Clin. exp. Immunol. (1979) 38, 531-538.
In vitro proliferation of macrophage depleted human peripheral blood lymphocytes A. J. TREVE S, V. BARAK & Z. FUKS Department ofRadiation and Clinical Oncology, Sharett Institute of Oncology, the Hebrew University-Hadassah Medical School, Jerusalem, Israel
(Acceptedfor publication 8 June 1979) SUMMARY
The incorporation of thymidine by normal human peripheral blood lymphocytes was tested in vitro following various culture conditions. A significant increase of thymidine uptake was observed in cultures depleted of plastic adherent, nylon wool adherent, or phagocytic cells. This proliferative activity occurred in the presence of various sera but was not due to a blastogenic response to a foreign protein, since it was also observed when autologous plasma was the only source of protein in the culture medium. The similarities and differences between this 'spontaneous' proliferative phenomenon and other blastogenic responses which are regulated by macrophages are discussed.
INTRODUCTION The blastogenic response of lymphocytes to foreign antigens is one of the in vitro cell mediated immune responses most extensively explored. Increased incorporation of thymidine in vitro occurs in mixed lymphocyte cultures (MLC) (Dutton, 1966), following secondary interaction with previously encountered antigens (Dutton, 1967) and following exposures to nonspecific mitogens (Ling, 1968). In addition, it has been shown that blastogenic and cytotoxic responses of human lymphocytes may occur following their in vitro incubation in the presence of foetal calf serum (FCS) (Johnson & Russel, 1965; Zielske & Golub, 1976). Ortaldo, Bonnard & Herberman (1977) and Treves et al. (1978) have recently reported that culture induced cytotoxicity occurs following incubation of lymphocytes in the presence of FCS but not in human serum (HS), and that this cytotoxicity is probably different from the natural killing activity (Treves et al., 1978). These proliferative and cytotoxic reactions are initiated following the exposure of the responding lymphocytes to foreign antigens. On the other hand, it has recently been shown that in vitro autoreactivity of T lymphocytes against non-T cells may occur in an MLC response. The autologous MLC usually occurs following recombination of pre-separated populations of T and non-T lymphocytes or lymphoblastoid cell lines, and is not caused by an exposure to a foreign antigen (Green & Sell, 1970; Birnbaum, Siskind & Weksler, 1972; Opelz et al., 1975; Kuntz, Innes & Weksler, 1976; Smith, 1978; Weksler et al., 1978). In the present study, we report that in vitro enhanced incorporation of thymidine and blastogenic response by human peripheral blood lymphocytes (PBL) may occur following the removal of macrophages from the culture and in the absence of extrinsic antigenic stimulation. MATERIAL AND METHODS Lymphocytes. Peripheral blood mononuclear cells (PBM) were obtained from healthy male and female donors 18 to 40 years old. Venous blood was drawn and mixed with 50 iu/ml sodium heparin (Leo Pharmaceutical Products, Ballerup,
Correspondence: A. J. Treves, Department of Radiation and Clinical Oncology, Sharett Institute of Oncology, the Hebrew University-Madassah Medical School, Jerusalem, Israel. 0099-9104/89/1200-0531$02.00 (© 1979 Blackwell Scientific Publications
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Denmark). The PBM were separated on Ficoll-Hypaque gradients (Boyum, 1968). The separated mononuclear cells were washed once with phosphate-buffered saline (PBS) pH 7-2. Cell separation procedures. Nylon wool adherent cells were removed by the method of Julius, Simpson & Herzenberg (1973). In various experiments 60%+ 17 (47-72) non-adherent cells were recovered following this separation procedure. Plastic adherent cells were removed by culturing 20-30x 106 PBM in 7 ml of complete RPMI-1640 media with 5% foetal calf serum (FCS) (Microbiological Associates, Bethesda, Maryland; aseptically collected) in 10 cm plastic petri dishes (Nunc, Roskilde, Denmark) for 1 hr at 37 C. The culture media were supplemented with GIBCO MEM vitamin solution (1: 100); Penicillin-Streptomycin 50 ju/ml and 50 1u/ml respectively; L-glutamine 2 mM; Sodium Pyruvate 1 mM; and Hepes buffer 10-2 M. Following separation 80% + 15 (70-91) non-adherent cells were recovered from the original cell population. Phagocytic cells were removed following incubation with carbonyl iron. Approximately 30-40 x 106 PBM were incubated with 50 mg of carbonyl iron in a 17 x 100 mm test tube for 30 min at 37 C. The carbonyl iron fed macrophages were attracted to the bottom of the tube by a magnet and the cells remaining in the supernatant were collected. In different experiments 67%+ 12 (48-80) of the cells were recovered from the original populations. Each of the three procedures resulted in reduction in the percentage of macrophages to 1-4% as shown by phagocytosis of latex particles. FCS was replaced with 5% pooled human AB serum (HS) during the separation procedures whenever cells were prepared for experiments in which sera other than FCS were present in the culture medium. In vitro cultures. PBM or macrophage depleted populations of PBM were cultured in different concentrations in round bottom microtitre plates (Nunc) in 0-1 ml of supplemented RPMI-1640 with either 10% of FCS (Microbiological Associates) or human pooled AB sera or autologous plasma (AP). All sera and the autologous plasma were heat inactivated. Unless otherwise indicated, the cells were incubated for six days in 5% C02 in air in a humidified incubator. On the fifth day of incubation, 1 pUCi of 3H-thymidine (Nuclear Research Center, Negev, Israel; specific activity 5 Ci/mM) in 10 puLi of PBS was added to each well. At the end of the sixth day the cells were harvested on glass fibre filters by an automatic cell harvester (Titertek Cell Harvester, Flow Laboratories, Oslo, Norway). The filters were dried out, immersed in toluene scintillation fluid and the radioactivity was counted in a fi scintillation counter. In some experiments, thymidine uptake was replaced by radioactive leucine. In these experiments the cells were cultured in leucine free medium for five days and then 1 pUCi of 3H-leucine (Nuclear Research Center, Negev, Israel; specific activity 30 Ci/mm) in leucine-free media was added to each well. Mitotic activity was also measured by three hours accumulation of mitoses using 1 mg/ml of Colcemid. After this procedure, the cells were collected, stained with methanol-Giemsa and 500 cells in each experimental group were scored for mitotic and blast figures. Phytohaemaglutinin (PHA) (Wellcome Research Laboratories, Beckenham, England) when used was added to the same cultures to a final concentration of 1 uLi pet well. In other experiments concanavalin A (Con A) (Miles Yeda, Rehoxvot, Israel) crystallized three times was added to a final concentration of 15 ug/ml. The mitogens were added to the culture either on the first day or on the third day of the culture. Two days later, the cultures were labelled by 1 pUCi of 3H-thymidine and harvested as described above. Viable cell counting was performed by the trypan-blue (0-04%) exclusion test. Each experimental group consisted of four duplicates and results were expressed as the average counts per minute+ one standard deviation (ct/min+ s.d.). In some experiments, the activity index was calculated by dividing the ct/min of the depleted PBM cultures by the ct/min of the non-depleted PBM culture. Each experimental procedure was performed with PBM obtained from at least three different donors. The figures presented in this manuscript show the results observed with a single donor, but consistent phenomena were always observed with the cells obtained from the other donors as well.
RESULTS Fig. 1 shows the incorporation of 3H-thymidine by normal PBM during the first seven days of culture. Enhanced incorporation occurred in cultures depleted of nylon wool adherent cells. The highest incorporation occurred in cultures depleted of nylon wool adherent cells. The highest incorporation occurred during the fifth day of incubation when the concentration of cells in the culture was high (7 5 x 105 cells/OK1 ml) and on the seventh day when the concentration of cells in the culture was decreased (2 5 x 105 cells/O01 ml). Some incorporation of 3H-thymidine was observed in non-depleted PBM but this occurred only when the concentration of cells in the culture was low, and even under these conditions the absolute activity was significantly lower compared to the activity of the nylon wool depleted cells of the same cell concentration. It can be concluded that either lowering of cell concentration, or removal of nylon wool adherent cells from PBM results in similar phenomena of enhanced thymidine uptake. Fig. 2 compares the enhanced proliferation of nylon wool depleted PBM with that occurring following stimulation of whole PBM by PHA. The peak of the mitogenic response to PHA occurred two days earlier than that of the spontaneous proliferation of nylon wool depleted cells. It was also observed that removal of nylon wool adherent cells significantly enhanced the mitogenic response of PBM or PHA. In addition to removal of nylon wool adherent cells, the removal of plastic adherent or phagocytic cells from the cultures also resulted in enhanced lymphocyte proliferation (Fig. 3). These results suggest
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0 x
.E U-
3 4 5 6 Time after culturing (days)
FIG. 1. Effect of cell number and duration of culture on incorporation of 3H-thymidine by whole and macrophage depleted PBM. Different numbers of PBM (o a) or of nylon wool depleted PBM (e 0) were incubated for 1-7 days in supplemented RPMI-1640 with 10% FCS, pulsed with 3H-thymidine and assayed for radioactivity.
300
A
240 x
180 _ 120 A
60
2
4 6 Time after culturing (days)
FIG. 2. Spontaneous and PHA induced incorporation of 3H-thymidine by whole and macrophage depleted PBM. 5 x 105 PBM (open figures) or nylon wool depleted PBM (closed figures) were assayed for 3H-thymidine incorporation at different times following initiation of culture. Spontaneous incorporation (circles), and the mitogenic response to PHA (triangles) are shown. PHA was added at initiation of culture.
that macrophages present naturally in PBM, inhibit the proliferation of PBL in culture. To further test this hypothesis, we examined the proliferation of macrophage depleted PBM before and after reconstitution with plastic adherent cells. When phagocytic cells depleted PBM were plated on wells containing plastic adherent cells (prepared by pre-incubation of PBM of the same donor for 60 min at 370C followed by removal of the non-adherent cells), the mitogenic response to PHA or Con A was significantly reduced
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to0 r-
E L).
4.0 6-0 Number of cells (x0-5)
FIG. 3. Effect of mode of macrophage depletion on the incorporation of 3H-thymidine by PBM. PBM were depleted by plastic adherence (e 0), removal of nylon wool adherent cells (A A), or by removal of U = Carbonil iron) and assayed for 3H-thymidine incorporation. (x x ) Indicates phagocytic cells (U no separation. 260 240
220 200 0 x
+1 c U)
._
180 160 140
120 100
80
60 40
20
Ii
-Vwith with -- with - with iwith I1with monocytes monocytes ! monocytes No mitogen Con A PHA "
FIG. 4. Effect of reconstitution with plastic adherent cells on spontaneous or PHA induced 3H-thymidine incorporation, by whole or macrophage depleted PBM. 6x 106 whole PBM (0) or phagocytic cells depleted PBM (a) were plated in round bottom microtitre wells, or in similar wells which had been coated previously with autologous plastic adherent cells derived from 6 x lo0 whole PBM. PHA or Con A were added to some wells after four days in culture. 3H-thymidine incorporation was assayed after six days in culture.
following reconstitution of PBM depleted of phagocytic cells with plastic adherent cells (Fig. 4). It was also noted that with low cell concentrations in the culture, the responsiveness to these mitogens was considerably less inhibited by macrophages (results not shown). Similar to the enhanced incorporation of 3H-thymidine, there was also augmentation in the incorporation of 3H-leucine by macrophage depleted PBM, which was significantly higher than that observed with whole PBM (Table 1). In all of the experiments described above, the culture media contained 10% FCS. Table 2 shows that significant proliferative activity was also observed in the presence of other sera in the culture medium,
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TABLE 1. The incorporation of radioactive thymidine or leucine by macrophage deleted PBM*
3H-thymidine
3H-leucine
Donor name
Whole PBM
Phagocytic Cells Depleted PBM
Activity index
Whole PBM
Phagocytic cells depleted PBM
Activity
SA SC
853+ 196 3144+ 1357
25080+ 5900 209205+ 19321
29-4 66-5
7160+ 1079 16019+ 1559
48052+ 10658 51151+4673
3-2
index 6-7
* 6x 105 whole PBM or phagocytic cell depleted PBM were incubated in leucine free RPMI-1640 with 10% dialysed-FCS for five days. Radioactive thymidine or leucine were then added for 24 hr. followed by cell harvesting and counting of radioactivity.
TABLE 2. Effect of various sera on the incorporation of 3H-thymidine by macrophage depleted PBM*
Source of serum
Autologous plasma Human AB serum (1) Human AB serum (2) Human cord blood serum Horse serum Rabbit serum Calf serum Foetal calf serum
Whole PBM
Nylon wool depleted PBM
918+ 110 1717+393 943+ 556 2566+ 319 1316+ 833 6728+3511 2460+ 1330 1692+ 494
15830+46 26115+6942 17141+ 1946 28840+ 2902 59379+ 13350 35230+4098 60338+ 4709 63785+ 10946
Activity index 17-2
15-2 18-2 11-2 45 1 52 24 5 37-7
* 6 x IO1 whole PBM or nylon wool depleted PBM were incubated in the presence of different sera for 5 days; pulsed for 24 hr with 3H-thymidine and radioactivity assayed. Separation and elution procedures were performed with medium containing 5% human AB serum.
including autologous plasma. The degree of spontaneous proliferation in the presence of autologous plasma was similar to that observed in the presence of FCS (Table 3). The total ct/min of macrophage depleted PBM was more than tenfold higher than that observed with the corresponding whole PBM in 6/10 donors when FCS was present in the medium culture compared to 5/10 in the presence of autologous plasma. Blast formation and mitotic activity were also observed in the phagocytic cells depleted PBM in the presence of autologous plasma and in the absence of any foreign components in the culture media. No such activity was found in the undepleted populations (Table 4). DISCUSSION In this study we have shown that proliferation of human lymphocytes can be initiated in culture by the removal of macrophages. A complete removal of macrophages was not necessary since the depleted populations ot PBM in our experiments still contained 1-4% of phagocyte cells. Furthermore, lowering the total number of macrophages by merely reducing the number of undepleted PBM in the cultures to 2 5 x 105 cells/0-1 ml also resulted in a weak but still detectable proliferative response (Fig. 1). Furthermore, the presence of macrophages in certain proportions was necessary to suppress the proliferation of lymphocytes since the addition of macrophages to macrophage depleted cell populations inhibited the in vitro proliferation (Fig. 4). The addition of macrophages to phagocytic cells depleted PBM also suppressed the responses of T cells to mitogens (Fig. 4).
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TABLE 3. Effect of FCS or autologous plasma on the incorporation of 3H-thymidine by macrophage depleted PBM*
Autologous plasma
FCS Whole PBM
Depleted PBM
3091+693 100+ 30 174+ 54 6238+ 1127 1804+900 1728+ 123 918+ 110 223+29 1512+ 423 2655+1442
9663+3032 7929+ 1764 8348+ 708 30386+5235 14396+4196 21530+4218 15830+46 3349+248 5694+ 834 5667 +567
Activity index
Donor name
Whole PBM
Depleted PBM
EK JX HB MS MH TZ IC AB AH TT
1788+499 1444+ 172 133+ 54 212+2 285+58 1338+269 1692+494 1884+225 3317+401 2750±757
12009+ 1515 10142+ 2005 12768+ 1348 27411+8817 65192+7011 37409+ 1902 63785+ 10946 23057+3804 12332+ 2030 11713+892
6*7 7*0 96-0 129 0 228*0
28*0 37-7 12-1 3-7 4-3
Activity index 31
7913 48-0 49 8-0 12 5 17-2 15 0 3-8 21
* Phagocytic cells (first four donors) or nylon wool adherent cells (other donors) were removed from PBM and 5-6x 105 cells were incubated for five days in the presence of 10% FCS or autologous plasma pulsed for 24 hr with 3H-thymidine and assayed for radioactivity. Separation and elution procedures were performed with media containing 5% human AB serum. TABLE 4. Mitotic and blastogenic activity of phagocytic cells depleted PBM*
Phagocytic cells depleted PBM
PBM
Experiment number
Donor name
Sera
ct/min+ s.d.
Mitotic index
Blastogenic index
ct/min+ s.d.
Mitotic index
Blastogenic index
1
AH AH AB HB VB VB SR SR
AP FCS AP FCS AP FCS AP FCS FCS
432+ 36 124+ 2 112+5 243+27 469+ 18 627+ 138 2444+426 5091+471 2931+590
0 0 0 0 0 0 0 0 0
2 1 0 1 0 1 0 0 1
33158± 3898 40456± 3078 4703±764 11157+630 33525±4769 47475± 6448 89396+ 16035 68042+ 13914 87071+3853
1 1 1 3 5 4 1 3 4
10 6 7 13 15 7 8 9 20
2
3
JG
* Phagocytic cells were removed from PBM and 6 x 105 cells were incubated for five days in the presence of either 10% FCS or autologous plasma, pulsed for 24 hr with 3H-thymidine and assayed for radiolable incorporation. Part of the cultures were treated with colcemid for 3 hr and then collected and stained. The percent of mitotic figures and blast cells were calculated by counting 500 cells in each experimental group.
The in vitro proliferation of macrophage depleted lymphocytes in this study is unlikely to represent merely a blastogenic response to foreign proteins, such as FCS or other heterologous sera, since it also occurred in the presence of human AB serum and in the presence of autologous plasma (Tables 2, 3, 4). On the other hand, it is possible that the higher proliferative responses observed in the presence of FCS or horse serum (Table 2) could be attributed to an additional mitogenic response to the foreign antigens present in these cultures. A similar phenomenon of a mitogenic reaction to FCS has been described previously (Johnson & Russell, 1965; Zielske & Golub, 1976). Johnson & Russell (1965) have also reported that mitotic activity and blast formation may occur in the presence of autologous sera. In these experiments, however, the lymphocyte cultures were treated with carbonyl iron prior to their in vitro incubation and no attempt was made to investigate the role of macrophages in the proliferative response.
In vitro human lymphocyte proliferation 537 Macrophage mediated suppression of proliferative responses of lymphocytes has been well documented (Keller, 1975). It has been shown that macrophages secrete soluble mediators which regulate the blastogenic response of lymphocytes (Nelson, 1973; Calderone & Unanue, 1975). Macrophages were also found to regulate haematopoiesis in vitro (Cline, 1979). Some investigators have attributed the macrophage related inhibition of blastogenic response in the mouse to the presence of large quantities of 'cold' thymidine secreted by the macrophages which competitively inhibit the uptake of radioactive thymidine used to assay the blastogenic response (Opitz, Naethammer & Jackson, 1975). However, it is unlikely that 'cold' thymidine competition is the explanation for the macrophage related inhibition of the augmented proliferation of human lymphocytes shown in our study since in the absence of macrophages, proliferation has also been demonstrated by 3H-leucine incorporation (Table 1). Furthermore, actual blast formation and enhanced mitotic activity were observed in the macrophage depleted PBM cultures but not in whole PBM cultures, even in the absence of foreign proteins in the culture media (Table 4). These observations cannot be explained by selective inhibition of 3H-thymidine uptake by 'cold' thymidine. They also stress the point that actual blast formation and cell division, and not just thymidine uptake, were induced by the removal of macrophages even in the presence of autologous plasma. In vitro proliferation of lymphocytes as a result of antigenic or mitogenic stimulation has been described extensively (Dutton, 1967; Ling, 1968; Keller, 1975). It has also been shown that chemical modification of the culture medium, such as the addition of mercaptoethanol (Goodman & Weigle, 1977) or pretreatment of lymphocytes with periodate (Novogrodsky, 1975) also resulted in in vitro proliferation of lymphocytes. In vitro proliferation of mouse thymocytes has been demonstrated following the addition of soluble factors secreted by macrophages. 'Spontaneously' proliferating mouse T cells were also suggested to mediate suppression of graft-vs-host response (Moorhead, 1978; Calderon & Unanue, 1975). However, all these experiments were performed in the presence of FCS which may contribute some antigenic stimulation. There has been only one situation in which in vitro proliferation of human lymphocyte occurred in the absence of a foreign substance in the medium (Opelz et al., 1975; Weksler et al., 1978; Smith, 1978). It has been shown that human T cells proliferate following their recombination with autologous B cells (the autologous MLC reaction) and that this reaction occurs in the absence of an extrinsic antigenic stimulation. The nature of the enhanced proliferation of human peripheral blood lymphocytes occurring after the removal of macrophages described in this paper is still unclear. It does not seem to represent an autologous MLC reaction since preliminary experiments performed in our laboratory have shown that the cell proliferating in our experiments is not a T cell, and that this cell population is different from the cells responding to T cell mitogens such as PHA and Con A (data not shown). It is possible that the cells proliferating in our experiments have been artificially modified by one of the experimental procedures and are therefore undergoing a blastogenic response. On the other hand, such a modified population may itself serve as a stimulus for blastogenic response of another subset of lymphocytes in the human peripheral blood. Although such an artificial modification is possible, it is very difficult to prove or disprove it for the obvious methodological limitations of the currently available testing systems. Alternatively, it is possible to speculate that lymphocyte proliferation in vitro may occur 'spontaneously', namely, without any antigenic stimulation, but only as a result of the reduction in the number of macrophages. It is possible that the apparently 'spontaneous' proliferation described in our study represents a physiological process of human lymphocyte proliferation which seems to be regulated by macrophages. Any interference in the balance between the growth regulatory activity of macrophages and the proliferative capacity of certain subpopulations of lymphocytes may result in excessive proliferation of these cells. Such an imbalance may in fact exist in certain states of disease. It has recently been shown that spontaneous incorporation of thymidine and spontaneous blast formation occur in lymphocytes separated from blood of patients with Hodgkin's disease (Shiftan, Caviles & Mendelson, 1978), infectious mononucleosis (Nikoskelainen & Stevens, 1979) and rheumatoid arthritis (Slaughter et al., 1978). It has also been shown that certain abnormalities of macrophage function, such as increased suppressor cell activity occur in Hodgkin's disease (Zembala et al., 1977; Hillinger & Herzig, 1978), although this abnormality may be at least in part attributed to suppressor lymphocyte dusfunction (Twomey et al., 1975). Further-
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more, Kaplan & Gartner (1977) have recently suggested that the Reed-Sternberg cell of Hodgkin's disease originates from macrophages, a phenomenon which represents another macrophage related abnormality in a lymphoproliferative disorder. However, this hypothesis on the interaction between macrophage activity and lymphocyte proliferation under normal conditions and in certain lymphoproliferative diseases should be regarded at the present time as highly speculative and further investigations will be necessary to test its validity. This work was supported in part by the Joint Research Fund of the Hebrew University and Hadassah, and by the Philip Lash and Dr Edward D. Lash Research Endowment Fund.
REFERENCES BIRNBAUM, G., SISKIND, G.W. & WEKSLER, M.E. (1972) Autologous and allogeneic stimulation of peripheral human leukocytes. Cell Immunol. 3, 44. BOYUM, A. (1968) Separation of lymphocytes from blood and bone marrow. Scand. J. Clin. Lab. Invest. 21 (Suppl. 97), 1. CALDERONE, J. & UNANUE, E.R. (1975) Two biological activities regulating cell proliferation found in cultures of peritoneal exudate cells. Nature, 253, 359. CLINE, M.J. (1979) Cellular interactions in hoematopoiesis. Nature, 277, 177. DUTTON, R.W. (1966) Symposium on in vitro studies of the immune response. II. Significance of the reaction of lymphoid cells to homologous tissue. Bacteriol. Rev. 30, 397. DUTTON, R.W. (1967) In vitro studies of immunological responses of lymphoid cells. Adv. Immunol. 6, 253. GOODMAN, M.G. & WEIGLE, W.P. (1977) Non-specific activation of murine lymphocytes. 1. Proliferation and polyclonal activation induced by 2-mercaptoethanol and a thioglycerol. J. Exp. Med. 145, 473. GREEN, S.S. & SELL, K.W. (1970) Mixed leukocyte stimulation of normal peripheral leukocytes by autologous lymphoblastoid cells. Science, 170, 989. HILLINGER, S.M. & HERZIG, G.P. (1978) Impaired cellmediated immunity in Hodgkin's disease mediated by suppressor lymphocytes and monocytes. J7. Clin. Invest. 61, 1620. JOHNSON, G.J. & RUSSELL, P.S. (1965) Reaction of human lymphocytes in culture to components of the medium. Nature, 208, 343. JULIUS, M.H., SIMPSON, E. & HERZENBERG, L.A. (1973) A rapid method for the isolation of functional thymusderived murine lymphocytes. Eur. J. Immunol. 3, 645. KAPLAN, H.S. & GARTNER, S. (1977) 'Sternberg-Reed' giant cells of Hodgkin's disease: Cultivation in vitro, heterotransplantation, and characterization as neoplastic macrophages. Int. J. Cancer, 19, 511. KELLER, R. (1975) Major changes in lymphocyte proliferation evoked by activated macrophages. Cell Immunol. 17, 542. KuNTZ, M.M., INNES, J.B. & WEKSLER, M.E. (1976) Lymphocyte transformation induced by autologous cells. IV. Human T-lymphocyte proliferation induced by autologous or allogeneic Non-T lymphocytes. J. Exp. Med. 143, 1042. LING, N.R. (1968) Lymphocyte Stimulation. Wiley, New York. MOORHEAD, J.W. (1978) Subpopulations of mouse T lymphocytes. II Suppression of graft-vs-host reactions by naturally proliferating splenic T cells. Eur. J. Immunol. 8, 163. NELSON, D.S. (1973) Production by stimulated macrophages
of factors depressing lymphocyte function. Nature, 246, 306. NIKOsKELAINEN, J. & STEVENS, D.A. (1979) Cellular immunity in infectious mononucleosis: I. Spontaneous and phytohaemagglutinin-induced blast transformation in infectious mononucleosis, with special reference to depressed cellular reactivity and stimulation-blocking serum factors. Oncogenesis and Herpes virus. IARC Scientific Publications. (In press.) NOVOGRODSKY, A. (1975) Induction of lymphocyte cytotoxicity by modification of the effector or target cells with periodate or with neuraminidase and galactose oxidase. 3. Immunol. 114, 1089. OPELZ, G., KIUCHI, M., TAKASUGI, M. & TERASAKI, P.I. (1975) Autologous stimulation of human lymphocyte subpopulations. 3. Exp. Med. 142, 1327. OPITZ, H.G., NIETHAMMER, D. & JACKSON, R.C. (1975) Biochemical characterization of a factor released by macrophages. Cell. Immunol. 18, 70. ORTALDO, I.R., BONNARD, G.D. & HERBERMAN, R.B. (1977) Cytotoxic reactivity of human lymphocytes cultured in vitro. 3. Immunol. 119, 1351. SHIFTAN, T.A., CAVILES JR. A.P. & MENDELSOHN, J. (1978) Spontaneous lymphocyte proliferation and depressed cellular immunity in Hodgkin's disease. Clin. exp. Immunol. 32, 144. SLAUGHTER, L., CARSON, D.A., JENSEN, F.C., HOLBROOK, T.L. & VAUGHAN, J.H. (1978) In vitro effects of EpsteinBarr virus on peripheral blood mononuclear cells from patients with rheumatoid arthritis and normal subjects. 3. Exp. Med. 148, 1429. SMITH, J.B. (1978) Stimulation of autologous and allogeneic human T-cells by B-cells occurs through separate Bcell antigen system. Cell. Immunol. 36, 203. TREVES, A.J., HEIDELBERGER, E., FELDMAN, M. & KAPLAN, H.S. (1978) In vitro sensitization of human lymphocytes against histiocytic lymphoma cell lines: 11. Characterization of three different effector activities and suppressor cells. 3. Immunol. 121, 86. TWOMEY, J.J., LAUGHTER, A.H., FARROW, S. & DOUGLASS, C.D. (1975) Hodgkin's disease: An immunodepleting and immunosuppressive disorder. 3. Clin. Invest. 56, 467. WEKSLER, M.E., KUNTz, M.M., BIRNBAUM, G. & INNEs, J.B. (1978) Lymphocyte transformation induced by autologous cells. Fed. Proc. 37, 2373. ZEMBALA, M., MYTAR, B., POPIELA, T. & ASHERSON, G.L. (1977) Depressed in vitro peripheral blood lymphocyte response to mitogens in cancer patients: The role of suppressor cells. Int. 3. Cancer, 19, 605. ZIELSKE, J.V. & GOLUB, S.H. (1976) Fetal calf-seruminduced blastogenic and cytotoxic responses of human lymphocytes. Cancer Res. 36, 3842.
In vitro proliferation of macrophage depleted human peripheral blood lymphocytes A. J. TREVE S, V. BARAK & Z. FUKS Department ofRadiation and Clinical Oncology, Sharett Institute of Oncology, the Hebrew University-Hadassah Medical School, Jerusalem, Israel
(Acceptedfor publication 8 June 1979) SUMMARY
The incorporation of thymidine by normal human peripheral blood lymphocytes was tested in vitro following various culture conditions. A significant increase of thymidine uptake was observed in cultures depleted of plastic adherent, nylon wool adherent, or phagocytic cells. This proliferative activity occurred in the presence of various sera but was not due to a blastogenic response to a foreign protein, since it was also observed when autologous plasma was the only source of protein in the culture medium. The similarities and differences between this 'spontaneous' proliferative phenomenon and other blastogenic responses which are regulated by macrophages are discussed.
INTRODUCTION The blastogenic response of lymphocytes to foreign antigens is one of the in vitro cell mediated immune responses most extensively explored. Increased incorporation of thymidine in vitro occurs in mixed lymphocyte cultures (MLC) (Dutton, 1966), following secondary interaction with previously encountered antigens (Dutton, 1967) and following exposures to nonspecific mitogens (Ling, 1968). In addition, it has been shown that blastogenic and cytotoxic responses of human lymphocytes may occur following their in vitro incubation in the presence of foetal calf serum (FCS) (Johnson & Russel, 1965; Zielske & Golub, 1976). Ortaldo, Bonnard & Herberman (1977) and Treves et al. (1978) have recently reported that culture induced cytotoxicity occurs following incubation of lymphocytes in the presence of FCS but not in human serum (HS), and that this cytotoxicity is probably different from the natural killing activity (Treves et al., 1978). These proliferative and cytotoxic reactions are initiated following the exposure of the responding lymphocytes to foreign antigens. On the other hand, it has recently been shown that in vitro autoreactivity of T lymphocytes against non-T cells may occur in an MLC response. The autologous MLC usually occurs following recombination of pre-separated populations of T and non-T lymphocytes or lymphoblastoid cell lines, and is not caused by an exposure to a foreign antigen (Green & Sell, 1970; Birnbaum, Siskind & Weksler, 1972; Opelz et al., 1975; Kuntz, Innes & Weksler, 1976; Smith, 1978; Weksler et al., 1978). In the present study, we report that in vitro enhanced incorporation of thymidine and blastogenic response by human peripheral blood lymphocytes (PBL) may occur following the removal of macrophages from the culture and in the absence of extrinsic antigenic stimulation. MATERIAL AND METHODS Lymphocytes. Peripheral blood mononuclear cells (PBM) were obtained from healthy male and female donors 18 to 40 years old. Venous blood was drawn and mixed with 50 iu/ml sodium heparin (Leo Pharmaceutical Products, Ballerup,
Correspondence: A. J. Treves, Department of Radiation and Clinical Oncology, Sharett Institute of Oncology, the Hebrew University-Madassah Medical School, Jerusalem, Israel. 0099-9104/89/1200-0531$02.00 (© 1979 Blackwell Scientific Publications
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Denmark). The PBM were separated on Ficoll-Hypaque gradients (Boyum, 1968). The separated mononuclear cells were washed once with phosphate-buffered saline (PBS) pH 7-2. Cell separation procedures. Nylon wool adherent cells were removed by the method of Julius, Simpson & Herzenberg (1973). In various experiments 60%+ 17 (47-72) non-adherent cells were recovered following this separation procedure. Plastic adherent cells were removed by culturing 20-30x 106 PBM in 7 ml of complete RPMI-1640 media with 5% foetal calf serum (FCS) (Microbiological Associates, Bethesda, Maryland; aseptically collected) in 10 cm plastic petri dishes (Nunc, Roskilde, Denmark) for 1 hr at 37 C. The culture media were supplemented with GIBCO MEM vitamin solution (1: 100); Penicillin-Streptomycin 50 ju/ml and 50 1u/ml respectively; L-glutamine 2 mM; Sodium Pyruvate 1 mM; and Hepes buffer 10-2 M. Following separation 80% + 15 (70-91) non-adherent cells were recovered from the original cell population. Phagocytic cells were removed following incubation with carbonyl iron. Approximately 30-40 x 106 PBM were incubated with 50 mg of carbonyl iron in a 17 x 100 mm test tube for 30 min at 37 C. The carbonyl iron fed macrophages were attracted to the bottom of the tube by a magnet and the cells remaining in the supernatant were collected. In different experiments 67%+ 12 (48-80) of the cells were recovered from the original populations. Each of the three procedures resulted in reduction in the percentage of macrophages to 1-4% as shown by phagocytosis of latex particles. FCS was replaced with 5% pooled human AB serum (HS) during the separation procedures whenever cells were prepared for experiments in which sera other than FCS were present in the culture medium. In vitro cultures. PBM or macrophage depleted populations of PBM were cultured in different concentrations in round bottom microtitre plates (Nunc) in 0-1 ml of supplemented RPMI-1640 with either 10% of FCS (Microbiological Associates) or human pooled AB sera or autologous plasma (AP). All sera and the autologous plasma were heat inactivated. Unless otherwise indicated, the cells were incubated for six days in 5% C02 in air in a humidified incubator. On the fifth day of incubation, 1 pUCi of 3H-thymidine (Nuclear Research Center, Negev, Israel; specific activity 5 Ci/mM) in 10 puLi of PBS was added to each well. At the end of the sixth day the cells were harvested on glass fibre filters by an automatic cell harvester (Titertek Cell Harvester, Flow Laboratories, Oslo, Norway). The filters were dried out, immersed in toluene scintillation fluid and the radioactivity was counted in a fi scintillation counter. In some experiments, thymidine uptake was replaced by radioactive leucine. In these experiments the cells were cultured in leucine free medium for five days and then 1 pUCi of 3H-leucine (Nuclear Research Center, Negev, Israel; specific activity 30 Ci/mm) in leucine-free media was added to each well. Mitotic activity was also measured by three hours accumulation of mitoses using 1 mg/ml of Colcemid. After this procedure, the cells were collected, stained with methanol-Giemsa and 500 cells in each experimental group were scored for mitotic and blast figures. Phytohaemaglutinin (PHA) (Wellcome Research Laboratories, Beckenham, England) when used was added to the same cultures to a final concentration of 1 uLi pet well. In other experiments concanavalin A (Con A) (Miles Yeda, Rehoxvot, Israel) crystallized three times was added to a final concentration of 15 ug/ml. The mitogens were added to the culture either on the first day or on the third day of the culture. Two days later, the cultures were labelled by 1 pUCi of 3H-thymidine and harvested as described above. Viable cell counting was performed by the trypan-blue (0-04%) exclusion test. Each experimental group consisted of four duplicates and results were expressed as the average counts per minute+ one standard deviation (ct/min+ s.d.). In some experiments, the activity index was calculated by dividing the ct/min of the depleted PBM cultures by the ct/min of the non-depleted PBM culture. Each experimental procedure was performed with PBM obtained from at least three different donors. The figures presented in this manuscript show the results observed with a single donor, but consistent phenomena were always observed with the cells obtained from the other donors as well.
RESULTS Fig. 1 shows the incorporation of 3H-thymidine by normal PBM during the first seven days of culture. Enhanced incorporation occurred in cultures depleted of nylon wool adherent cells. The highest incorporation occurred in cultures depleted of nylon wool adherent cells. The highest incorporation occurred during the fifth day of incubation when the concentration of cells in the culture was high (7 5 x 105 cells/OK1 ml) and on the seventh day when the concentration of cells in the culture was decreased (2 5 x 105 cells/O01 ml). Some incorporation of 3H-thymidine was observed in non-depleted PBM but this occurred only when the concentration of cells in the culture was low, and even under these conditions the absolute activity was significantly lower compared to the activity of the nylon wool depleted cells of the same cell concentration. It can be concluded that either lowering of cell concentration, or removal of nylon wool adherent cells from PBM results in similar phenomena of enhanced thymidine uptake. Fig. 2 compares the enhanced proliferation of nylon wool depleted PBM with that occurring following stimulation of whole PBM by PHA. The peak of the mitogenic response to PHA occurred two days earlier than that of the spontaneous proliferation of nylon wool depleted cells. It was also observed that removal of nylon wool adherent cells significantly enhanced the mitogenic response of PBM or PHA. In addition to removal of nylon wool adherent cells, the removal of plastic adherent or phagocytic cells from the cultures also resulted in enhanced lymphocyte proliferation (Fig. 3). These results suggest
In vitro human lymphocyte proliferation
533
0 x
.E U-
3 4 5 6 Time after culturing (days)
FIG. 1. Effect of cell number and duration of culture on incorporation of 3H-thymidine by whole and macrophage depleted PBM. Different numbers of PBM (o a) or of nylon wool depleted PBM (e 0) were incubated for 1-7 days in supplemented RPMI-1640 with 10% FCS, pulsed with 3H-thymidine and assayed for radioactivity.
300
A
240 x
180 _ 120 A
60
2
4 6 Time after culturing (days)
FIG. 2. Spontaneous and PHA induced incorporation of 3H-thymidine by whole and macrophage depleted PBM. 5 x 105 PBM (open figures) or nylon wool depleted PBM (closed figures) were assayed for 3H-thymidine incorporation at different times following initiation of culture. Spontaneous incorporation (circles), and the mitogenic response to PHA (triangles) are shown. PHA was added at initiation of culture.
that macrophages present naturally in PBM, inhibit the proliferation of PBL in culture. To further test this hypothesis, we examined the proliferation of macrophage depleted PBM before and after reconstitution with plastic adherent cells. When phagocytic cells depleted PBM were plated on wells containing plastic adherent cells (prepared by pre-incubation of PBM of the same donor for 60 min at 370C followed by removal of the non-adherent cells), the mitogenic response to PHA or Con A was significantly reduced
A. J. Treves, V. Barak & Z. Fuks
534
to0 r-
E L).
4.0 6-0 Number of cells (x0-5)
FIG. 3. Effect of mode of macrophage depletion on the incorporation of 3H-thymidine by PBM. PBM were depleted by plastic adherence (e 0), removal of nylon wool adherent cells (A A), or by removal of U = Carbonil iron) and assayed for 3H-thymidine incorporation. (x x ) Indicates phagocytic cells (U no separation. 260 240
220 200 0 x
+1 c U)
._
180 160 140
120 100
80
60 40
20
Ii
-Vwith with -- with - with iwith I1with monocytes monocytes ! monocytes No mitogen Con A PHA "
FIG. 4. Effect of reconstitution with plastic adherent cells on spontaneous or PHA induced 3H-thymidine incorporation, by whole or macrophage depleted PBM. 6x 106 whole PBM (0) or phagocytic cells depleted PBM (a) were plated in round bottom microtitre wells, or in similar wells which had been coated previously with autologous plastic adherent cells derived from 6 x lo0 whole PBM. PHA or Con A were added to some wells after four days in culture. 3H-thymidine incorporation was assayed after six days in culture.
following reconstitution of PBM depleted of phagocytic cells with plastic adherent cells (Fig. 4). It was also noted that with low cell concentrations in the culture, the responsiveness to these mitogens was considerably less inhibited by macrophages (results not shown). Similar to the enhanced incorporation of 3H-thymidine, there was also augmentation in the incorporation of 3H-leucine by macrophage depleted PBM, which was significantly higher than that observed with whole PBM (Table 1). In all of the experiments described above, the culture media contained 10% FCS. Table 2 shows that significant proliferative activity was also observed in the presence of other sera in the culture medium,
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535
TABLE 1. The incorporation of radioactive thymidine or leucine by macrophage deleted PBM*
3H-thymidine
3H-leucine
Donor name
Whole PBM
Phagocytic Cells Depleted PBM
Activity index
Whole PBM
Phagocytic cells depleted PBM
Activity
SA SC
853+ 196 3144+ 1357
25080+ 5900 209205+ 19321
29-4 66-5
7160+ 1079 16019+ 1559
48052+ 10658 51151+4673
3-2
index 6-7
* 6x 105 whole PBM or phagocytic cell depleted PBM were incubated in leucine free RPMI-1640 with 10% dialysed-FCS for five days. Radioactive thymidine or leucine were then added for 24 hr. followed by cell harvesting and counting of radioactivity.
TABLE 2. Effect of various sera on the incorporation of 3H-thymidine by macrophage depleted PBM*
Source of serum
Autologous plasma Human AB serum (1) Human AB serum (2) Human cord blood serum Horse serum Rabbit serum Calf serum Foetal calf serum
Whole PBM
Nylon wool depleted PBM
918+ 110 1717+393 943+ 556 2566+ 319 1316+ 833 6728+3511 2460+ 1330 1692+ 494
15830+46 26115+6942 17141+ 1946 28840+ 2902 59379+ 13350 35230+4098 60338+ 4709 63785+ 10946
Activity index 17-2
15-2 18-2 11-2 45 1 52 24 5 37-7
* 6 x IO1 whole PBM or nylon wool depleted PBM were incubated in the presence of different sera for 5 days; pulsed for 24 hr with 3H-thymidine and radioactivity assayed. Separation and elution procedures were performed with medium containing 5% human AB serum.
including autologous plasma. The degree of spontaneous proliferation in the presence of autologous plasma was similar to that observed in the presence of FCS (Table 3). The total ct/min of macrophage depleted PBM was more than tenfold higher than that observed with the corresponding whole PBM in 6/10 donors when FCS was present in the medium culture compared to 5/10 in the presence of autologous plasma. Blast formation and mitotic activity were also observed in the phagocytic cells depleted PBM in the presence of autologous plasma and in the absence of any foreign components in the culture media. No such activity was found in the undepleted populations (Table 4). DISCUSSION In this study we have shown that proliferation of human lymphocytes can be initiated in culture by the removal of macrophages. A complete removal of macrophages was not necessary since the depleted populations ot PBM in our experiments still contained 1-4% of phagocyte cells. Furthermore, lowering the total number of macrophages by merely reducing the number of undepleted PBM in the cultures to 2 5 x 105 cells/0-1 ml also resulted in a weak but still detectable proliferative response (Fig. 1). Furthermore, the presence of macrophages in certain proportions was necessary to suppress the proliferation of lymphocytes since the addition of macrophages to macrophage depleted cell populations inhibited the in vitro proliferation (Fig. 4). The addition of macrophages to phagocytic cells depleted PBM also suppressed the responses of T cells to mitogens (Fig. 4).
A. 9. Treves, V. Barak L Z. Fuks
536
TABLE 3. Effect of FCS or autologous plasma on the incorporation of 3H-thymidine by macrophage depleted PBM*
Autologous plasma
FCS Whole PBM
Depleted PBM
3091+693 100+ 30 174+ 54 6238+ 1127 1804+900 1728+ 123 918+ 110 223+29 1512+ 423 2655+1442
9663+3032 7929+ 1764 8348+ 708 30386+5235 14396+4196 21530+4218 15830+46 3349+248 5694+ 834 5667 +567
Activity index
Donor name
Whole PBM
Depleted PBM
EK JX HB MS MH TZ IC AB AH TT
1788+499 1444+ 172 133+ 54 212+2 285+58 1338+269 1692+494 1884+225 3317+401 2750±757
12009+ 1515 10142+ 2005 12768+ 1348 27411+8817 65192+7011 37409+ 1902 63785+ 10946 23057+3804 12332+ 2030 11713+892
6*7 7*0 96-0 129 0 228*0
28*0 37-7 12-1 3-7 4-3
Activity index 31
7913 48-0 49 8-0 12 5 17-2 15 0 3-8 21
* Phagocytic cells (first four donors) or nylon wool adherent cells (other donors) were removed from PBM and 5-6x 105 cells were incubated for five days in the presence of 10% FCS or autologous plasma pulsed for 24 hr with 3H-thymidine and assayed for radioactivity. Separation and elution procedures were performed with media containing 5% human AB serum. TABLE 4. Mitotic and blastogenic activity of phagocytic cells depleted PBM*
Phagocytic cells depleted PBM
PBM
Experiment number
Donor name
Sera
ct/min+ s.d.
Mitotic index
Blastogenic index
ct/min+ s.d.
Mitotic index
Blastogenic index
1
AH AH AB HB VB VB SR SR
AP FCS AP FCS AP FCS AP FCS FCS
432+ 36 124+ 2 112+5 243+27 469+ 18 627+ 138 2444+426 5091+471 2931+590
0 0 0 0 0 0 0 0 0
2 1 0 1 0 1 0 0 1
33158± 3898 40456± 3078 4703±764 11157+630 33525±4769 47475± 6448 89396+ 16035 68042+ 13914 87071+3853
1 1 1 3 5 4 1 3 4
10 6 7 13 15 7 8 9 20
2
3
JG
* Phagocytic cells were removed from PBM and 6 x 105 cells were incubated for five days in the presence of either 10% FCS or autologous plasma, pulsed for 24 hr with 3H-thymidine and assayed for radiolable incorporation. Part of the cultures were treated with colcemid for 3 hr and then collected and stained. The percent of mitotic figures and blast cells were calculated by counting 500 cells in each experimental group.
The in vitro proliferation of macrophage depleted lymphocytes in this study is unlikely to represent merely a blastogenic response to foreign proteins, such as FCS or other heterologous sera, since it also occurred in the presence of human AB serum and in the presence of autologous plasma (Tables 2, 3, 4). On the other hand, it is possible that the higher proliferative responses observed in the presence of FCS or horse serum (Table 2) could be attributed to an additional mitogenic response to the foreign antigens present in these cultures. A similar phenomenon of a mitogenic reaction to FCS has been described previously (Johnson & Russell, 1965; Zielske & Golub, 1976). Johnson & Russell (1965) have also reported that mitotic activity and blast formation may occur in the presence of autologous sera. In these experiments, however, the lymphocyte cultures were treated with carbonyl iron prior to their in vitro incubation and no attempt was made to investigate the role of macrophages in the proliferative response.
In vitro human lymphocyte proliferation 537 Macrophage mediated suppression of proliferative responses of lymphocytes has been well documented (Keller, 1975). It has been shown that macrophages secrete soluble mediators which regulate the blastogenic response of lymphocytes (Nelson, 1973; Calderone & Unanue, 1975). Macrophages were also found to regulate haematopoiesis in vitro (Cline, 1979). Some investigators have attributed the macrophage related inhibition of blastogenic response in the mouse to the presence of large quantities of 'cold' thymidine secreted by the macrophages which competitively inhibit the uptake of radioactive thymidine used to assay the blastogenic response (Opitz, Naethammer & Jackson, 1975). However, it is unlikely that 'cold' thymidine competition is the explanation for the macrophage related inhibition of the augmented proliferation of human lymphocytes shown in our study since in the absence of macrophages, proliferation has also been demonstrated by 3H-leucine incorporation (Table 1). Furthermore, actual blast formation and enhanced mitotic activity were observed in the macrophage depleted PBM cultures but not in whole PBM cultures, even in the absence of foreign proteins in the culture media (Table 4). These observations cannot be explained by selective inhibition of 3H-thymidine uptake by 'cold' thymidine. They also stress the point that actual blast formation and cell division, and not just thymidine uptake, were induced by the removal of macrophages even in the presence of autologous plasma. In vitro proliferation of lymphocytes as a result of antigenic or mitogenic stimulation has been described extensively (Dutton, 1967; Ling, 1968; Keller, 1975). It has also been shown that chemical modification of the culture medium, such as the addition of mercaptoethanol (Goodman & Weigle, 1977) or pretreatment of lymphocytes with periodate (Novogrodsky, 1975) also resulted in in vitro proliferation of lymphocytes. In vitro proliferation of mouse thymocytes has been demonstrated following the addition of soluble factors secreted by macrophages. 'Spontaneously' proliferating mouse T cells were also suggested to mediate suppression of graft-vs-host response (Moorhead, 1978; Calderon & Unanue, 1975). However, all these experiments were performed in the presence of FCS which may contribute some antigenic stimulation. There has been only one situation in which in vitro proliferation of human lymphocyte occurred in the absence of a foreign substance in the medium (Opelz et al., 1975; Weksler et al., 1978; Smith, 1978). It has been shown that human T cells proliferate following their recombination with autologous B cells (the autologous MLC reaction) and that this reaction occurs in the absence of an extrinsic antigenic stimulation. The nature of the enhanced proliferation of human peripheral blood lymphocytes occurring after the removal of macrophages described in this paper is still unclear. It does not seem to represent an autologous MLC reaction since preliminary experiments performed in our laboratory have shown that the cell proliferating in our experiments is not a T cell, and that this cell population is different from the cells responding to T cell mitogens such as PHA and Con A (data not shown). It is possible that the cells proliferating in our experiments have been artificially modified by one of the experimental procedures and are therefore undergoing a blastogenic response. On the other hand, such a modified population may itself serve as a stimulus for blastogenic response of another subset of lymphocytes in the human peripheral blood. Although such an artificial modification is possible, it is very difficult to prove or disprove it for the obvious methodological limitations of the currently available testing systems. Alternatively, it is possible to speculate that lymphocyte proliferation in vitro may occur 'spontaneously', namely, without any antigenic stimulation, but only as a result of the reduction in the number of macrophages. It is possible that the apparently 'spontaneous' proliferation described in our study represents a physiological process of human lymphocyte proliferation which seems to be regulated by macrophages. Any interference in the balance between the growth regulatory activity of macrophages and the proliferative capacity of certain subpopulations of lymphocytes may result in excessive proliferation of these cells. Such an imbalance may in fact exist in certain states of disease. It has recently been shown that spontaneous incorporation of thymidine and spontaneous blast formation occur in lymphocytes separated from blood of patients with Hodgkin's disease (Shiftan, Caviles & Mendelson, 1978), infectious mononucleosis (Nikoskelainen & Stevens, 1979) and rheumatoid arthritis (Slaughter et al., 1978). It has also been shown that certain abnormalities of macrophage function, such as increased suppressor cell activity occur in Hodgkin's disease (Zembala et al., 1977; Hillinger & Herzig, 1978), although this abnormality may be at least in part attributed to suppressor lymphocyte dusfunction (Twomey et al., 1975). Further-
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more, Kaplan & Gartner (1977) have recently suggested that the Reed-Sternberg cell of Hodgkin's disease originates from macrophages, a phenomenon which represents another macrophage related abnormality in a lymphoproliferative disorder. However, this hypothesis on the interaction between macrophage activity and lymphocyte proliferation under normal conditions and in certain lymphoproliferative diseases should be regarded at the present time as highly speculative and further investigations will be necessary to test its validity. This work was supported in part by the Joint Research Fund of the Hebrew University and Hadassah, and by the Philip Lash and Dr Edward D. Lash Research Endowment Fund.
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