELL
LINE
I. Morphology G. KAPLAN
MONOCYTIC
TUMOR
J-774 IN VITRO and Endocytosis and B. M@RLAND
institute of Medical Biology, University of Tromsti, Tromsr, Norway
SUMMARY A murine monocytic tumor cell line J-774 was maintained in culture in the presence or absence of endotoxin, in an attempt to induce differentiation similar to that found in activated peritoneal macrophages. The morphology and Fc and Cs receptor functions of attachment and ingestion were compared in the treated and untreated cultures. J-774 cells maintained in culture for 72 h seemed to resemble endotoxin-activated macrophages rather than normal peritoneal macrophages. A striking amount of ruffhng was observed on the surface of the cells cultured for 34 days both in the presence and in the absence of endotoxin. As compared to the peritoneal macrophage where particles attached to the C, receptors are not ingested unless the cells are activated, J-744 cells attached and ingested particles via the C, receptor even without stimulation. The presence of endotoxin in the culture medium of these cells gave rise to more efficient phagocytosis but did not effect the temperature sensitivity of the phagocytic receptors. Both in treated and untreated cultures attachment to the Fc receptor was less dependent on the temperature than that of the C3 receptor while ingestion was more sensitive in the Fc receptor as compared with the C, receptor.
Normal mouse mononuclear phagocytes ture conditions was compared with that of have been shown to differentiate during in- murine peritoneal macrophages. Endotoxin has been shown to effectively flammation as a result of stimulation by factors available in the extravascular compart- inhibit the proliferation of the J-774 cells in ment at the inflammatory site [ 11. A similar vitro [7]. It was of interest to establish differentiation of murine peritoneal macro- whether endotoxin at concentrations which phages can be obtained both in vivo and in are cytostatic but not cytotoxic could induce in these cells any differentiated propvitro by exposure of the cells to endotoxin [2], and other chemical compounds. This erties similar to those found in the actidifferentiation is expressed by changes in vated peritoneal macrophages. Phase contrast and scanning electron mimorphology, phagocytic capacity, cytotoxicity of the cells, and increase in their croscopy (SEM) were carried out on the cells. The Fc and C, receptor functions of lysosomal enzyme activity [l-5]. A murine reticulum cell sarcoma (J-774) attachment and internalization of opsonized has been described, which in ascites form particles were investigated. has the macrophage properties of adherMATERIALS AND METHODS ence, morphology, receptors for immunoglobulins and complement, and antibody- Mouse cell lines dependent lysis of target cells [6]. The J-774 tumor was obtained from Dr P. Ralph from The J-774 cells’ response to in vitro cul- the Sloan Kettering Institute for Cancer Research, Exp Ceil Res I15 (1978)
54
Kaplan and M&and
New York. The ascites tumor was passaged intraperitoneally in BALB/c mice (0.2-0.4 ml). Ascites cells with diameter about 30 urn were reckoned as tumor cells [6]. They constituted about 90% of the cellular elements in the J-774ascites, the remaining 5-10% were smaller cells with a diameter about lo-pm. Experiments were performed with ascites J-774 A cells, showing greater than 90% viability by trypan blue exclusion.
Macrophage culture conditions Mouse oeritoneal macrouhages were obtained from outbred*NMRI mice. Hat!ves&g and culture methods were essentially those of Cohn & Benson [l]. Cells were cultured in MEM Earle (G&co, Grand Island, N.Y.) with 20% heat-inactivated fetal calf serum (FCS) (Gibco), 100 W/ml of penicillin, and 100 wg/ ml streptomycin, The cells were seeded in Linbro plates (Linbro them. Co., New Haven) with glass coverslips (14 mm 0), in 5% CO* in air, at 37°C. After 2 h the cultures were washed and new medium added. Cells were seeded to give 2.5~ 105cells/culture.
J-774 culture conditions Harvesting and culture conditions were the same as above. Cells were seeded to give 2.5~ 105cells/culture.
Sensitivity to endotoxin assay Lyophilized endotoxin (Escherichin coli 026 : B6, Boivin preparation, Difco Laboratories, Detroit, Mich.) was suspended in sterile saline and stored frozen at a concentration of 1 n&ml. Different concentrations of endotoxin were added to 6 and 24 h normal macrophage and J-774 cell cultures in MEM Earle 20% FCS [8]. The cultures were subsequently incubated for 3 days, and the extent of cell death in each culture was recorded. A cell which was rounded up, detached from the surface or showed signs of disintegration was recorded as dead.
and used within a week. Rabbit anti-sheep red cell immunoalobulin M (IeM) and rabbit anti-sheen red cell imm\noglobulin G(IgG) were obtained from Cordis Lab., Miami, Fla. A 5% solution of SICr-labelled E [9] was incubated with 5 pg/ml IgG or 50 pg/ml IgM antibodies for 15 min at 37°C in serum-free MEM Hepes (Gibco) giving E-IgG and E-IgM, respectively. The E-IeM were incubated with a 1 : 20 dilution of C, deficienrfresh mouse serum from AKR mice in Verol r&buffered glucose with Ca2+, Mg*+ and 0.1% gelatin, for 30 min at 37°C giving E-IgMC. The cells were washed and suspended in MEM Hepes.
Phagocytosis of erythrocytes For attachment of E-IgG and E-IgMC to macrophages, 2.5 x lo5 phagocytes and 0.2 ml of 0.05 % red cells were incubated in MEM Hepes for varying lengths of time. Controls containina E and E-IaM did not attach to the macrophages, and-sheep red cells used for absorbing the AKR serum were not attached or internalized. Some of the cultures were washed and kept to calculate the attachment, while others were washed and further incubated for internalization of the attached particles. At the end of the incubation period a 0.14 M solution of NH,Cl was added to lyse attached, noninternalized red cells. The percentage of phagocytes that attached or internalized red cells was calculated. At least 300 cells were scored in each culture. In other cases the attachment and internalization were estimated by counting the S’Cr content of the cultures (Packard, autogamma scintillation spectrometer).
Phase contrast microscopy After washing, the cultures were fixed in 2% &araldehyde in 0.1 M cacodylate buffer @H 7.3) with 0.1 M sucrose. Coverslips were washed and mounted on a drop of Aqua-mount (Edward Gurr Ltd., London). The slides were sealed and examined with a Zeiss phase contrast photomicroscope. Kodak Panatomic-X black and white film was used for photography.
Autoradiography
Scanning electron microscopy (SEM)
An overnight pulse (12 h) of 1 /.&i/ml [3H]thymidine (thymidine(methyl)-3H), Institutt for Atomenergi, Kjeller, Norway) was added to cultures. The cells were washed well and fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer with 0.1 M sucrose (pH 7.3). The coverslips were dipped in a 1 : 3 dilution in water, emulsion in gel form (Ilford Ltd) and dried. The slides were developed after 1 week with D-19 developer (Kodak) and fixed-with Ranid fixer (HVDXI fixer, Ilford). The cells were examined in a phase contrast and light microscope and 300 cells were scored for rH]thymidine incorporation. A cell with 10 grains or more over the nucleus was counted as positive.
The cultures were fixed as described above. The cells were dehydrated in alcohol, transferred to amyl acetate, and critical point dried (Hitachi CPI, Tokyo), in carbon dioxide. The specimens were coated with gold (Polaron SEM Coating Unit E SOOO) and examined with a high resolution Hitachi SEM (HHS/;?R) at 20 kV and a tilt angle of 20”. Pictures were taken on Ilford FP4 film.
Opsonization of sheep red blood cells Sheep red blood cells (E) stored in Alsever’s solution were obtained from National Institute of Health, Oslo, Exp Cell Res~ll5 (1978)
RESULTS Toxic effect of endotoxin on cells in culture In order to establish the toxic effect endotoxin had on the J-774 cells, 24 h old cultures were incubated with varying concen-
Murine monocytic tumor cell line J-774. I a
Table 1. Cytotoxic effect of endotoxin on macrophages and J-774 cells in vitro
55
300
Results expressed as extent of viability after 72 h of incubation with the agent in MEM Earle with 20% FCS; + + +, all cells viable; + + , more than 2/3 of the cells viable; + -, less than half of the cells viable; - , all cells dead
260 220 1 180.
Endotoxin cont. (&ml) Cells
0.1
0.5
1
10
30
140.
Macrophages J-774
+++ +++
+++ +++
+++ ++
+++ +-
++ -
100
y
oi-?---1
0
trations of the agent for 3 days. The cultures were maintained in medium with serum. Damaged J-774 cells resuspend from the surface very easily and the cultures could not be washed or disturbed before the killing effect was evaluated. These two factors made it necessary to use direct morphological methods for the evaluation of cell death. Endotoxin was found to kill a large number of J-774 cells, at concentrations which do not damage normal macrophages (table 1). At concentrations below 1 pglml most of the cells were undamaged. Attempts to activate these cells with endotoxin were therefore carried out at low concentrations (la. 1 pg/ml). Eflect of in vitro cultivation on cell number
As tumor cells have higher and irregular amounts of both DNA and protein/cell than normal macrophages, these parameters were considered inaccurate for the estimation of cell numbers. We have rather used the number of cells per culture, calculated from the cell counts of 10 random microscopic fields. Fig. 1 shows the results for J-774 and normal macrophage cultures under the different conditions. The J-774 cultures maintained in culture
6
24
48
72
b
300 I 260.
I
/”
220iao-
,4o-c 100 1
i 04 0
I 6
24
48
, 72
Fig. 1. Abscissa: time (hours); ordinate: no. of cells/ field. Average cell members per microscopic field calculated from counts of 10 random fields, of in vitro cultures of (a) normal macrophages and (b) J-774 cells. All results varied by about +lO% of counts. (a) n , No endotoxin; 0, 0.1 &nl endotoxm; 0, 1 &nl endotoxin; 0, 10 &ml endotoxin; (b) A, no endotoxin; A, 0.5 &ml endotoxin.
for 72 h in the presence of serum showed a rise in cell number. The presence of 0.5 &ml endotoxin caused a net cell loss. The normal macrophages showed a decrease in cell number after 72 h cultivation. This decrease was independent of the culture conditions used. The differences in cell numbers in the J774 cultures with and without endotoxin Exp Cell Res 115 (1978)
56
Kaplan and M&and
Table 2. Percent of cells incorporating
[3H)-
thymidine At 24 or 72 h, a 12 h pulse was given to the cultures. Results expressed as percent of cells with grains fS.E.M. Time in culture (hours)
Incubation with endotoxin (0.5 dml)
J-774 24 J-774 24 J-774 72 J-774 72 J-774 72
None 6-24 h None 6-72 h 6-48h
% of cells incorporating thymidine 12+2 3L4 2 39+2
were found to be a result of two effects. A drop in the net number of cells was observed during the first 24 h, and an inhibition of DNA synthesis was apparent as long as endotoxin was present with the cells (table 2). The inhibition of thymidine incorporation by endotoxin was completely reversed by the removal of the agent from the 2. SEM micrographs of J-774 cells. The cells medium. Normal macrophages do not in- Fig. were cultured for (a) 6 h in MEM Earle 20% FCS; corporate thymidine in the presence or ab- (b) 72 h in MEM Earle 20% with 0.5 fig/ml endotoxin. x27.50. sence of endotoxin. Effect of in vitro cultivation cell morphology
on
After adherence to the glass the J-774 cells started to spread out very quickly. By 6 h in culture in medium with FCS, many of the cells had aquired an elongated bipolar shape and had flattened out on the glass. The surface of the cells had a very irregular shape. Many ridges and villi were observed, much longer and more pronounced than those found in normal macrophages (fig. 2a). Some ruffling was observed. With time in culture the cells flattened and spread much more but never lost the numerous ridges and villi. Endotoxin treatment of the cells in culture produced larger and even more spread out cells. The amount of ruffling Exp Cell Res 115 (1978)
went up and appeared to be the same at 0.1 pg/ml endotoxin as at 0.5 or 1 pg/ml (fig. 2b). The density of the cultures had some effect on the morphology. When the cells were seeded at a low density they were bigger, more spread out and with more ruffling on their surface. Dense cultures gave more rounded cells with more villi and ridges. Phagocytosis
Attachment to the Fc receptor of J-774 was found to be very effkient in all cultures tested, as can be seen from table 3 A. The percent of cells which internalized the attached particles went up from 6 to 24 h in
Murine monocytic tumor cell line J-774. I
57
Table 3. Phagocytosis mediated by the Fc and C3 receptors of (A) J-774 cells; and (B) mouse peritoneal macrophages Opsonized particles (0.1 ml of 0.5%) were added to 0.25~ IV cells for 1 h at 4°C for attachment and 1.5 h at 37°C for internalization. The results are expressed as percent of cells that attached or internalized particles (calculated by counting 300 cells/culture in the phase microscope) + S.E.M. J-774
CS
Fc
A
Culture conditions
6h 24 h 4 days 4d-ays
MEM + 20 % FCS MEM + 20 % FCS MEM + 20 % FCS MEM+20%FCS+ 0.1 pg/ml endotoxin 4 days MEM + 20 % FCS + 0.5 /.&nl endotoxin. 4 days MEM + 20 % FCS + 1 pg/ml endotoxin 4 days MEM + 20 % FCS + 10 pg/ml endotoxin
Attachment, % of cells
Intemalization, % of cells
Attachment Internalization ~ ~ % of Average no. % of Average no. cells particles cells particles
91f5 98 95+4
59f9 87+4 82+3
9Of6 97+3 95fl
9.5k2.7 11.7f1.7 9.5+ 1.0
40+ 12 47+12 64f7
3.2kO.6 3.3f0.4 3.7f0.3
99
89fl
98
>20
67+7
4.4k0.6
99
9Of2
98
>20
65f5
6.3f0.5
99
89+ 1
98
>20
56f 15
6.0f0.7
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
Macrophages Fc B 6h 24h 4 days 4 days
Culture conditions
MEM + 20 % FCS MEM + 20 % FCS MEM + 20% FCS MEM + 20 % FCS + 0.1 &ml endotoxin 4 days MEM+ZO% FCS + 0.5 pg/ml endotoxin 4 days MEM + 20% FCS + 1 &ml endotoxin 4 days MEM + 20 % FCS + 10 pg/ml endotoxin
G
Attachment, % of cells
Internalization, % of cells
Attachment, % of cells
Internalization, % of cells
N.T. 97kO.4 98f0.3
sN;+T;,3 9520:4
N.T. 97+0.9 98kO.3
N.T. 20+ 1.7 22k1.9
98kO.4
97f0.3
98kO.3
42k3.0
N.T.
N.T.
N.T.
N.T.
99f0.3
99
99
51+20
99
99f0.3
99
80f2.1
culture from 59 to 87 % respectively and remained at the same level after 4 days regardless of the presence of endotoxin in the medium. In the case of the C3 receptor a different pattern was found. The percentage of cells which attached particles was very high in all cultures, but the average number of parti-
cles attached to each cell was dependent on the culture conditions. The presence of endotoxin in the medium caused a significant rise in the efficiency of attachment, while the time in culture had very little effect (table 3 A). Endotoxin-treated and nontreated cells not only attached but also internalized particles by the Cbreceptor (fig. Exp CeNRes 115(1978)
58
Kaplan and M&and a
Fc Attachment
b
C3 Attachment
Fc Internalization
d
C3 Internalization
60
b
0
30
60
'90
120
Fig. 3. Abscissa: time (min); or&are: %. (a) Attachment 25°C and (b) internalization 37°C of l , E-IgG and A, E-IgMC by J-774 cells maintained in culture for 3 days in the presence of 0.5 pg/ml endotoxin. Results expressed as percent of maximum Wr internalized + S.D.
Fig. 4. Ordinate: %. The effect of temperature on attachment and internalization of E-I& (Fc) and E-IgMC (Cd by J-774 cells maintained in culture 3 days with 0.5 pg/ml endotoxin. Results expressed as percent of attachment or internalization at 37°C + SD.
Sa). The percent of cells which showed internalization of the attached particles went up with time while the average number of internalized particles seemed to be independent of the time in culture but dependent on the concentration of endotoxin in the medium. Normal peritoneal macrophages were found to attach opsonized particles at about the same efficiency under all culture conditions, both in the Fc and in the C3 (table 3B). Unstimulated macrophages gave no significant C3 mediated internalization. The 20 and 22 % of cells that did internalize particles (table 3B) only internalize an average of l-2 particles/cell, which was less than 5 % of the attached particles. A triggering of efficient internalization of the parti-
cles was observed in the presence of endotoxin. Medium supplemented with FCS and 10 pg/ml gave macrophage cultures where 80% of the cells were found to internalize the opsonized particles attached to the C3 receptor (45% of total 51Cr-labelled particles [2]). This was therefore the concentration of endotoxin we used routinely for macrophage activation [2].
Exp
Cell
Res
115 (1978)
The function of the Fc and the C3 receptors All observations were made on 3 day old J-774 cells cultured in the presence or absence of endotoxin. Essentially the same results were obtained regardless of the presence of endotoxin in the medium (unless otherwise mentioned). The results shown
Murine monocytic tumor cell line J-774. I
59
Fig. 5. Phase and SEM micrographs of phagocytosis by J-774 cells maintained in culture for 3 days. (a) Phase contrast micrograph of E-IgMC internalized via the C, receator of unstimulated 72 h old J-774 cells in culkre; 6) SEM of E-IgMC (E) being internalized by a J-774 cell (M), via the C3 receptors (30 min at 25°C). The particles are sinking down into the cell cytoplasm. A large amount of ruffling is observed
adjacent to the site of ingestion, but not necessarily in contact with the particle being ingested @rows); (c) SEM of E-IgG internalized via the Fc receptors, The particles are being internalized by a creeping up of the phagocyte membrane over them (30 min at 37°C). Close contact between the membrane and the particles is observed (arrows). (a) x665; (b) x 11000; (c) x9000.
here are for 3 day old cells cultured in the presence of 0.5 pg/ml endotoxin. When the kinetics of attachment and internalization by the Fc and the C3 receptors were looked at an obvious difference between the two receptors is observed. Attachment to the Fc receptor at all temperatures is very fast. At room temperature a high percentage of the particles attach within the first 30 min (fig. 3~) and thereafter a levelling out of the rate of attachment is observed. The limiting factor seems to be the surface area of the cells. Attachment to the C, receptor at 25°C is linear with time and at about 60 min a large percent of the attachment has taken place. The sedimentation rate of the particles is
not a rate limiting factor as most of the particles have sedimented by 10-15 min. Attachment levels out when the entire cell surface is covered by particles. This is not the case with cells cultured without endotoxin, where attachment seems to be less efficient (table 3A). Internalization of the attached particles at 37°C follows very similar kinetics. As can be seen from fig. 3b, by 30 min internalization mediated by the Fc receptor is close to maximal. In the case of the C, receptor it takes over 60 min for maximal intemalization to take place. The number of attached particles is not the rate limiting factor here as only about l&20% of the attached particles are internalized by the cells. Exp Cell Res 115 (1978)
60
Kaplan and Msrland
As mentioned before attachment to the Fc receptor is very efficient at all temperatures and is not affected much by a drop in temperature from 37 to 4°C (fig. 4a). On the other hand attachment to the C, receptor goes down significantly as the temperature goes down (fig. 4b). Internalization of the attached particles is different. Here the Fc receptor shows a high sensitivity to a drop in temperature from 37 to 25°C (fig. 4c) while the C3 receptor is not much affected (fig. 4d). Internalization of particles attached to the C, receptor at 25°C is almost as effective as at 37°C. The morphology of attachment and internalization Morphological observations on the attachment of opsonized particles to the Fc and the C3 receptors shows random attachment over the cell surface of the cell. Particles attached to the Fc receptor cover the entire surface of cultures maintained with or without endotoxin. As the cultures in the presence of endotoxin were less dense and therefore the cells were larger the attachment per cell was higher here. Attachment to the C3 receptor is random too. In cells cultured with endotoxin most of the cell surface is covered, less. so in cells cultured without endotoxin. A difference is observed at the extreme periphery of the cells and on long thin cell extension where attachment to the Fc receptor is much more common than that to the C3 receptor. This is the case with internalization of the particles too. The mode of internalization of particles attached to the two types of receptors is different and resembles that reported for Kupffer cells [lo] and activated macrophages [ 111.In the case of the Fc the membrane can be seen to creep up over the Exp Cell Res I IS (1978)
particles (fig. 5b). Particles attached to the C3 receptor sink directly into the cell cytoplasm, even at 25°C (fig. 5 c). DISCUSSION Attempts to activate the J-774 cells in vitro with endotoxin gave rise to some quantitative rather than qualitative changes. Untreated cells seemed to resemble endotoxin activated macrophages rather than normal peritoneal macrophages. A striking amount of ruffling was observed on the surface of the cells cultured for 34 days in the presence or the absence of endotoxin. Ruffling has been found to be a morphological indication of macrophage activation [l-2]. The presence of endotoxin gave a somewhat larger amount of ruffling. This could be as a result of the cytostatic effect on the cells which stopped the density of the cultures from going up with time, and gave larger, more spread out cells. The phagocytic properties of the cells showed some resemblance to those of activated macrophages too. Unstimulated J774 cells internalized particles attached to the C3 receptor. This is not the case with normal macrophages where internalization mediated by the C3 receptor is dependent on cell activation [12]. In the presence of endotoxin attachment and internalization were more efficient but no difference in the temperature sensitivity and kinetics was found whether endotoxin was present in the incubation medium or not. The J-774 system was therefore considered to be useful for a comparative study of the Fc and the C3 receptor functions. Both attachment and internalization mediated by the two receptor types could be studied under normal unstimulated culture conditions. Differences between the two phagocytic systems could not be attributed to the effect endo-
Murine monocytic tumor cell line J-774. I
toxin had on the cells but rather seen as an inherent difference. Griffin et al. [13] reported on a requirement for circumferential attachment of particle-bound ligands to the receptors on the macrophage plasma membrane. Michl et al. [1415] showed that the two immunological receptors are inhibited by 2-deoxyglucose in a similar manner. However, other evidence has been accumulating in favour of a different role and function for the two receptors. The Fc and C3 receptors,of mouse macrophages have been shown to be not only separate molecules with a distinct specificity [l&18] but to function independently of one another during attachment and internalization of opsonized particles [ 19201. We have previously presented observations on the morphology of phagocytosis by the two receptors that point to a different mode of internalization of the attached particles in the two systems [ 111.The observations in this paper provide more evidence in favour of a difference in the function of the two receptors. The difference in sensitivity to temperature during attachment and even more so, during internalization could indicate a different dependence on the fluidity of the plasma membrane in the ingestion phase mediated by the two receptors. Internalization via the Fc receptor drops proportionally to the drop in temperature. In the case of the C3 receptor ingestion is not affected much by lowering the temperature to 25°C or even 20°C. A sudden drop in activity was observed at temperatures of 17-20°C (preliminary results, unpublished). The differences in the mode of internalization by the two types of receptors were the same as those reported for activated macrophages [ll] and Kupffer cells [lo]. Ingestion mediated by the Fc receptors in-
61
volved creeping up of the cell membrane over the attached particles in circumferential attachment. The C&-mediatedingestion seemed to involve a sinking down of the particles directly into the cell cytoplasm. Creeping up of the phagocyte membrane over the attached particle did not seem to be essential for ingestion. We are deeply indebted to Professor Rolf Seljelid for critical reading and stimulating discussion of the manuscript. We are gratelul to Dr P. Ralph for making this work possible by. providing us with the tumor _ ceil line. This investigation was supported in part by a grant from Landsforeningen mot Kreft, Norway.
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2. 3. 4. 5. 6. 7. 8. 9.
Cohn, Z A &Benson, B, J exp med 121(1%5) 153. M&and, B t Kaplan, G, Exp cell res 108 (1977) 279. Cohn, Z A, Hirsch, J G & Fedorko, M E, J exp med 123(1966) 747. Gordon, S, Unkeless, J C & Cohn, Z A, J exp med 140 (1974) 995. Alexander, P & Evans, R, Nature new biol 232 (1971) 76. Ralph, P, Richard, J & Cohn, M, J immunol 114 (1975) 898. Ralph, P & Nakoinz, I, Cancer res 37 (1977) 546. Wahl, L M, Wahl, S M, Mergenhagen, S E & Martin, G R, Proc natlacad sci US 71(1974) 3598. E71som, H L SelJehd, R, J exp med 137 (1973)
10. Mu&he-Kaas, A, Kaplan, G & Seljelid, R, Exp cell res 103 (1976) 201. 11. Kaplan, G, Stand j immunol6 (1977). In press. 12. Bianco, C, GriIEn, F M & Silverstein, S C, J exp med 141(1975) 1278. 13. GrilBn, F M, Griflin, J A, Leider, J E & Silverstein, SC, J exp med 142 (1975) 1263. 14. Michl, J, Ohlbaum, D J & Silverstein, S C, J exp med 144 (1976) 1465. - J exp med 144 (1976) 1484. :2: Lay, W H & Nussenzweig, V, J exp med 128 (1968) 991. 17. Holland, P, Holland, N H & Cohn, Z A, J exp med 135 (1972) 458. 18. Mantovani, B, Rabinovitch, M & Nussenzweig, V, J exp med 135(1972) 780. 19. Griffin, F M & Silverstein, S C, J exp med 139 (1974) 323. 20. Griffin, F M, Bianco, C & Silverstein, S C, J exp med 141(1975) 1269. Received June 29, 1977 Revised version received November 24, 1977 Accepted February 24, 1978 Exp Cell Res 115 (1978)