Scand, J. Immtmol. 9, 53-60. 1979
Evidence that Soluble Products Released by PHA-stimulated Human Lymphoid Cells Activate Immunosuppressive Monocytes E.-L. LARSSON & H. BLOMGREN Department of Biochemistry, Arrhenius Laboratot7, University of Stockholm, and Departmetil of Tumour Biology, Karolinska Institute, Slockholm, Sweden
Larsson, B.-L. & Blomgren, H. Evidence ihat Soluble Products Released by PHA-stimulated Human Lymphoid Cells Activate Immunosuppressive Monocytes. Seand. J. Immunol. 9, 5.1-60, 1979.
Soluble mediators, lyniphokines, released by stimulated lymphoid cells can modiry immunological responses in several ways. In this investigation we have examined whether ihe supernaiants of phytohaemaggluiinin (PHAl-aciivated human lymphocytes (active SUPs) contain factors ihat can suppress proliferalivc responses of lymphocytes in vilro. The results have shown ihat crude preparations of peripheral lymphoid cells incubated for lA h in active SUPs can suppress ihe responses of cocullured autulogoiis lympiiocytes to PPD tuberculin in vitro. Their suppressive activity was not abolished by mitomycin treatment. Some reduction of phytomitogen responses was also noted. Ma.ximal suppressive activity was obtained within 24 h of incubation in active SUPs and it could noi be induced in cell preparations depleted of monocytes macrophages. Similar results were obtained by treating lymphoid cells wilh tipopolysuccharide from Esclwrichia co/i, wbich is a known activator of monocytes. These results thus show that lymphokines released by stimulated lymphoid cells can aclivale monocytes macrophages in such u way that ihey become immunosuppressive. E.-L. Larsson. Basel InstittUe for Immtmology. Crenzachersirasse 487. Postfaeh 400S. Basel 3, Sii'ttzerland.
Soluble products of non-gammag!obulin nature, lyniphokines, generated by antigen- or phytomitogen-activated lymphocytes are considered Important regulators of immune respon.ses [21], There is evidence that certain lymphokines enhance proliferative responses of lymphocyies [10] whereas others may exert a suppressive effect [5, 10, 14]. The mode of action of these regulator substances is largely unknown. Previous experiments have shown that PHAactivated human lymphoid cells release factors which are highly mitogenic for sheep red blood cell rosetted (SRBC) T-cells [3, 13], Biochemical characterization of those mitogenic factors (M F) has also been performed [15]. Furthermore, circumstantial evidence has been presented that lymphokines released by such activated lymphoid cells can stimulate inhibitor cells capable of terminating proliferation of lymphocytes [5, 14]. 0300-9475/79/0100-0053 $02.00
In this article we present results showing that PHA-activated human lymphoid cells release soluble factors that stimulate monocytesmacrophages in such a way that they can inhibit proliferative responses of lymphocytes in vitro.
MATERIALS AND METHODS Preparation of lymphocytes. Heparinized venous blood was obtained from healthy donors ot" botb sexes. Lymphoid cells were separated by centrifugation on Ficoll-lsopatjuc |12). These preparations of lymphoid cells, which will be termed tion-purified. served as a source of non-adherent. Fe-piiri/ied cell preparations. They were oblained by incubating 5 • 10* cells for 1 h in a plastic Petri dish (with a diameter of 50 mml in 5 ml of Eagle's minimal essential medium supplemented with Earle's salts (MEM) containing 20" „ of heatinactivated human scrum (HS). The non-adherent cells were then incubated with iron powder followed by
.Q 1979 Blackwell Scientific Publications
53
54
E.-L. Larsson & H. Blomgren
treatment with a magnet to remove phagocjtic cells [2], T cells were separated by rosetling them wiih sheep er>tbrocytes (SRBC) followed by centrifugation on Ficoil-lsopaquu" (11]. Approximately 90-95"n of ihe T-cell-cnriched preparations formed spontaneous rosetles with SRBC. The corresponding figure for the other two cell preparations ranged between S0°o and 60%. Lymphocyte stimulants. Concanavalin A (Con A) was obtained from Sigma Chemical Co., St I.ouis, Mo., USA, II was dissolved in MPM and ils concentration expressed as [Jg/ml. Pbytohaemaggiulinin (I'HA) was purchased from Difco Lah., Detroit. Mich.. USA (Bacio Ph>tobaemagglutinin Ml. Tbe contents of viaLs containing lyophilized PHA were dissolved in 5 ml of MEM. This solution is referred to as \W\, PHA. Puritied protein derivative oi luberculin IPPD) was obtained from Statens Serum Institute, Copenhagen, Denmark. The concentration of this antigen is expressed as i^g'ml, Lipopolysaccbaride (LPS) was purchased from Difco Lab. (Lipopolysaccharide W. Escheriehia coli 055:B5). The concentration is e.xpressed as [Jg/ml. Production of active .supernatants (SUPs). Tbis procedure has been described in detail previously [3). Briefly, non-purified cell preparations were suspended in MEM containing antibiotics and 10" „ beat-inactivated HSin the presence of PHA at a final concentration of 3°o. Tbe cells were then incubated ft>r 24 h al 37 C in a humidified atmosphere containing 5" „ COj, wasbed several times, and reincubated in fresh MEM containing HS for another 24 b in the absence of PHA. TTie supernatants of Ihese cultures, hereafter termed active SUPs, were pooled and stored al - 20 C. Preliminary experiments showed that control SUPs obtained from lymphocytes not pulsed with PHA did not exhibit any detectable activity in our test system. Mitomycin treatment of cells. Approximately 10' lymphoid cells were suspended in 3 ml of MFM containing 25 jjg of mitomycin C/ml (Nutritional Biochemicals Corp.. Cleveland. Ohio. USA). After 35 min of incubation at 37 C tbe cells were wasbed t\%lce by cenirifugation. Inctdxition of eell.i in active SUPs. Various preparations of lymphoid cells were suspended in active SUP al a concentration of I • 10" cells, ml. Control cells were incubated in fresh MEM containing 10" „ HS and antibiotics. After incubation for various periods at 37 C in a humidified 5°,, CO, atmosphere the cells were washed twice by centrifugation. In most experiments the cells were mitomycin-trcaied before use. LPS treatment of celts. LPS was dissolved in MEM, containing 10° „ HS and antibiotics, at a concentration of 1.0 yg ml. Lymphoid celts were incubaled in this solution for 24 b at 37 C and control cells were incubated in medium wiihout LPS. Detertiifnation of ^H-thymidine incorporatinn. A previously described microassay was used [16]. Brieiiy. various numbers of untreated l>mpboid cells were incubated alone or together wiih preincubated mitomycin-treated autologous lymphocytes in the wells of microlest plates containing (1.2 ml of MLM supplemented with 10",, H,S and antibiotics. The cells were stimulated with PPD, PHA or Con A at concentrations indicated in the text. Control cullures received no
stimulants. In some tests 0,1 ml of tbe culture medium consisted of active SUP ami 0.1 ml of fresh M t M , The cultures were incubated for various periods at 37 C in a bumidilied atmosphere of 5",, CO^. 24 b before termination of ihe cultures 'H-tbymidine was added (1.0 |jCi per culture: specific activity. 5 Ci/mMI. Incorporated radioactivity of triplicate cultures was determined by liquid scintillation counting and e.xpressed as counts per minute (cpm). Variabilily within the triplicates did not e.xceed 10",,. Isotope uplukes of control cultures and milogeii-cxposcd mitomycin-trcated cells was negligible and did not exceed 500 cpm.
RESULTS Iti/h/etice of active SUPs on the PPD response of tymphoid cells PPD re.spon.ie of lymphoid cells preincuhated in active SUPs. Non-purified cell preparations were iiicubaled in active SUPs or medium for 24 h. They were then examined for responses to 1,0 Jig of PPD/ml during a cuiture period of 9 days. Fig. 1, which depicts the results of a representative experiment, shows thai the control cells that were pre-incubated in medium exhibited a continuous increase of DNA synthesis during the first 7 days, followed by a decline. In contrast, the cells that were treated with active SUPs exhibited maximal response on day 3. followed by a steady state or a decline.
100 H
3
5 7 Days of mcubohon
9
FIG. I. PPD lubercuiin response of 10'' non-purified cells preincubaied in active SUP (•) or medium (O) for 24 h.
Lymphokine Induced Jmmunosuppression
55
TABLH I. PPD tuberculin responses of 5 >; 10' non-purified lympboid cells coculiured witb 10-^ aulologous non-purilied lympboid cells preincubaied Tor 1 ^ days in active SUP or in nn;diuni. The preincubated cells were cither luitreaied or blocked with mitomycin C. ^H-thymidine incorporations, expressed as cpm, determined on day 7 are presented.
Days of preincubation
Type of preincubation
3
1
Medium Mit.* Active SUP 1- Mit.
8,190 1.520 (81)t
Medium Active SUP
6.050 1.300 (78)
14,250 6.450 (551 24,220 6,330 (79)
4
9,680 5.280 (45) 33,840 4,230 (87)
21.820 7.440 (66) 12,250 2,440 (80)
* Mil. means ibal ihe cells were mitoinyciii-irealed afier preincubation. t Figures within parentheses sbow Ihe reduciioiis of stimulations, expressed as percent, caused by cells preincubaied in active SUP compared lo corresponding cells preincubated in medium.
PPD respon.ses oJ lymphocytes cocultured with lymphoid cells preincuhated in active SUPs. One explanation ol" the above results could be that the active SUP had activated suppressor cells capable of inhibiting the PPD response of other lymphocyies. To test this possibility, nonpurified cell preparations were incubated in active SUPs or medium for 1 ^ days and thereafter examined for capacity to modify the responses of fresh autologous lymphocytes to 1.0 [ig of PPD/ml. Table I shows that iymphocytes coculturcd with cells preincubated in active SUPs exhibited lower PPD responses than those cocultured with cells preincubated in medium. Such a dilTcrence was noted with both untreated and mitomycin-treated preincubated cells. The apparent suppressor activity induced by active SUPs reached its peak on day 1. and there was no significant change by prolonging the incubation period.
FIG, 2. PPD tuberculin response of 5 x 10' non-purified cells cullured alone ( • ) or togetber with 10' mitomycin-ireaied, autologous. non-purified cells preincubated in active SUP (•) or in medium (O) for 24 h.
Fig, 2 shows an experiment where 5x10^ fresh non-purified cells were tested for PPD responses (1.0 jig/ml) when cultured alone or together wilh 10^ mitomycin-treated, autologous non-purified cells preincubated for 24 h in active SUPs or medium. The control lymphocytes enhanced the PPD response, whereas those that were pretreated with active SUPs suppressed it. To further verify the above findings, a series of experiments was conducted in which nonpurified and T-cell-enrichcd preparations were
incubated for 24 h in active SUPs or medium. They were then mitomycin-treated and tested for capacity to modify the PPD responses (1,0 i/g/ml) of fresh, autologous non-purified cells. Table M shows that the PPD response of cultures containing non-ptirified cells pretreated with active SUPs was sirongly suppressed compared to control cultures. Such a suppression, although lower and statistically non-significant, was observed by adding T-ce 11-enriched preparations pretreated with active SUPs.
H
3
5 7 Days of incubation
9
56
E.-L. Larsson & H. Blomgrctt II. PPD tuberculin responses of 5- 10' non-purified lymphoid cells cocultured with 10* mitomycin-treated, autologous, non-purified or T-cell-enriched preparations preincubated for 24 h in active SUP or medium. ^H-tbyniidrne incorporations determined on day 7 are presented. Mean values ! SE calculated from five or six separate experiments using dilTerent cell donors are presented.
Type of preincubation
No. of tests
Non-purified in medium Non-purified in active SUP T-cell enriched in medium T-cell enriched in active SUP
6 6 5 5
epm {mean ± SE) 21,290 5.9.10 19.940 12,6.10
i 4,610 -^ 1.770 ± 4.630 -1- 2,660
reduction by active SUPs* (mean •; SE)
68.3 4- l5.a(P
Evidence that Soluble Products Released by PHA-stimulated Human Lymphoid Cells Activate Immunosuppressive Monocytes E.-L. LARSSON & H. BLOMGREN Department of Biochemistry, Arrhenius Laboratot7, University of Stockholm, and Departmetil of Tumour Biology, Karolinska Institute, Slockholm, Sweden
Larsson, B.-L. & Blomgren, H. Evidence ihat Soluble Products Released by PHA-stimulated Human Lymphoid Cells Activate Immunosuppressive Monocytes. Seand. J. Immunol. 9, 5.1-60, 1979.
Soluble mediators, lyniphokines, released by stimulated lymphoid cells can modiry immunological responses in several ways. In this investigation we have examined whether ihe supernaiants of phytohaemaggluiinin (PHAl-aciivated human lymphocytes (active SUPs) contain factors ihat can suppress proliferalivc responses of lymphocytes in vilro. The results have shown ihat crude preparations of peripheral lymphoid cells incubated for lA h in active SUPs can suppress ihe responses of cocullured autulogoiis lympiiocytes to PPD tuberculin in vitro. Their suppressive activity was not abolished by mitomycin treatment. Some reduction of phytomitogen responses was also noted. Ma.ximal suppressive activity was obtained within 24 h of incubation in active SUPs and it could noi be induced in cell preparations depleted of monocytes macrophages. Similar results were obtained by treating lymphoid cells wilh tipopolysuccharide from Esclwrichia co/i, wbich is a known activator of monocytes. These results thus show that lymphokines released by stimulated lymphoid cells can aclivale monocytes macrophages in such u way that ihey become immunosuppressive. E.-L. Larsson. Basel InstittUe for Immtmology. Crenzachersirasse 487. Postfaeh 400S. Basel 3, Sii'ttzerland.
Soluble products of non-gammag!obulin nature, lyniphokines, generated by antigen- or phytomitogen-activated lymphocytes are considered Important regulators of immune respon.ses [21], There is evidence that certain lymphokines enhance proliferative responses of lymphocyies [10] whereas others may exert a suppressive effect [5, 10, 14]. The mode of action of these regulator substances is largely unknown. Previous experiments have shown that PHAactivated human lymphoid cells release factors which are highly mitogenic for sheep red blood cell rosetted (SRBC) T-cells [3, 13], Biochemical characterization of those mitogenic factors (M F) has also been performed [15]. Furthermore, circumstantial evidence has been presented that lymphokines released by such activated lymphoid cells can stimulate inhibitor cells capable of terminating proliferation of lymphocytes [5, 14]. 0300-9475/79/0100-0053 $02.00
In this article we present results showing that PHA-activated human lymphoid cells release soluble factors that stimulate monocytesmacrophages in such a way that they can inhibit proliferative responses of lymphocytes in vitro.
MATERIALS AND METHODS Preparation of lymphocytes. Heparinized venous blood was obtained from healthy donors ot" botb sexes. Lymphoid cells were separated by centrifugation on Ficoll-lsopatjuc |12). These preparations of lymphoid cells, which will be termed tion-purified. served as a source of non-adherent. Fe-piiri/ied cell preparations. They were oblained by incubating 5 • 10* cells for 1 h in a plastic Petri dish (with a diameter of 50 mml in 5 ml of Eagle's minimal essential medium supplemented with Earle's salts (MEM) containing 20" „ of heatinactivated human scrum (HS). The non-adherent cells were then incubated with iron powder followed by
.Q 1979 Blackwell Scientific Publications
53
54
E.-L. Larsson & H. Blomgren
treatment with a magnet to remove phagocjtic cells [2], T cells were separated by rosetling them wiih sheep er>tbrocytes (SRBC) followed by centrifugation on Ficoil-lsopaquu" (11]. Approximately 90-95"n of ihe T-cell-cnriched preparations formed spontaneous rosetles with SRBC. The corresponding figure for the other two cell preparations ranged between S0°o and 60%. Lymphocyte stimulants. Concanavalin A (Con A) was obtained from Sigma Chemical Co., St I.ouis, Mo., USA, II was dissolved in MPM and ils concentration expressed as [Jg/ml. Pbytohaemaggiulinin (I'HA) was purchased from Difco Lah., Detroit. Mich.. USA (Bacio Ph>tobaemagglutinin Ml. Tbe contents of viaLs containing lyophilized PHA were dissolved in 5 ml of MEM. This solution is referred to as \W\, PHA. Puritied protein derivative oi luberculin IPPD) was obtained from Statens Serum Institute, Copenhagen, Denmark. The concentration of this antigen is expressed as i^g'ml, Lipopolysaccbaride (LPS) was purchased from Difco Lab. (Lipopolysaccharide W. Escheriehia coli 055:B5). The concentration is e.xpressed as [Jg/ml. Production of active .supernatants (SUPs). Tbis procedure has been described in detail previously [3). Briefly, non-purified cell preparations were suspended in MEM containing antibiotics and 10" „ beat-inactivated HSin the presence of PHA at a final concentration of 3°o. Tbe cells were then incubated ft>r 24 h al 37 C in a humidified atmosphere containing 5" „ COj, wasbed several times, and reincubated in fresh MEM containing HS for another 24 b in the absence of PHA. TTie supernatants of Ihese cultures, hereafter termed active SUPs, were pooled and stored al - 20 C. Preliminary experiments showed that control SUPs obtained from lymphocytes not pulsed with PHA did not exhibit any detectable activity in our test system. Mitomycin treatment of cells. Approximately 10' lymphoid cells were suspended in 3 ml of MFM containing 25 jjg of mitomycin C/ml (Nutritional Biochemicals Corp.. Cleveland. Ohio. USA). After 35 min of incubation at 37 C tbe cells were wasbed t\%lce by cenirifugation. Inctdxition of eell.i in active SUPs. Various preparations of lymphoid cells were suspended in active SUP al a concentration of I • 10" cells, ml. Control cells were incubated in fresh MEM containing 10" „ HS and antibiotics. After incubation for various periods at 37 C in a humidified 5°,, CO, atmosphere the cells were washed twice by centrifugation. In most experiments the cells were mitomycin-trcaied before use. LPS treatment of celts. LPS was dissolved in MEM, containing 10° „ HS and antibiotics, at a concentration of 1.0 yg ml. Lymphoid celts were incubaled in this solution for 24 b at 37 C and control cells were incubated in medium wiihout LPS. Detertiifnation of ^H-thymidine incorporatinn. A previously described microassay was used [16]. Brieiiy. various numbers of untreated l>mpboid cells were incubated alone or together wiih preincubated mitomycin-treated autologous lymphocytes in the wells of microlest plates containing (1.2 ml of MLM supplemented with 10",, H,S and antibiotics. The cells were stimulated with PPD, PHA or Con A at concentrations indicated in the text. Control cullures received no
stimulants. In some tests 0,1 ml of tbe culture medium consisted of active SUP ami 0.1 ml of fresh M t M , The cultures were incubated for various periods at 37 C in a bumidilied atmosphere of 5",, CO^. 24 b before termination of ihe cultures 'H-tbymidine was added (1.0 |jCi per culture: specific activity. 5 Ci/mMI. Incorporated radioactivity of triplicate cultures was determined by liquid scintillation counting and e.xpressed as counts per minute (cpm). Variabilily within the triplicates did not e.xceed 10",,. Isotope uplukes of control cultures and milogeii-cxposcd mitomycin-trcated cells was negligible and did not exceed 500 cpm.
RESULTS Iti/h/etice of active SUPs on the PPD response of tymphoid cells PPD re.spon.ie of lymphoid cells preincuhated in active SUPs. Non-purified cell preparations were iiicubaled in active SUPs or medium for 24 h. They were then examined for responses to 1,0 Jig of PPD/ml during a cuiture period of 9 days. Fig. 1, which depicts the results of a representative experiment, shows thai the control cells that were pre-incubated in medium exhibited a continuous increase of DNA synthesis during the first 7 days, followed by a decline. In contrast, the cells that were treated with active SUPs exhibited maximal response on day 3. followed by a steady state or a decline.
100 H
3
5 7 Days of mcubohon
9
FIG. I. PPD lubercuiin response of 10'' non-purified cells preincubaied in active SUP (•) or medium (O) for 24 h.
Lymphokine Induced Jmmunosuppression
55
TABLH I. PPD tuberculin responses of 5 >; 10' non-purified lympboid cells coculiured witb 10-^ aulologous non-purilied lympboid cells preincubaied Tor 1 ^ days in active SUP or in nn;diuni. The preincubated cells were cither luitreaied or blocked with mitomycin C. ^H-thymidine incorporations, expressed as cpm, determined on day 7 are presented.
Days of preincubation
Type of preincubation
3
1
Medium Mit.* Active SUP 1- Mit.
8,190 1.520 (81)t
Medium Active SUP
6.050 1.300 (78)
14,250 6.450 (551 24,220 6,330 (79)
4
9,680 5.280 (45) 33,840 4,230 (87)
21.820 7.440 (66) 12,250 2,440 (80)
* Mil. means ibal ihe cells were mitoinyciii-irealed afier preincubation. t Figures within parentheses sbow Ihe reduciioiis of stimulations, expressed as percent, caused by cells preincubaied in active SUP compared lo corresponding cells preincubated in medium.
PPD respon.ses oJ lymphocytes cocultured with lymphoid cells preincuhated in active SUPs. One explanation ol" the above results could be that the active SUP had activated suppressor cells capable of inhibiting the PPD response of other lymphocyies. To test this possibility, nonpurified cell preparations were incubated in active SUPs or medium for 1 ^ days and thereafter examined for capacity to modify the responses of fresh autologous lymphocytes to 1.0 [ig of PPD/ml. Table I shows that iymphocytes coculturcd with cells preincubated in active SUPs exhibited lower PPD responses than those cocultured with cells preincubated in medium. Such a dilTcrence was noted with both untreated and mitomycin-treated preincubated cells. The apparent suppressor activity induced by active SUPs reached its peak on day 1. and there was no significant change by prolonging the incubation period.
FIG, 2. PPD tuberculin response of 5 x 10' non-purified cells cullured alone ( • ) or togetber with 10' mitomycin-ireaied, autologous. non-purified cells preincubated in active SUP (•) or in medium (O) for 24 h.
Fig, 2 shows an experiment where 5x10^ fresh non-purified cells were tested for PPD responses (1.0 jig/ml) when cultured alone or together wilh 10^ mitomycin-treated, autologous non-purified cells preincubated for 24 h in active SUPs or medium. The control lymphocytes enhanced the PPD response, whereas those that were pretreated with active SUPs suppressed it. To further verify the above findings, a series of experiments was conducted in which nonpurified and T-cell-enrichcd preparations were
incubated for 24 h in active SUPs or medium. They were then mitomycin-treated and tested for capacity to modify the PPD responses (1,0 i/g/ml) of fresh, autologous non-purified cells. Table M shows that the PPD response of cultures containing non-ptirified cells pretreated with active SUPs was sirongly suppressed compared to control cultures. Such a suppression, although lower and statistically non-significant, was observed by adding T-ce 11-enriched preparations pretreated with active SUPs.
H
3
5 7 Days of incubation
9
56
E.-L. Larsson & H. Blomgrctt II. PPD tuberculin responses of 5- 10' non-purified lymphoid cells cocultured with 10* mitomycin-treated, autologous, non-purified or T-cell-enriched preparations preincubated for 24 h in active SUP or medium. ^H-tbyniidrne incorporations determined on day 7 are presented. Mean values ! SE calculated from five or six separate experiments using dilTerent cell donors are presented.
Type of preincubation
No. of tests
Non-purified in medium Non-purified in active SUP T-cell enriched in medium T-cell enriched in active SUP
6 6 5 5
epm {mean ± SE) 21,290 5.9.10 19.940 12,6.10
i 4,610 -^ 1.770 ± 4.630 -1- 2,660
reduction by active SUPs* (mean •; SE)
68.3 4- l5.a(P
