A differential interaction in vitro of mouse macrophages with normal lymphocytes and malignant lymphoma cells

Europ. J. Cancer Vol. 13, pp. 605-615. Pergamori Press 1977. Printed inGreat Britain

A Differential Interaction In Vitro of

Mouse Macrophages with Normat Lymphocytes and Malignant Lymphoma Cells AVRAHAM RAZ, MICHAEL .INBAR. and RACHEL GOLDMAN Department of Membrane Research, The Weizmann Institute of Science, Rehovot, Israel A b a l a ' a c t - - I n vitro studies have shown that non-activated mouse peritoneal macrophages establish a differential interaction with mouse normal and malignant lymphoqyte~ from syngeneic animzds. The number of nuzlignant lymphoma cells (YAC) bound to macropbages after 60 rain of incubation at 37°C is 3-5fold higher than that of normal lymphocytes. The bimting of lymphema cells to macrophagespersistsfor at le~t 72 hr after cell-cell contactformation, whereas the ~ormal lymphoc.ytes detachfrom macrophages during the first 24 hr "of.incubati.on. Incubation of macrophage-lymphema cell rosettesfor 72 hr results in an extensive phag,ocytosis of about 50% oft he bound lymphoma cells. However, replacementof the cv[ture medium with fresh medium every 24 hr bolished the ingestion of the lymphoma cells Without changing their attachment to sop ahages.

14]. While several authors report on spontaneous nonspecific cytotoxicity towards tumour cells in "normal" macrophages derived from unstimulated animals [8, 15]. others report on development of such activities only in macrophages infected with bacteria and parasites [10, 16, 17]. Immunologically specific macrophage cytotoxicity depending on T-cell arming factors has also been extensively studied [18, 19] An unexpected observation of Nathan and Terry [20] adds to the already confused situation in that macrophages were shown to exert a differential stimulation of murine lymphoma cells. Close contacts between guinea pig macrophages and lymphocytes have been shown to develop via an immunological species and strain specific mechanism as well as via a non immunological species but not strain specific mechanism [21-23], Lymphoma cell growth in vitro was shown to be inhibited by normal macrophages [15] and stable cell-cell contact between lymphoma cells and normal macrophages have also been described [24]. In both cases specific conditions could be found where immunological activated macrophages were more efficient. The following study was undertaken to determine (a) whether there is a differential attachment of normal and malignant lympho-

INTRODUCTION

DIV~RS~ functions have been ascribed to mononuclear phagocytes. Among t h e most extensively studied are those involving phagocytosis of invading organisms and senescent self cells, killing of tumour cells,, interaction with immunocompetent lymphocytes, elimination of cell debris in inflammation and secretion of various macromolecules [1-4]. There is no doubt that expre.,~ion of several of these functions demands an intimate contact between the macrophages and the cell on which the function is exerted. Evidence is accumtilating, however, of the release of soluble factors from macrophages, either under normal conditions or due to external nonspecific or immunologic stimulation that may replace the macrophage [5, 6]. A wide range of reports on macrophage cytotoxicity seems to establish contradictory concepts. Accordkng to the different experimental designs, cytotoxicity was found to be both non specific in some systems [7-10] and specific in others [11, 12]; killing was observed in some [9, 10, 13] whereas in otb_~rs macrophages brought about cytostatic effects [7, 8,

Accepted 26 October 1976. 605

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Avraham Raz, Micigtel Inbar and Rachel Goldman

cytes to mouse peritoneal macrophages and (b) whether the attachment of the two cell types to macrophages is followed by phagocytic events. MATF_AtIKL AND M E T H O D S

Media Sterile phosphate buffered saline (PBS), Dulbecco's modified Eagle's medium (EM) supplemented with 100u/1 penicillin and 100/~g/l streptomycin and heat inactivated newborn calf serum (NCS) were obtained from Grand Island Biological Co. (N.Y.). Ce//s Peritoneal macrophages were aseptically collected from A and BALB/c strain mice (4-6 weeks old), following essentially the method of Cohn and Benson [25]. Peritoneal exudate cells suspended in medium were allowed to attach (1 hr, 37°C) on 25 mm diameter Coming cover-glasses, placed in Flacon plastic tissue culture dishes (35 × 10 mm, Falcon Plastics Div. Bioquest, Oxnard, Calif.). Peritoneal exudate cells containing about 0.5 x 106 macrophages were applied in 0"2 ml medium to the cover-glasses. The attached cell monolayers were thoroughly rinsed with PBS in order to remove the nonadhering cells. The cultures were then cultivated for 24 hr at 37°C in 2 ml of 20% serum in medium, in a CO2incubator (5% CO2-air mixture). Thymus derived lymphocytes, splenocytes and lymph node cells of A mice were collected by teasing the respective organs apart. Tissue pieces and visible cell clumps that settle to the bottom of the tube by gravity were discarded. Malignant lymphoma cells, an ascites form of a Moloney virus-induced lymphoma (YAC) [26] were grown in A strain mice by intraperitoneal inoculation of 105 cells per animal, and collected for experiments 11-14 days after inoculation. For the experiments normal lymphocyte and malignant lymphoma cells were freshly collected from animals in PBS, washed twice and resuspended in medium before use. _Interaction of macrophages with normal and ~mphoma cells Macrophage monolayers were washed twice with PBS and exposed to 30× 106 normal lymphocytes or lymphoma cells in 2 ml of medium for 60 min at 37°C. Culture plates were subsequently washed twice in PBS and were either fixed (2% glutaraldehyde 30 rain,

4°C) or reincubated in 20% serum in medium for 72 hr and fixed. Culture plates were then stained (Giemsa) and cells attached per 100 macrophages were enumerated in tetraplicate cultures. Attached cells were readily distinguished from ingested cells by the colour changes and irregular contour of the latter. The stained cultures were examined and photographed with Karl Zeiss Ultraphot microscope. RESULTS Attachment of normal lymphocytes and malignant lymphoma cells to macrophages Thymus derived lymphocytes establish few stable cell-cell contacts with the macrophages (Fig. lb) whereas lymphoma cells form stable rosettes at the periphery of the macrophages (Fig. lc). Q.uantitation of the differential interaction of thymus derived lymphocytes and lymphoma cells with macrophages established a 3-5 fold higher interaction for the latter (Table 1). Macrophages are free of any interaction with extraneous cells unless incubated with thymus derived lymphocytes or lymphoma cells (Fig. 1a). Binding of YAC lymphoma cells to macrophages was not affected even by in vitro growing of the lymphoma cells for two passages. Table 1. Malignant lymphoma cell and normal lym. phocyte attachment to mozrophages* Exp. No.

Lymphoma cells attached/100 macrophages + S.E.

Normal lymphocytes attached/100 macrophages + S.E.

1 2 3

256 + 11.2 220+ 14.8 292 _+22.8

68 _+7.6 48+4.0 80 _+9"0

*Macrophages were incubated with 30 x 106 lymphoma cells or thymus-derived normal lymphocytes in medium for 60 w.ln at 37°C.

Macrophage-lymphocyte rosette formation following macrophage incubation with either lymph node lymphocytes or spleen ' cells was of the same extent as that established with thymus-derived lymphocytes. Analysis of the distribution curve of lymphoma cells attached to macrophages indicate that 88% of macrophages establish stable contacts with lymphoma cells and the number of attached cells per macrophage appears to follow a Gaussian distribution. Thus the whole macrophage population is apparently capable

Interaction of Macrophages with Normal and Malignant Lymphocytes

Fig. 1. Physical interaction of macrophages with thymus derived lymphocytes and malignant lymphoma cells. Macrophages were incubated for 60 min at 37°C with either (a)-medium, (b)-thymocytes and (c)-lymphoma cells at a concentration of 30 x 106 cells/ml, x 850.

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Avraham Raz, Michael Inbar aud Rachel Goldman

Fig. 3. Lymphoma cell ingestion by macrophages. Preformed rosettes of macrophage lymphoma cells (see Fig. lc) were cultured for 72 h (a)--without culture medium replacement and (b)--with culture medium replacement every 24 h. (c) and (d)--macrophages that were not challenged with lymphoma cells cultured under conditions of (a) and ( b) , respectively, x 850.

Interaction of Macrophages with Normal and Malignant Lymphocytes

609

Fig. 4. Electron microscopy of YAC lymphoma cell-macrophage interactions. Macrophages YAC lymphoma cells after 60 min of ~interaction at 37°C (a and a'); ingestion of lymphoma cells by macrophages 72hr after incubation (b and b'); ingestion of YAC lymphoma cells abolished by culture medium replacement(c and c'), (Y-YAC lymphoma cells; M-macrophages). Scanning elec~!ron microscopy cells left grown on 13 mm coming cover glasses were fixed with 2% glutaraldehyde in 0 . 1 M Na-cacodylate buffer (pH 7.4) for 1 hr at 24"C. After washing three times in the buffer, dehydration in increasing concentrations of' ethanol was followed by acetone. The preparations were dried with a critical point drying apparatus (Polaron Equipment Ltd., Watford, Herts). The dried specimens were coated with gold using rotating stage vacuum evaporator. A steroscan Cambridge ,5'-180 scanning electron microscope was used at an acceleration voltage of 30 kV and tilt angle of 30 °. x 1900. Electron microscopy cells (right) grown on parlodin sheets were prepared for visualization by electron microscopy according to Spnrr [39] as described by Raz and Goldman [40]. Thin sectioning was obtained with SorveU, PorterBlum MT-2 ultramicrotone and analyzed in a Philips EM-300 electron microscope operated at 80 kV. x 6300.

Interaction of Macrophages with .Normal and Malignant Lymphocytes of forming cell-cell connections and the adherence phenomenon is not limited to a small subclass of macrophages (Fig. 2). A small subclass of macrophages incapable of binding of lymphoma cells is however not excluded. 50

I

I

I

I

I

l

I

I

7

8

4O

Normol

lymphocytes

2 30 o

~ 2o I0

I

2

3

4

5

6

No. of lymphocytes attached per rnacrophage

Fig. 2. Distribution curves of thymocyte and lymphoma cell attachment to macrop~ages. Based on experiments 1-3 in Table 1.

To assess whether the macrophage-lymphoma cell association is strain dependent, macrophages derived from BALB/c mice were cultivated and interacted with lymphoma cells under conditions comparable to the above. BALB/c macrophages were found to establish 10% of the cell-~:ell association observed with macrophages derived from A mice.

Ingestion of malignant lymphoma cells by macrophages Most of the studies reporting nonimmunological cytotoxic effects of nonspecifically activated peritoneal macrophages on malignant target ceils were carried out under conditions of prolonged incubation of the interacting cells. Destruction of target cells is readily observable at 60-72 hr of ceil-cell interaction and does not involve phagocytic events though cell debris has been shown to be disposed of within the macrophages [24]. Incubation of lymphoma cell-macrophage rosettes for 72 hr in 20% serum-medium results in up to 50% irLteriorization of the attached lymphoma cells ('Fable 2). The total number oI lymphoma ceils associated with macrophages (ingested - - attached) at 72 hr incubation is equivalent to the number associated with macrophages at rile beginning of the incubation, namely after a 6(3 rain incubation with lymphome cells and x 2 wash in PBS. It is worth

6/1

mentioning at this point that thymus derived lymphocytes detach from the macrophages in the course of the first 24 hr of incubation and therefore no comparable studies with these cells could be carried out. The light micrographs of macrophagelymphoma cells cultured for 72 hr (Fig. 3a) suggest that normal macrophages are capable of both ingestion and digestion of lymphoma cells. The morphological observations further indicate that whole lymphoma cells are ingested and not just fragments of debris derived from degenerating cells. Instead of the strong homogeneous staining pattern of attached lymphoma cells, those ingested stain by Giemsa in a faded colour and non-homogeneously. Macrophage--lymphoma cell rosettes incubated for 24 hr show a very low index of phagocytosis (about 5% of attached lymphoma cells are ingested). Particles or cells that are phagocytozible by macrophages are usually ingested within a period of minutes of the time of their attachment. Macrophage maturation in culture involves an increase in phagocytic capacity [27]. On the other hand continuous secretion of biologically active macromolecules including various enzymes from macrophages as well as from the lymphoma cells may modulate both the macrophage and the attached lymphoma cell plasma membrane and metabolism. In order to establish whether the maturation of macrophages per se, the aging oflymphoma cells or components accumulated in the culture medium within the 72 hr are detrimental in the delayed phagocytic response (most effective only after 48 hr incubation of rosettes) the following experimental procedure was adopted macrophage-lymphoma cell rosettes were incubated for 72hr (37°C) in 20% serummedium as above but culture medium was replaced by a fresh medium at 24 hr intervals. A striking difference in the fate of the lymphoma cells in indicated in Fig. 3(b). Practically no ingestion was detected under this condition, the: cells remain adherent to macrophages and seem to form clusters over the entire area of the macrophage. Additional evidence to support the results obtained with the light microscopy techniques (Fig. 1, 3) was gathered by the aid of scanning and transmission electron microscopy (Fig. 4). The differential interaction of macrophagelymphoma cells with and without serummedium replacement excludes the maturation of macrophages as the detrimental factor for the observed phagocytosis at 72 hr as opposed to lack of phagocytic activity at 24 hr. It should

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~Avrakam Rb.z, Midmel Ikbarand Rachel GOldman

be emphasized L~~however~ that. ,mae~ophages cultured for 72 hr :with and without serum= medium replacement may~diffe~ ~to some extent in their metabolic :activity and endOcytlc activities [27]. Figs 3c . a n d 3d indicate a morphologiCal clifference tander the two culturing conditions expressed i n abundance of :minute phase lucent vesicles in cultures incubated in serum±medium for 72 h r ~ t h o u t replacement as compared t o moderate, vesicle formation in cells incubated with fresh serummedium every 24 hr. -

DISCUSsioN

,

%

,"

•

Normal macrophages have been shown to establish a differential interaction pattern wi'th lymphocytes and malignant lymphoma cells. The interaction 'with lymphoma cells involves both a high degree of rosette formation (Table 1) and the development of stable-cell-cell contacts] i.e., t h e number of attached lymphoma cells is not reduced with a 72 hr incubation period (Fig.i 3b). The degree-of rosette formation with normal lymphocytes derived from the thymus, spleen and lymph nodes is several times lower than that with lymphoma cells and the attached :lymphocytes detach within the first 24 hr of incubation, : Viral induction of malignant transformation, ochanges various parameters of,cell physiology. One of the mos t extensively studied parameters is that involving changes in plasma membrane composition, organization and function [2829]. : The high degree of macrophage'interaction with lymphoma cells reported :in the above Could reflect a recognition by the macrophage of alterations in surface properties of the attached cell. It is of interest that this interaction depends on syngeneity of macrophages and lymphoma cells; namely macrophages from BALB/c mice that do not serve as hosts for YAC lymphoma Cell transplantation do not form extensive interactions with the lymphoma cells. Activated mouse macrophages exhibit a cytotoxic effect on tumorigenic 3T12 and SV 40 3T3 transformed fibroblasts and are noncytotoxic to their nontransformed parental cell-line 3T3 fibroblasts [9]. Lipsky and Rosenthal [21] report however that two normal lymphoid cell populations, thymus and lymph node lymphocytes, were bound to syngeneic guinea pig macrophages in significantly larger numbers than L 2C leukemia cells. In the guinea pig macrophage-thymocyte interaction is exceptionally high, amounting to up to 300 thymocytes per 100 macrophages

[2 I, 22, ,30]:. In the~A mome~our result~ indicate a much lower degree of interaction of ths,mmderived lymphocytes with macrophages, :: Stable cell-cell associations of lymphocytes and macrophages have been frequently ob' served during in vitro cultivation of these cells [31-33], as well as in fixed preparations of lymphoid organs [34, 35]. Under in vivo conditions clusters of lymphocytes and blast cells arOund macrophages are formed in response to immunization procedures [34, 35]. Likewise the in vitro induction of primary antibody responses and antigen-mediated in vitro proliferation of immune lymphocytes [32, 33..] has been shown to involve direct physical" contact between lymphocytes and antigen~containing macr0Phages. Lymphocytes have also been found to adhere to macrophages both in vivoand in vitro in the absence of specific immunization [21; 22]. When macrophage-lymphocyte associations involve antigen [2] or lectin [6] presentation, the result of the association is lymphocyte proliferation. Very rarely have phagocytic events been observed.' :'~' The association of nonimmune and nonactxvated peritoneal macrophages with a variety of lymphoma cells has been reported to result in inhibition of proliferation and (3H)-thymidine uptake [15]. Many of the studies suggest the need for macrophage-lymphoma contact and indicate that though immune and non= specifically activated macrophages are more efficient in their growth inhibition capacity, normal macrophages are also endowed with cytostatic as well as cytotoxic capacity [14, 24]. Most experimental systems studied use macrophage-target Cell ratios of 100: 1, 40: 1, 10:1, 5:1 and rarely a ratio of l : l [7, 14, 17, 24]. The differential activity of activated vs normal macrophages is mostly evident under conditions of a low ratio of macrophage to target cell [ 14, 24]. • In our studies we have used a ratio of 1:60 of macrophages to target ceUs. This ratio en, abled the establishment of extensive inter: actions of macrophages and lymphoma cells; o n the average 2.5 lymphoma cells were associated with each macrophage in the culture. A n incubation of macrophage-lymphoma cell rosettes for 72 hr without culture medium replacement results in an extensive phagocytosis of adherent lymphoma cells (Table 2). Up to 50% of macrophages were involved in phagocytosis oflymphoma cells and up to 50% of the adherent ceils were ingested. In contra-distinction when culture medium was changed every 24 hr for fresh culture medium the macrophage adherent lymphoma cells preserved their stain-

Interaction ,of :Macrophages. witk. dVormat a~l,Mulignant Lymphocytes

61S

Table 2. Ingestion* of lymphoma cells attached to macrophages ..

Exp. No. 1 2 3

i

ml

|

Percentage of macrophages with at least one ~ingested lymph0ma cell + S.E.

No. of ingested lymphoma cells/ 100 macrophages _ S.E.

No, of attached lymphoma cells/ 100 macrophages + S.E.

50 :/: 3 48:]:2 36_+2

116 + 12 108+ 10 44.+3

121 5:14 158+22 218+4.

Total No. of macrophage associated lymphoma cells + S.E. 237 + 17 . 266+24 262+ 16

*Preformed rosettes of macrophages and lymphoma cells (for conditions and quantitation see. Exp. 1-3 in Table 1, respectively) were incubated for 72 hr in serum-medium at 37°C.

ing characteristics with the Giomsa stain, and were all attached to macrophage surface. Extensive ingestion of lymphoma c'ells has not been reported hitherto though Lejeune and Evans have obserx~ed engulfment by macrophages of lymphoma debris [24]. An early report by Bennett et al. [36] suggests that even peritoneal macr0phages from immunized mice do not exhibit an inherent specific ability to phagocytose turn our cells in vitro, unless the medium contained a hum.oral antibody against the tumor. The differential interaction of macrophagelymphoma cells under conditions of culture medium replacement and continuous culture in the same medium is not fully understood. Several possible processes may b e involVed in the phenomenort, either 'of which can be the major responsible factor or they may exert synergistic effects. During the extended incubation time of macrophage-lymphoma cell rosettes both cell types may undergo metabolic as well as surface changes. Recent studies in our laboratory indicate a continuous maturation process of macrophages in culture, during which the phagocytic activity increases and surface morphology changes [27]. The functional changes during maturation depend on whether or not the culture was incubated continuously in the same serum medium or whether culture medium was replaced every 24 hr. Figures 3(c) and 3(d) amply show the differential morphology of macrophages under the abovementioned conditions. Moreover, macrophages release factors and various enyzmes to the medium [1, 37]. Their accumulation within 72 hr of incubation may act on both the macrophages as well as on the adherent lymphocytes and render them more susceptible to phagocytosis. Metabolic changes, cytostatic or cyto-

toxic effectors may serve as signals for macrophage scavenging capacity. Bennett et al. [36] have in fact shown that under certain conditions killed tumor c e l l s a r e readily phagocytosed even without opsonization with' isoantibody. If mediated by products released from macrophages, the fact that almost no phagocytosis is observed under conditions of culture-medium replacement means that a concentration factor for the product has to be involved. Either its secretion within 24 hr is not sufficient for triggering phagocytic events or the enriched aged medium enhances in itself product secretion. Macrophage challenge with tumor cells has been shown to cause rapid changes in lysosomal morphology and content [24]. It is not impossible that rosette formation serves in itself as a challenge for macrophage activation. That this would be more effective without culture medium replacement can stem from need of factors released by either macrophages, that upon activation release more enyzmes and biologically active products [28], or By lymphocytes. So that in addition to macrophage involvement in the phagocytic event, lymphocytes could mediate potentiation of macrophage function. The various aspects of in vitro lymphoma cell interaction with macrophages and the phagocytic events that conclude it are currently under investigation. It is hoped that studies of the in vitro macrophage-lymphoma cell interaction would help to understand the in vivo situation where 103 lymphoma cells injected i.p. escape in vivo the resident peritoneal macrophage population (1-2 x 106 ceils), proliferate and kill the mouse within 20-25 days.

A c i m o w l e c t g e m e t t t s - - W e express our thanks to Mrs. N. H a r p a z for skilful technical assistance.

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Avraham Raz, Michael Inbar and Raxhel Goldman REFERENCES

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