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Immunology 1991 73 120-122
Inhibition of IL-1 activity induced with allogeneic transfusion of UV-irradiated blood B. HORVAT, M. POLJAK-BLAZI* & M. HADIJA* Department of Physiology, School of Medicine, Zagreb University, and *Department of Experimental Biology and Medicine, Institute Ruder Boskovic, Zagreb, Yugoslavia
Acceptedfor publication 8 January 1991
SUMMARY Treatment with UV-irradiated donor-specific blood transfusion is known to induce specific unresponsiveness in recipient animals and prolong allograft survival. Mixed lymphocyte response in transfused mice was decreased towards spleen cells of the blood donor strain, but was not altered to third-party cells. Sera from treated mice showed significantly lower interleukin-1 (IL-I) activity, which was increased with higher dilutions of sera, indicating the presence of IL-1 inhibitor. Furthermore, sera decreased rIL-1-induced cell proliferation in dose-dependent manner, while the response to rIL-2 neither depended on the concentration of sera, nor differed between non-treated controls and treated mice. These results indicate that UV-irradiated allogeneic blood transfusion could induce an inhibitor, specifically directed to IL-l activity, which may be involved in the generation of immunological unresponsiveness in treated animals.
Effects of blood transfusion on the success of allograft survival have been widely discussed since the beginning of clinical transplantation.' UV-irradiated donor-specific blood transfusions (DST), given before transplantation, decrease the risk of sensitization by allogeneic cells and lead to prolonged, even permanent, survival of allografts of neonatal pancreatic tissue and islets2'3 or heart.4'5 The mechanism involved in the improved graft survival may include induction of suppressor mechanisms6 and generation of specific serum suppressor factor and anti-idiotipic antibodies.4 We have shown that DST of UV-irradiated blood provokes specific immunological unresponsiveness to bone marrow or tissue antigens of the blood donor strain type.7 In order to further elucidate mechanisms responsible for the generation of this unresponsiveness, we have investigated the role of interleukin-1 (IL-1), a hormone-like protein with a wide range of biological properties, including the ability to alter immunological, neuroendocrine and metabolic functions.8 In this study we have analysed IL-1 activity in the sera of mice specifically immunosuppressed by transfusion of UV-irradiated allogeneic blood. CBA, BALB/c and C57BL/6 mice were bred and maintained at the Ruder Boskovic Institute, Zagreb, Yugoslavia. Whole blood for transfusion was obtained from C57BL/6 mice by jugular vein bleeding. Whole blood was diluted 1:50 in Correspondence: Dr B. Horvat, Dept. of Physiology, School of Medicine, Zagreb University, Salata 3, P.O. 978, Zagreb 41000, Yugoslavia.
phosphate-buffered saline (PBS), and irradiated from unfiltered Phillips lamps (12,000 J/m2) in thin layer (2 mm). Irradiated blood cells were centrifuged and pellet was resuspended in PBS in the original volume. Each CBA mouse received 0-5 ml of this material, three times at 7-day intervals. Sera were taken 7 days after the third transfusion and tested individually, or pooled before testing. Mixed lymphocyte response was generated by culturing 5 x 105 responder spleen cells (CBA) per well with the same number ofX-irradiated (3000 rads) stimulator cells (C57BL/6 or BALB/c) in 200 p1 of RPMI- 1640, supplemented with 5 % foetal calf serum (FCS). After 96 hr the cultures were pulsed with 1 1iCi of low activity [3H] thymidine for the additional 18 hr and then harvested by automated sample harvester. Thymidine uptake was determined in a scintillation counter (Backman LP, Minchen, Germany). IL-I activity in sera was tested on cloned T-cell line DIO.G4.1 (D1O). DIO is a AKR/J-derived cloned helper T-cell line specific for the hen egg white protein conalbumin in the context of I-Ak.9 The assay is based on the ability of IL-l to induce proliferation of D10 cells, stimulated with the monoclonal antibody 3D3 directed to the antigen-specific receptor on these cells.9 D10 cells were added to assay cultures at 2 x 104 cells/well in a 100-pl volume of RPMI medium with 5% of FCS. Tested sera were titrated in several dilutions and added to the cultures in 50 pi volumes. All cultures received 3D3 antibody in the final concentration 1:100, and some of them recombinant IL-1 (rIL-l; Hoffmann-La Roche, Nutley, NJ; 10-4 U/ml, where 1 unit of IL-I is defined in thymocyte co-stimulatory
120
121
IL-I inhibitor and UV-irradiated blood transfusion Table 1. Mixed lymphocyte response of CBA mice treated with UV-irradiated C57BL/6 blood
assay, as described elsewhere)."I Triplicate cultures of cells and factors were incubated in a volume of 200 p1 for 72 hr. [3H]thymidine was added during the final 4 hr of incubation. Cultures were harvested and radioactivity was measured as described above. All lymphokines were used in the concentration determined to induce half maximal proliferation of the tested cells. IL-2-dependent cell proliferation was measured using cell lines D10 and CTLD. D1O cells were used identically as described above, and rIL-2 (Amgen, Biologicals, Thousand Oaks, CA) was added to the cultures, in addition to the different dilutions of sera. CTLD cells, an IL-2-dependent cell line, were used in the concentration of 5 x 103 cells in 100 pl of RPMI medium with 5% of FCS, with the addition of 50 p1 of diluted sera per well and 50 p1 of rIL-2 (lU/ml, where one unit is defined as an amount of IL-2 which causes half maximal incorporation of [3H] thymidine in cells in culture). Cultures were incubated for 48 hr and pulsed for the final 6 hr with 1 pCi of [3H] thymidine per well, and harvested as described above. CBA mice were treated with C57BL/6-derived UV-irradiated blood and 7 days after the last transfusion spleen cells were taken and MLR was performed with C57BL/6 or BALB/c stimulatory cells. Table 1 shows that spleen cells obtained from treated animals respond significantly less to the stimulatory cells of the blood donor type (C57BL/6), while the response to the third party (BALB/c) is not affected, indicating donor-specific decrease in MLR. The ability of spleen cells from treated mice to
Responder treatment Stimulator cells C57BL/6 BALB/c
UV-blood transfusion UV-blood transfusion UV-blood transfusion
C57BL/6 BALB/c
C.p.m. + SE 806+ 150 20,230 + 4320
15,820+2340 1250 + 835 6831 +2360* 12,230 + 1250
*P
Immunology 1991 73 120-122
Inhibition of IL-1 activity induced with allogeneic transfusion of UV-irradiated blood B. HORVAT, M. POLJAK-BLAZI* & M. HADIJA* Department of Physiology, School of Medicine, Zagreb University, and *Department of Experimental Biology and Medicine, Institute Ruder Boskovic, Zagreb, Yugoslavia
Acceptedfor publication 8 January 1991
SUMMARY Treatment with UV-irradiated donor-specific blood transfusion is known to induce specific unresponsiveness in recipient animals and prolong allograft survival. Mixed lymphocyte response in transfused mice was decreased towards spleen cells of the blood donor strain, but was not altered to third-party cells. Sera from treated mice showed significantly lower interleukin-1 (IL-I) activity, which was increased with higher dilutions of sera, indicating the presence of IL-1 inhibitor. Furthermore, sera decreased rIL-1-induced cell proliferation in dose-dependent manner, while the response to rIL-2 neither depended on the concentration of sera, nor differed between non-treated controls and treated mice. These results indicate that UV-irradiated allogeneic blood transfusion could induce an inhibitor, specifically directed to IL-l activity, which may be involved in the generation of immunological unresponsiveness in treated animals.
Effects of blood transfusion on the success of allograft survival have been widely discussed since the beginning of clinical transplantation.' UV-irradiated donor-specific blood transfusions (DST), given before transplantation, decrease the risk of sensitization by allogeneic cells and lead to prolonged, even permanent, survival of allografts of neonatal pancreatic tissue and islets2'3 or heart.4'5 The mechanism involved in the improved graft survival may include induction of suppressor mechanisms6 and generation of specific serum suppressor factor and anti-idiotipic antibodies.4 We have shown that DST of UV-irradiated blood provokes specific immunological unresponsiveness to bone marrow or tissue antigens of the blood donor strain type.7 In order to further elucidate mechanisms responsible for the generation of this unresponsiveness, we have investigated the role of interleukin-1 (IL-1), a hormone-like protein with a wide range of biological properties, including the ability to alter immunological, neuroendocrine and metabolic functions.8 In this study we have analysed IL-1 activity in the sera of mice specifically immunosuppressed by transfusion of UV-irradiated allogeneic blood. CBA, BALB/c and C57BL/6 mice were bred and maintained at the Ruder Boskovic Institute, Zagreb, Yugoslavia. Whole blood for transfusion was obtained from C57BL/6 mice by jugular vein bleeding. Whole blood was diluted 1:50 in Correspondence: Dr B. Horvat, Dept. of Physiology, School of Medicine, Zagreb University, Salata 3, P.O. 978, Zagreb 41000, Yugoslavia.
phosphate-buffered saline (PBS), and irradiated from unfiltered Phillips lamps (12,000 J/m2) in thin layer (2 mm). Irradiated blood cells were centrifuged and pellet was resuspended in PBS in the original volume. Each CBA mouse received 0-5 ml of this material, three times at 7-day intervals. Sera were taken 7 days after the third transfusion and tested individually, or pooled before testing. Mixed lymphocyte response was generated by culturing 5 x 105 responder spleen cells (CBA) per well with the same number ofX-irradiated (3000 rads) stimulator cells (C57BL/6 or BALB/c) in 200 p1 of RPMI- 1640, supplemented with 5 % foetal calf serum (FCS). After 96 hr the cultures were pulsed with 1 1iCi of low activity [3H] thymidine for the additional 18 hr and then harvested by automated sample harvester. Thymidine uptake was determined in a scintillation counter (Backman LP, Minchen, Germany). IL-I activity in sera was tested on cloned T-cell line DIO.G4.1 (D1O). DIO is a AKR/J-derived cloned helper T-cell line specific for the hen egg white protein conalbumin in the context of I-Ak.9 The assay is based on the ability of IL-l to induce proliferation of D10 cells, stimulated with the monoclonal antibody 3D3 directed to the antigen-specific receptor on these cells.9 D10 cells were added to assay cultures at 2 x 104 cells/well in a 100-pl volume of RPMI medium with 5% of FCS. Tested sera were titrated in several dilutions and added to the cultures in 50 pi volumes. All cultures received 3D3 antibody in the final concentration 1:100, and some of them recombinant IL-1 (rIL-l; Hoffmann-La Roche, Nutley, NJ; 10-4 U/ml, where 1 unit of IL-I is defined in thymocyte co-stimulatory
120
121
IL-I inhibitor and UV-irradiated blood transfusion Table 1. Mixed lymphocyte response of CBA mice treated with UV-irradiated C57BL/6 blood
assay, as described elsewhere)."I Triplicate cultures of cells and factors were incubated in a volume of 200 p1 for 72 hr. [3H]thymidine was added during the final 4 hr of incubation. Cultures were harvested and radioactivity was measured as described above. All lymphokines were used in the concentration determined to induce half maximal proliferation of the tested cells. IL-2-dependent cell proliferation was measured using cell lines D10 and CTLD. D1O cells were used identically as described above, and rIL-2 (Amgen, Biologicals, Thousand Oaks, CA) was added to the cultures, in addition to the different dilutions of sera. CTLD cells, an IL-2-dependent cell line, were used in the concentration of 5 x 103 cells in 100 pl of RPMI medium with 5% of FCS, with the addition of 50 p1 of diluted sera per well and 50 p1 of rIL-2 (lU/ml, where one unit is defined as an amount of IL-2 which causes half maximal incorporation of [3H] thymidine in cells in culture). Cultures were incubated for 48 hr and pulsed for the final 6 hr with 1 pCi of [3H] thymidine per well, and harvested as described above. CBA mice were treated with C57BL/6-derived UV-irradiated blood and 7 days after the last transfusion spleen cells were taken and MLR was performed with C57BL/6 or BALB/c stimulatory cells. Table 1 shows that spleen cells obtained from treated animals respond significantly less to the stimulatory cells of the blood donor type (C57BL/6), while the response to the third party (BALB/c) is not affected, indicating donor-specific decrease in MLR. The ability of spleen cells from treated mice to
Responder treatment Stimulator cells C57BL/6 BALB/c
UV-blood transfusion UV-blood transfusion UV-blood transfusion
C57BL/6 BALB/c
C.p.m. + SE 806+ 150 20,230 + 4320
15,820+2340 1250 + 835 6831 +2360* 12,230 + 1250
*P
