Journal of Clinical Immunology, Vol. 12, No. 2, 1992
Suramin Inhibits the Mixed Lymphocyte Reaction by Suppressing Lymphokine Production MOHAN SHENOY, 1 BRUCE MacPHERSON, 2 and PREMKUMAR CHRISTADOSS 1' 3
Accepted: October 22, 1991
suppressed in vitro secondary immune response (1). The authors suggested that suramin-induced immunosuppression was not related to a defect in the macrophage or the B-cell functions but may be due to suppressor T-cell activation. Suramin was also effective in suppressing delayed-type hypersensitivity (DTH) to SRBC when administered before or at the time of antigen sensitization (2). Suramin also suppressed complement fixation and cellular infiltration and destroyed vessel walls during arthus reaction in experimental animals (3). Suramin treatment also caused a reduction in clinical and pathological expression of experimental allergic encephalomyelitis (EAE) in Lewis rats (4). Further, multiple cell lines in culture, particularly lymphoid cells, were growth inhibited upon exposure to suramin (5). In mice, suramin caused profound and prolonged thymic atrophy and splenic lymphocyte depletion in a dose-dependent manner. However, this phenomenon is reversible upon stopping suramin treatment (5). The above immunosuppressive properties of suramin prompted us to evaluate the immunomodulatory effect of this drug in graft rejection and graft-vs-host disease (GVHD). The mixed lymphocyte reaction (MLR) is an in vitro test to measure lymphocyte recognition and proliferation, in response to histocompatibility antigens, and an indicator of cellular immunity and the degree to which the cells proliferate correlates with the prognosis of a transplanted organ. Therefore, as a first step, we studied the immunosuppressive effect of suramin in the H-2- and HLA-incompatible MLR. In this report, we document for the first time the in vitro immunosuppressive effect of suramin in the I-A-, H-2-, and HLA-incompatible MLR. In addition, we document that the suppression induced by suramin is mediated by reduced lymphokine production,
New compounds with a greater potency than cyclosporin A (CyA) for thwarting host rejection of organ transplantation are being sought. Suramin sodium may be a novel drug to prevent or delay graft rejection and graft-vs-host disease (GVHD), because of its in vitro and in vivo immunosuppressive properties. Since the allogeneic mixed lymphocyte reaction (MLR) is considered to be the in vitro counterpart of the initial T-lymphocyte recognition and response to allogeneic histocompatibility antigens on grafted tissue or organ and to GVHD, we initially evaluated the in vitro suppressive effect of suramin in the allogeneic MLR. Suramin inhibited the H-2- and HLAincompatible MLR in a dose-dependent manner. The suppressive effect was observed both in the primary and in the secondary MLR. The suppression of the MLR by suramin is due predominantly to the inhibition of interleukin-2 (IL-2) production by the responding T cells. KEY WORDS: suramin;immunosuppression;mixedlymphocyte reaction; lymphokineproduction.
INTRODUCTION In organ transplantation, rejection still remains the greatest single cause of graft loss. Therefore, a search is still being made for ways to control the immune response in order to protect the foreign grafts from rejection. Suramin sodium, a polyanionic drug used in the treatment of trypanosomiasis (especially of the East African type) and onchocerciasis, is immunosuppressive, both in vitro and in vivo. Administration of suramin in vivo during priming with sheep red blood cells (SRBC) led to a modification of some T,cell functions, resulting in a 1Department of Microbiology, University of Texas Medical Branch, Galveston, Texas 77550. ZDepartmentof Pathology, Universityof Vermont, Burlington, Vermont05405. 3To whomcorrespondenceshouldbe addressed. 122
0271-9142/92/0300-0122506.50/0 © 1992 Plenum Publishing Corporation
SURAMIN INHIBITSLYMPHOKINEPRODUCTION
probably interleukin 2 (IL-2), by responding T lymphocytes in the MLR.
MATERIALS AND METHODS
Mice C57BL6/J (B6), C57BL10/Srd (B10), AKR/J, and Balb/c mice breeding pairs were purchased from the Jackson Laboratory, Bar Harbor, ME. Strains B10.F, BI0.Q, B10.A(4R), B10.S, and B6.C-H2 bml2 (bml2) were provided by Dr. C. S. David (Mayo Clinic, Rochester, MN 55905) and Dr. Stimpfling (McLaughlin Research Institute, Great Falls, MT 59401).
Suramin Sodium Suramin sodium was obtained through the National Institutes of Health. Suramin is a trisodium salt, 8,8'-(3",3" '-ureylenebis(3" "benzamido-4" "methylbenzamido)bis-l,3,5-naphthalenetrisulfonic acid. It is a pinkish white flocculent powder and is hygroscopic. Therefore, it should be stored in the dark and under dry conditions. Suramin is poorly absorbed from the intestines. As it causes intense irritation when given by the subcutaneous or intramuscular route, it is always administered intravenously. Most of it combines with serum proteins and circulates in the blood, whereas some of it is taken up by the reticuloendothelial system. Suramin gets excreted slowly in the urine, mainly because it is bound to serum proteins of all kinds. However, chemical estimations of this drug have shown that no depot was formed in any tissue.
123
The Mouse MLR Responder splenic lymphocytes at 5 × 105/100 V.1 were cultured with I06/100 ~1 irradiated (2000 R) allogeneic or syngeneic stimulator lymphocytes in triplicate wells of a 96-well fiat-bottom microtiter plates. The cells were cultured in the presence or absence of suramin sodium dissolved in RPMI 1640 medium at varied concentrations and incubated for 4 days at 37°C, in humidified 5% CO 2enriched air. The cells were pulse labeled with 3H-thymidine (Amersham, Arlington Heights, IL) for the last 18 hr of the culture and then harvested in a Mini-Mash Harvester (M.A. Bioproducts, Walkersville, MD). The 3H-thymidine uptake was determined in a beta scintillation counter and the Specific incorporation of 3H is expressed as counts per minute (cpm).
Secondary Mixed Lymphocyte Culture (MLC) Primary MLC were set up with 4 x 106/ml responder splenic lymphocytes and 8 × 106/ml irradiated (2000 R) allogeneic or syngeneic stimulator lymphocytes in 24-well tissue culture plates. The responding cells were harvested on day 10, and viable cells were separated through Ficoll-Hypaque (Sigma, St. Louis, MO) density gradients, then washed in medium, and 5 × 104/100 ixl responder cells were recultured for 2-3 days with 4 × 105/100 vd irradiated syngeneic or allogeneic splenic lymphocytes in triplicate wells of a 96-well fiat-bottommicrotiter plate, in the presence or absence of various concentrations of suramin sodium. The 3H-thymidine uptake was determined as described above in the primary MLR.
The Human MLR Preparation of Splenic Lymphocytes Spleens were crushed in cold (4°C) phosphate buffered saline (PBS), and splenocytes were pelleted by low-speed centrifugation. Red blood cells were lysed with Tris-ammonium chloride buffer. Cells were then washed in RPMI 1640 medium two or three times and resuspended in complete culture medium [RPMI 1640 enriched with 2 mM L-glutamine, 1% Hepes buffer (25 mM), I% normal mouse serum, 2-mercaptoethanol (5 × 10-5 M), penicillin G (100 U/ml), and streptomycin (I00 ~g/ml] at the appropriate cell concentration. Journal of Clinical Immunology, Vol. 12, No. 2, 1992
Peripheral blood mononuclear cells (PBMC) were separated from defibrinated whole blood from normal healthy blood donors by Ficoll-Hypaque (Pharmacia Chemicals, Piscataway, NJ) density-gradient centrifugation. Cells were washed twice with RPMI 1640 and resuspended in RPMI 1640 supplemented with 10% heat-inactivated human AB serum, penicillin G (100 U/ml), streptomycin (100 ixg/ml), 2 mM L-glutamine, 25 mM Hepes buffer, and 2-mercaptoethanol (5 × 10-5 M). PBMC (7.5 × 104/100 ~1) from individual M (HLA-A2/3, B8/17, DR3/7) were cultured either with 7.5 × 104/I00 txl irradiated (3000 R) autologous
124
SHENOY, MaCPHERSON, AND CHRISTADOSS
PBMC from individual M (autologous control) or with individual K-irradiated PBMC (HLA-A 3/2, B7/40, DR2/?), in triplicate wells of a 96-well fiatbottom microtiter plate. For two-way MLR, PBMC (7.5 × 104/100 ixl) from individual M were cultured with PBMC (7.5 x 104/100 I~1) from individual K. The cells were cultured in either the presence or the absence of different concentrations of suramin sodium diluted in RPMI and incubated under appropriate conditions as mentioned earlier. The cells were pulse labeled with 3H-thymidine on day 6 and harvested 18 hr later. 3 H uptake was evaluated as before.
20.
:~ EL L) t,3 I 0
•\
STIMULATORS
\
•B10 Q
\
,,B1oA(,m)
15. • 10
X 5
0
i
0
Lymphokine Assay The IL-2 activity was measured according to a previous method (6). In brief, the IL-2-dependent CT6 cell line was used to determine IL-2 activity present in the primary and in the secondary MLC supernatants. Undiluted culture supernatants (100 ~1) were added to 10 4 CT6 indicator cells in 96-well microtiter plates. The plates were incubated at 37°C for 24 hr in a humidified 5% CO 2 incubator. The cells were pulse labeled with 1 ~ci/well of 3Hthymidine for 18 hr, and thymidine incorporation was determined. RESULTS
Suramin Inhibits the H-2-1ncompatible MLR B10 (H-2b) lymphocytes were stimulated with irradiated B10.Q (H-2q) or B10.S (H-2 s) or B10.A (4R) (KkAkEbSbD b) stimulators. Suramin was added at various concentrations during the initiation of the MLR. As shown in Fig. 1, suramin significantly inhibited the above H-2-incompatible MLR at all concentrations tested using three stimulators in a dose-dependent manner (P < 0.006; Student's t test).
Suramin Inhibits the I-A-Incompatible MLR We made use of the I-AI3-chain mutant bml2 strain to study the effect of suramin on the I-Aincompatible MLR. B6 lymphocytes were stimulated with irradiated bml2 stimulators. Also, the bml2 lymphocytes were used as responders and stimulated with irradiated B6 stimulators. Suramin inhibited the above I-A-incompatible MLR in a dose-dependent manner (Fig. 2). A significant (P
Suramin Inhibits the Mixed Lymphocyte Reaction by Suppressing Lymphokine Production MOHAN SHENOY, 1 BRUCE MacPHERSON, 2 and PREMKUMAR CHRISTADOSS 1' 3
Accepted: October 22, 1991
suppressed in vitro secondary immune response (1). The authors suggested that suramin-induced immunosuppression was not related to a defect in the macrophage or the B-cell functions but may be due to suppressor T-cell activation. Suramin was also effective in suppressing delayed-type hypersensitivity (DTH) to SRBC when administered before or at the time of antigen sensitization (2). Suramin also suppressed complement fixation and cellular infiltration and destroyed vessel walls during arthus reaction in experimental animals (3). Suramin treatment also caused a reduction in clinical and pathological expression of experimental allergic encephalomyelitis (EAE) in Lewis rats (4). Further, multiple cell lines in culture, particularly lymphoid cells, were growth inhibited upon exposure to suramin (5). In mice, suramin caused profound and prolonged thymic atrophy and splenic lymphocyte depletion in a dose-dependent manner. However, this phenomenon is reversible upon stopping suramin treatment (5). The above immunosuppressive properties of suramin prompted us to evaluate the immunomodulatory effect of this drug in graft rejection and graft-vs-host disease (GVHD). The mixed lymphocyte reaction (MLR) is an in vitro test to measure lymphocyte recognition and proliferation, in response to histocompatibility antigens, and an indicator of cellular immunity and the degree to which the cells proliferate correlates with the prognosis of a transplanted organ. Therefore, as a first step, we studied the immunosuppressive effect of suramin in the H-2- and HLA-incompatible MLR. In this report, we document for the first time the in vitro immunosuppressive effect of suramin in the I-A-, H-2-, and HLA-incompatible MLR. In addition, we document that the suppression induced by suramin is mediated by reduced lymphokine production,
New compounds with a greater potency than cyclosporin A (CyA) for thwarting host rejection of organ transplantation are being sought. Suramin sodium may be a novel drug to prevent or delay graft rejection and graft-vs-host disease (GVHD), because of its in vitro and in vivo immunosuppressive properties. Since the allogeneic mixed lymphocyte reaction (MLR) is considered to be the in vitro counterpart of the initial T-lymphocyte recognition and response to allogeneic histocompatibility antigens on grafted tissue or organ and to GVHD, we initially evaluated the in vitro suppressive effect of suramin in the allogeneic MLR. Suramin inhibited the H-2- and HLAincompatible MLR in a dose-dependent manner. The suppressive effect was observed both in the primary and in the secondary MLR. The suppression of the MLR by suramin is due predominantly to the inhibition of interleukin-2 (IL-2) production by the responding T cells. KEY WORDS: suramin;immunosuppression;mixedlymphocyte reaction; lymphokineproduction.
INTRODUCTION In organ transplantation, rejection still remains the greatest single cause of graft loss. Therefore, a search is still being made for ways to control the immune response in order to protect the foreign grafts from rejection. Suramin sodium, a polyanionic drug used in the treatment of trypanosomiasis (especially of the East African type) and onchocerciasis, is immunosuppressive, both in vitro and in vivo. Administration of suramin in vivo during priming with sheep red blood cells (SRBC) led to a modification of some T,cell functions, resulting in a 1Department of Microbiology, University of Texas Medical Branch, Galveston, Texas 77550. ZDepartmentof Pathology, Universityof Vermont, Burlington, Vermont05405. 3To whomcorrespondenceshouldbe addressed. 122
0271-9142/92/0300-0122506.50/0 © 1992 Plenum Publishing Corporation
SURAMIN INHIBITSLYMPHOKINEPRODUCTION
probably interleukin 2 (IL-2), by responding T lymphocytes in the MLR.
MATERIALS AND METHODS
Mice C57BL6/J (B6), C57BL10/Srd (B10), AKR/J, and Balb/c mice breeding pairs were purchased from the Jackson Laboratory, Bar Harbor, ME. Strains B10.F, BI0.Q, B10.A(4R), B10.S, and B6.C-H2 bml2 (bml2) were provided by Dr. C. S. David (Mayo Clinic, Rochester, MN 55905) and Dr. Stimpfling (McLaughlin Research Institute, Great Falls, MT 59401).
Suramin Sodium Suramin sodium was obtained through the National Institutes of Health. Suramin is a trisodium salt, 8,8'-(3",3" '-ureylenebis(3" "benzamido-4" "methylbenzamido)bis-l,3,5-naphthalenetrisulfonic acid. It is a pinkish white flocculent powder and is hygroscopic. Therefore, it should be stored in the dark and under dry conditions. Suramin is poorly absorbed from the intestines. As it causes intense irritation when given by the subcutaneous or intramuscular route, it is always administered intravenously. Most of it combines with serum proteins and circulates in the blood, whereas some of it is taken up by the reticuloendothelial system. Suramin gets excreted slowly in the urine, mainly because it is bound to serum proteins of all kinds. However, chemical estimations of this drug have shown that no depot was formed in any tissue.
123
The Mouse MLR Responder splenic lymphocytes at 5 × 105/100 V.1 were cultured with I06/100 ~1 irradiated (2000 R) allogeneic or syngeneic stimulator lymphocytes in triplicate wells of a 96-well fiat-bottom microtiter plates. The cells were cultured in the presence or absence of suramin sodium dissolved in RPMI 1640 medium at varied concentrations and incubated for 4 days at 37°C, in humidified 5% CO 2enriched air. The cells were pulse labeled with 3H-thymidine (Amersham, Arlington Heights, IL) for the last 18 hr of the culture and then harvested in a Mini-Mash Harvester (M.A. Bioproducts, Walkersville, MD). The 3H-thymidine uptake was determined in a beta scintillation counter and the Specific incorporation of 3H is expressed as counts per minute (cpm).
Secondary Mixed Lymphocyte Culture (MLC) Primary MLC were set up with 4 x 106/ml responder splenic lymphocytes and 8 × 106/ml irradiated (2000 R) allogeneic or syngeneic stimulator lymphocytes in 24-well tissue culture plates. The responding cells were harvested on day 10, and viable cells were separated through Ficoll-Hypaque (Sigma, St. Louis, MO) density gradients, then washed in medium, and 5 × 104/100 ixl responder cells were recultured for 2-3 days with 4 × 105/100 vd irradiated syngeneic or allogeneic splenic lymphocytes in triplicate wells of a 96-well fiat-bottommicrotiter plate, in the presence or absence of various concentrations of suramin sodium. The 3H-thymidine uptake was determined as described above in the primary MLR.
The Human MLR Preparation of Splenic Lymphocytes Spleens were crushed in cold (4°C) phosphate buffered saline (PBS), and splenocytes were pelleted by low-speed centrifugation. Red blood cells were lysed with Tris-ammonium chloride buffer. Cells were then washed in RPMI 1640 medium two or three times and resuspended in complete culture medium [RPMI 1640 enriched with 2 mM L-glutamine, 1% Hepes buffer (25 mM), I% normal mouse serum, 2-mercaptoethanol (5 × 10-5 M), penicillin G (100 U/ml), and streptomycin (I00 ~g/ml] at the appropriate cell concentration. Journal of Clinical Immunology, Vol. 12, No. 2, 1992
Peripheral blood mononuclear cells (PBMC) were separated from defibrinated whole blood from normal healthy blood donors by Ficoll-Hypaque (Pharmacia Chemicals, Piscataway, NJ) density-gradient centrifugation. Cells were washed twice with RPMI 1640 and resuspended in RPMI 1640 supplemented with 10% heat-inactivated human AB serum, penicillin G (100 U/ml), streptomycin (100 ixg/ml), 2 mM L-glutamine, 25 mM Hepes buffer, and 2-mercaptoethanol (5 × 10-5 M). PBMC (7.5 × 104/100 ~1) from individual M (HLA-A2/3, B8/17, DR3/7) were cultured either with 7.5 × 104/I00 txl irradiated (3000 R) autologous
124
SHENOY, MaCPHERSON, AND CHRISTADOSS
PBMC from individual M (autologous control) or with individual K-irradiated PBMC (HLA-A 3/2, B7/40, DR2/?), in triplicate wells of a 96-well fiatbottom microtiter plate. For two-way MLR, PBMC (7.5 × 104/100 ixl) from individual M were cultured with PBMC (7.5 x 104/100 I~1) from individual K. The cells were cultured in either the presence or the absence of different concentrations of suramin sodium diluted in RPMI and incubated under appropriate conditions as mentioned earlier. The cells were pulse labeled with 3H-thymidine on day 6 and harvested 18 hr later. 3 H uptake was evaluated as before.
20.
:~ EL L) t,3 I 0
•\
STIMULATORS
\
•B10 Q
\
,,B1oA(,m)
15. • 10
X 5
0
i
0
Lymphokine Assay The IL-2 activity was measured according to a previous method (6). In brief, the IL-2-dependent CT6 cell line was used to determine IL-2 activity present in the primary and in the secondary MLC supernatants. Undiluted culture supernatants (100 ~1) were added to 10 4 CT6 indicator cells in 96-well microtiter plates. The plates were incubated at 37°C for 24 hr in a humidified 5% CO 2 incubator. The cells were pulse labeled with 1 ~ci/well of 3Hthymidine for 18 hr, and thymidine incorporation was determined. RESULTS
Suramin Inhibits the H-2-1ncompatible MLR B10 (H-2b) lymphocytes were stimulated with irradiated B10.Q (H-2q) or B10.S (H-2 s) or B10.A (4R) (KkAkEbSbD b) stimulators. Suramin was added at various concentrations during the initiation of the MLR. As shown in Fig. 1, suramin significantly inhibited the above H-2-incompatible MLR at all concentrations tested using three stimulators in a dose-dependent manner (P < 0.006; Student's t test).
Suramin Inhibits the I-A-Incompatible MLR We made use of the I-AI3-chain mutant bml2 strain to study the effect of suramin on the I-Aincompatible MLR. B6 lymphocytes were stimulated with irradiated bml2 stimulators. Also, the bml2 lymphocytes were used as responders and stimulated with irradiated B6 stimulators. Suramin inhibited the above I-A-incompatible MLR in a dose-dependent manner (Fig. 2). A significant (P
