Macrophage migration stimulation factor, migration inhibition factor and the role of suppressor cells in their production

CELLULAR

IMMUNOLOGY

47,69-78

(1979)

Macrophage Migration Stimulation Factor and the Role of Suppressor ROY A.Fox Department

of Medicine,

Dalhousie Received

Factor, Migration Inhibition Cells in Their Production

AND K. RAJARAMAN University, October

Haiifox,

Nova

Scotia,

Canada

I I, 1978

Guinea pig lymph node lymphocytes and human peripheral blood lymphocytes when stimulated by specific antigen or mitogen will release factors that atfect in vitro macrophage migration. Migration inhibition factor production appears to be under the control of suppressor cells which are T lymphocytes. When suppressor cells are generated by stimulation with Con A for 4 days, migration stimulation factor (M.St.F.) activity is found. In other situations where M.St.F. is found this is thought to be due to increased suppressor cell activity. For example, young adults produce this lymphokine when stimulated with Con A, whereas aged individuals produce MIF. Concanavalin A appears to be the mitogen of choice for M.St.F. production, and phytohemagglutinin for MIF production. The release of this putative factor M.St.F. from suppressor T cells helps to explain some of the difficulties that have existed in studies of macrophage migration inhibition.

INTRODUCTION The inhibition of in vitro macrophage migration is one of the most widely used measurements of delayed hypersensitivity. This test depends upon the release of a specific mediator or lymphokine from lymphocytes, known as macrophage migration inhibition factor (MIF). MIF is synthesized and released by small lymphocytes (1,2), both B and T (3-5). Furthermore, macrophages are essential for T-cell MIF (5,6) but not for B-cell MIF (5). Suppressor cells appear to control the production of MIF by both B and T lymphocytes (7). In both man and guinea pig in vitro aging of cells results in potentiation of MIF production in response to both specific antigen, PPD, or mitogen, Con A. It was suggested (7) that aging resulted in loss of suppressor cells and thus potentiation of MIF production. This phenomenon has been studied in greater depth and one of the purposes of this paper is to present experimental evidence that the suppressor function rests with the T lymphocyte. In performing these experiments new evidence has come forward with regard to another possible lymphokine, namely macrophage migration stimulation factor (M.St.F). The supernatants of antigen-stimulated cells may enhance macrophage migration (8). Fox et al. (9,lO) reported M.St.F. to be present in both fetal calf serum (FCS) and the supernatants of guinea pig lymphocytes. Weisbart et al. (11) described a migration enhancement factor in cultures of human lymphocytes. More recently Kuhner and David (12) confirmed the presence of a stimulatory material in the mouse system. It has also been well described and 69 0008-8749/79/l 10069-10$02.00/O Copyright All rights

0 1979 by Academic Press, Inc. of reproduction in any form reserved.

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partially characterized by Aaskov and Anthony (13) in the supematants of human lymphocytes. Thus, the result of a migration inhibition assay depends upon a number of factors which include the balance of inhibitory and stimulatory activities. Suppressor cells appear to be of great importance in determining which of these activities predominates. The nature of these suppressor cells and the conditions which predispose to the formation of M.St.F. have been investigated. The results of these experiments will be reported here. METHODS 1. Guinea pig M.St.F. Guinea pigs were injected with complete Freund’s adjuvant to induce tuberculin reactivity and 3 weeks later the lymph nodes were harvested. Single-cell suspensions were prepared and cultured according to the method of Bloom and Bennett (14). No serum. was added to these cultures, the concentration of lymphocytes being 2 x lO’/ml. The stimulated, lymphokine containing, supernatants were obtained by centrifugation following a 24-hr culture of the lymphocytes in the presence of PPD (50 pg/ml). The same amount of PPD was added to the control supematants immediately prior to centrifugation following the 24-hr incubation. 2. Human M.St.F. Healthy adult volunteers of various ages were used as the source of human peripheral blood lymphocytes. Mononuclear cells were separated from whole blood by the Ficoll-Isopaque method (15) and set up for culture in the same way as guinea pig lymphocytes. The cells were routinely stimulated with concanavalin A (10 pg/ml). However, the culture conditions were varied to determine optimal conditions for M.St.F. production. For example, the concentration and type of mitogen, the cell concentration, and the duration of culture. 3. Separation of subpopulations. T lymphocytes were identified and also removed by rosetting with sheep red blood cells (16). Removal of T lymphocytes provided a B-enriched population, and B cells were enumerated by counting cells fluorescing with anti-IgM (17). The T-enriched population was obtained by rosetting and red cells were removed by lysis with 0.81% ammonium chloride. Monocytes were removed by either phagocytosis with iron filings (17) or by adherence to petri plates. Monocytes were recovered after adherence by removal with a rubber policeman or addition of lidocaine (18). These techniques provided enriched but not purified populations. The T-enriched population contained between 40 and 70% T cells, and between 3 and 8% surface immunoglobulinpositive cells. The B cells were enriched to 30 to 50% surface immunoglobulinpositive cells. The adherent population was enriched to 40 to 70% monocytes determined by capability to phagocytize yeast. 4. Identification ofsuppressor cell. Peripheral blood mononuclear cells were set up in culture at a concentration at 107/ml in RPM1 1640. Cultures were aged by holding at 37°C for 24 hr. At the end of this time the donor received a second venepuncture, and T-, B-, or monocyte-enriched fractions were prepared by the methods described. These populations were then added to the aged populations at a ratio of 1: 1, giving a final concentration of IO7 cells/ml. MIF was generated by addition of Con A, 10 pg/ml, to the cultures which were then incubated at 37°C in 5%

SUPPRESSOR

CELLS

AND

LYMPHOKINES

71

CO, for 24 hr. The same concentration of Con A was added to control cultures at the end of the 24-hr incubation. In subsequent experiments T cells were removed by rosetting in the usual way and the monocytes were then obtained from the nonrosetted population by adherence. This provided a greater enrichment of the T and B populations, reducing the contamination by monocytes. 5. Enrichment of suppressor ceil population. Human peripheral blood mononuclear cells at a concentration of 5 x 10Yml in RPM1 1640 medium, 10% FCS, were divided into two aliquots. Activated cultures had Con A, 6 pg/ml, added at the beginning of the culture period, and for control cells the same concentration was added on completion of the culture. After 96 hr of culture, both control and activated cells were treated with mitomycin C, 50 pg/ml, for 30 min at 37”C, washed three times with 300 nG2 a-methyl-D-mannoside, twice with medium (RPM1 1640), and then resuspended in medium. Cell viability was assessed using eosin exclusion. The cells activated with Con A are referred to as suppressor cells, and the nonstimulated as control cells. Suppressor or control cells were then added to 2 x 10” aged, (24 hr) autologous, responder cells. The number of cells added ranged from 0.5 x 10Vml up to 10 x lO”/ml. Con A, 10 Fg/ml, was added to the aged plus suppressor cell population and incubated at 37°C for 1 day. The same concentration of Con A was added to the aged plus control cell population at the completion of the culture period. The supernatants were collected after centrifugation, then diluted 1:4 with medium, and assayed for their effect on migration of guinea pig peritoneal exudate cells. This technique is similar to that of Ante1 et al. (19). 6. Migration assay. The in vitro migration of guinea pig peritoneal macrophages was measured. An exudate was induced by the intraperitoneal injection of 20 ml sterile Marcel, and the macrophages after harvesting were packed into capillary tubes (7,9,10,20-24). Each migration chamber contained two capillaries, and each supernatant at 1 in 4 dilution in serum-free RPM1 1640 was set up in duplicate or triplicate. Each capillary tube contained 2 x 10fi peritoneal exudate cells and between 70 and 90% were macrophages. The migration area was measured, the area in stimulated supernatants was compared to the area in control supernatants, and results were expressed as migration index (M.I.). Supernatants were tested immediately on the day of harvesting. RESULTS 1. Detection

of Stimulatory

Activity

in Guinea Pig

Twenty-six guinea pigs were used to “manufacture” lymphokines 3 weeks after the injection of CFA. Each supernatant was tested in the macrophage migration assay at multiple dilution, and 16 (61%) contained significant inhibitory activity indicating the presence of MIF. In five of these supernatants no activity was detected at the first dilution tested, but was detectable as the sample was diluted further. This is interpreted as a prozone phenomenon. Three supernatants had no significant effect on macrophage migration. In the remaining eight stimulated supernatants there was significant stimulation of macrophage migration and this was the only detectable activity in at least one dilution. In only one of these eight supernatants was MIF detectable at any dilution when migration stimulation factor M.St.F. was present. On seven occasions the stimulated supernatants which

72

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contained MIF lost this activity on storage at -20°C and stimulatory activity (M.St.F.) became detectable (Table 1). Once present, M.St.F. remained and in three samples multiple testings were performed up to 6 weeks. M.St.F. activity remained and appeared to be stable. 2. Stimulatory

Activity

in Human

Lymphocyte

Supernatants

When human peripheral blood mononuclear cells are used as a source of lymphokine, M.St.F. is frequently found. A number of factors appears to contribute to this. (a) Nature and concentration of mitogen. When concanavalin A is used as mitogen, the supernatants are more likely to stimulate macrophage migration. M.St.F. production is less obvious when PHA is the mitogen. This is evident in Fig. 1. In this experiment the cells from one individual were cultured at a concentration of 5 x lo6 cells/ml and exposed to different concentrations of Con A or PHA. In the case of Con A the stimulated supernatant always contained M.St.F. (MI. > 1.20). When PHA is used as mitogen, MIF appears at the higher concentrations. Thus, the optimal concentration for M.St.F. production by Con A is 3 pg/ml and for MIF production by PHA, 50 pg/ml. The optimal cell density was also determined and found to be between 5 and 10 million cells per milliliter. Under optimal conditions the lymphokine production of five healthy young adults was studied. With Con A as mitogen, mean M.I. was 1.82 with a standard error of 0.16. All supernatants contained significant stimulatory activity, thus M.St.F. was produced and no MIF could be detected. In contrast, when PHA was mitogen, MIF was present in two of the five supernatants, and M.St.F. in one of four. Mean MI. was 1.04, the standard error was 0.14. (6) Age oflymphocyte donor. The technique used for lymphokine production is that widely used for routine MIF production. Yet in these diluted samples when TABLE

1

The Development of Macrophage Migration Stimulation in Supernatants That Once Contained MIF” MIF dilution (reciprocal) Experiment No. 1 2 3 4 5 6 7 Mean

2 0.58 0.87 0.66 0.76 0.59 0.69

’

4

8

Days in storage at - 20°C

0.78 0.54 0.76 0.99 0.77 0.77

0.80 0.96 0.99 1.03 0.71 0.79 0.65 0.79

5 J 14 2 32 12 3 11

Factor

M.St.F. dilution (reciprocal) 2

4

8

1.33 1.43 1.83 1.66 1.56

1.29 1.00 1.57 1.79 1.42 1.12 1.19 1.34

1.38 2.86 1.43 1.39 1.04 1.34 1.53 1.57

U The results are expressed as migration index-the stimulated supematants divided by the control supernatants. The normal range is 0.80 to 1.20. The supematants were assayed on the day of harvesting for lymphokine activity, the remainder was frozen and stored at’-20°C for varying lengths of time. The same supernatants were reassayed at the same dilutions.

SUPPRESSOR

CELLS

AND

73

LYMPHOKINES

2 .oCon

A

l.l?-

1.6-

50 DOSE

FIG. 1. Human peripheral blood mononuclear cells/ml and exposed to different concentrations migration index.

-

ml/m1

cells were cultured at a concentration of 5 x 10” of Con A and PHA. The results are expressed as the

Con A is used as a mitogen, we find preferential M.St.F. production. We looked at the effect of age of donor. The results are illustrated in Fig. 2. Supematants from Con A-stimulated lymphocytes of ten healthy young adults (20-38) are compared with nine healthy old adults (75 years and older). Lymphokine production was assessed using freshly harvested cells, and cells that had been allowed to age in vitro for 24 hr prior to stimulation. Six of the ten young adults produced M.St.F. in fresh cultures, and none produced MIF. When the lymphocytes were aged, M.I. decreased in all but two, and in two MIF could be detected. In contrast, seven of nine aged individuals showed significant MIF production, and none produced M.St.F. Aging these cells in vitro produced no significant change. 3. Identification

of Suppressor

Cell

When peripheral blood mononuclear cells of healthy young adult volunteers are aged in vitro, MIF activity appears, which confirms our previously published observations (7). The results of six experiments are shown (Table 2). When T-enriched or fresh whole populations are added to the aged cells MIF can no longer be detected in the supernatants. The mean M.I. for aged cells was 0.69, with added T cells 0.99, and for whole populations 0.87. These reconstituted populations were then behaving as fresh populations. In contrast, addition of a B-enriched population significantly increased MIF activity from a mean M.I. of 0.69 to 0.53. The effect of addition of adherent cells is more variable. In four experiments MIF was still produced after addition of adherent cells although slightly decreased in two. In the other two, no MIF was produced after adding adherent cells. The mean M.I. with adherent cells was not significantly different from that of the aged population alone.

74

FOX AND RAJARAMAN YOUNG

-I

I FRESH

&GED

Aging

FRESH

AGED

in Vitro

FIG. 2. Peripheral blood mononuclear cells from 10 healthy young adults, aged 20 to 38 years, and from 9 healthy old adults, over 75 years, were obtained. These cells, either fresh or aged for 24 hr, were stimulated with 10 pg Con A/ml and the supernatants collected. The supernatants were then assayed for lymphokine activity and the results are expressed as migration index.

Four further experiments were carried out wherein T cells were removed prior to preparing the adherent cell population, thus ensuring a more homogeneous monocyte population. The results are shown in Table 3. In these experiments three of the four fresh cultures produced migration stimulation factor. However, aging TABLE Characterization

2

of Suppressor Cell” Aged cells

Experiment No.

Mean SD P

Fresh cells

Alone

+T

+B

0.97 0.98 0.96 1.06 0.91 1.08

0.80 0.72 0.47 0.77 0.73 0.65

0.86 0.88 0.84 1.49 0.86 0.99

0.52 0.63 0.46 0.65 0.42 0.42

0.81 0.80 0.73 0.68 1.50 1.01

0.91 0.92 0.61 0.98 0.93 0.84

0.99 0.12 co.0025

0.69 0.06

0.99 0.25 co.025

0.53 0.09 co.01

0.92 0.30 N.S.

0.87 0.13 <0.0005

+Adherent

+ Whole population

a Mononuclear cells, lOr/ml, are stimulated with 10 pg Con A/ml, and the supematants collected and assayed for MIF activity using guinea pig peritoneal exudate cells. MIF activity was also measured after aging the cells in vitro for 24 hr. T-Enriched, B-enriched, monocyte/macrophage-enriched, and fresh whole mononuclear population were added to the aged cells and then cultured for 24 hr with Con A and MIF activity determined. Each result is compared with MIF activity in the aged population, using Student’s t test.

SUPPRESSOR

CELLS

AND

TABLE Further

Characterization

3 of the Suppressor

Cell”

Aged Experiment No.

75

LYMPHOKINES

cells

Fresh cells

Alone

+T-enriched

1 2 3 4

1.29 1.42 1.29 0.80

0.91 0.91 0.79 0.76

1.12 1.39 1.13 0.82

0.85 0.87 0.77 0.79

Mean SD P

1.20 0.23 <0.025

0.84 0.08

1.12 0.23 -co.05

0.82 0.05 N.S.

+Monocytes

a The procedure was identical to that used for the experiments in Table 2, with one difference. The whole population was rosetted with sheep erythrocytes and T cells removed prior to preparation of the adherent cell population.

resulted in a significant decrease in M.I., thus an increase in MIF production. The mean M.I. for fresh cells was 1.20 and for aged 0.84, which was significantly different. There was a significant change in M.I. from 0.84 to 1.12 when T-enriched cells were added to the aged population. Adherent cells produced no change in MIF activity. 4. Enrichment

of Suppressor Cell Population

Suppressor cell function is thought to decrease with age (19). Furthermore, in vitro aging results in depletion of suppressor cells. The results reported so far are consistent with the hypothesis that suppressor cells are important in M.St.F. production. It appears that M.St.F. production increases with the number of suppressor cells. To test this hypothesis the experiment was designed as described in the methods section. It can be seen (Table 4) that M.St.F. production increases with the number of suppressor cells added. This experiment was repeated on three occasions and similar results were obtained. In the second part of the experiment the question was asked if the M.St.F. was coming from the responder cells, or from TABLE Generation

of Suppressor

Cells

4 and M.St.F.

Production” Suppressor

Fresh cells Aged cells Aged + suppressor 1. 0.5 x 106 2. 1 x 106 3. 2 x 106 4. 4 x 106 5. 10 x 106

cells alone

0.90 0.79 cells 0.88 0.98 1.20 1.70 2.30

0.81 0.97 0.86 1.32 2.10

u Suppressor cells were generated by 96 hr of culture with Con A. Suppressor cells or control were added to 2 x lo6 responder cells and cultured for 24 hr with or without Con A.

cells

76

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the suppressor cells. When suppressor cells only are stimulated with Con A, the results are essentially the same. It is concluded that M.St.F. is being released from the suppressor cell population and not from the responder cells. Furthermore, M.St.F. is released from these cells when no further Con A is added (Table 5). These results show that the suppressor cells are already producing M.St.F., and addition of Con A causes only a slight increase. At the end of the 96-hr culture period the viable cells tend to be clumped. This makes it difficult to separate into subpopulations. However, in two experiments T-enriched and B-enriched populations were prepared. Lymphokine activity was only found in the T-enriched fraction, where there was significant stimulation of migration on both occasions. DISCUSSION There have been many reports in the literature of stimulation of macrophage migration. This was first reported in 1928 when studying migration from spleen and kidney fragments of tuberculin-positive animals (25). It appeared that stimulation occurred with low concentrations of antigen. This was first reported in human peripheral blood mononuclear cells in 1957 (25), and confirmed in guinea pig spleens (8,26). Soborg (27) claimed that stimulation might be considered to be very similar to inhibition in that it reflects weak sensitization. In his experiments and in some reported here, antigen recognition is used to produce M.St.F. Other workers, using human peripheral blood leukocyte migration (28-30) confirm that stimulation reflects a state of delayed hypersensitivity. Thus, stimulation of in virro migration appears to be a well-recognized phenomenon when spleen cells or peripheral blood leukocytes are used as target. It seems to occur as frequently when peritoneal macrophages are the indicator or target cells (9). As early as 1958 Waksman and Matoltsy found that peritoneal cells from sensitized animals were more motile when cultured for 48 hr in the presence of antigen (3 1). Presumably the sequence of events is similar to that found with MIF. That is to say sensitized lymphocytes release a factor (lymphokine) which in turn affects macrophage migration. Thus, in this two-stage assay Fox er al. (9) have postulated that a stimulation factor is released. Recent work (13) indicates that MIF is distinct from M.St.F. The latter is not chemotactic factor, it has a pZ of 6.5, and a molecular weight between 50,000 and 250,000. Fox er al. (9) also reached the conclusion that M.St.F. was distinct from MIF. TABLE Effect

of Con A on M.St.F.

5

Production

by Suppressor Con

Suppressor cells Control cells

Cells” A

+

-

1.68 1.29

1.44 1.09

LI After washing with 2-methyl-o-mannoside following the 4-day culture, the cells were placed in culture for a further 24 hr with or without Con A. Results expressed as migration index. The supernatants were compared with medium alone.

SUPPRESSOR

CELLS

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LYMPHOKINES

77

It is suggested that M.St.F. is a factor released from lymphocytes. This factor is released from sensitized guinea pig lymphocytes in the presence of antigen, and from human peripheral blood mononuclear cells when stimulated with antigen or mitogen. Con A appears to preferentially induce M.St.F. formation whereas PHA induces MIF. This suggests that M.St.F. is released from a specific subpopulation of cells. If we consider the results in individuals of different ages, then M.St.F. seems to be the predominant activity when lymphocytes from young healthy adults are stimulated with Con A. Conversely, no M.St.F. could be detected in older individuals. It is known that suppressor cell activity decreases with age (19). We have previously suggested that suppressor cells are important in controlling MIF production (7). These data support this conclusion, and furthermore suggest that the suppressor cell is the source of M.St.F. If this is true, then the increased suppressor cell activity probably accounts for the weak sensitization as described by Soborg (27) and others (28-30) as already discussed. We have suggested (7) that loss of suppressor cells with aging enhances MIF production. The results presented here support that hypothesis. Enhancement of MIF production might be due to the decrease and loss of an opposing activity, namely, M.St.F. Our results indicate that the suppressor cell is a T lymphocyte. It also appears to be adherent, since adherent cells did inhibit MIF production in some of the early experiments. When adherent T cells were removed there was no significant inhibition by monocytes. David (32) has commented upon the relationship between helper and suppressor factors and the lymphokines. What is this relationship? Clearly helper factors and MIF are produced by a nondividing lymphocyte population; suppressor factors and macrophage migration stimulation factor (M.St.F.) are produced by a different population. In the experiments reported here enrichment of the B-cell population resulted in increased MIF production. The synthesis of MIF by B cells is well recognized (4,5) and we interpret this increased production on enrichment as being due to the removal of the suppressor T cells. T lymphocytes have been reported to produce a soluble factor that inhibits MIF production. Cohen and Yoshida (33) refer to this as MIF inhibiting factor (MIFIF). We have not defined any relationship between MIFIF and M.St.F. It is possible that they are identical but more work needs to be done. The complexity of lymphokine production is reenforced by the data presented in this paper. There is enough evidence in the literature and from these experiments to suggest that the in vitro phenomenon of stimulation of macrophage migration is due to the production of a lymphokine, M.St.F. To be sure of this further work is needed, particularly with regard to biochemical characterization. However, this is now possible since optimal conditions for the production of this activity have been defined. The presence of M.St.F. and the conditions which predispose to its formation may explain the difficulty in obtaining reproducible MIF activity using human cells. ACKNOWLEDGMENTS This work was supported by the Medical Research Council of Canada Research Society. We gratefully acknowledge the help of the photography Hospital.

and the Canadian Geriatrics department at Camp Hill

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(Johansson,