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reperfusion injury: Inhibition by a selectin antagonist, P-selectin glycoprotein ligand-1
ELSEVIER
Early Cellular and Molecular Changes in Ischemia/Reperfusion Injury: Inhibition by a Selectin Antagonist, P-Selectin Glycoprotein Ligand-1 M. Takada, K.C. Nadeau, G.D. Shaw, and N.L. Tilney
I
SCHEMIA/reperfusion injury associated with organ transplantation may influence initial graft function, the development of acute rejection and late chronic changes.’ In this study, initial events surrounding both warm and cold ischemia/reperfusion have been examined. As the very early induced adhesion molecule, E-selectin (CD62E), is upregulated quickly and seems to trigger subsequent cellular events, we have assessed the inhibitory role of soluble P-selectin glycoprotein ligand (sPSGL) in the process. MATERIALS
AND
METHODS
The early (0 to 7 days) events surrounding this injury were examined serially by immunohistology and RT-PCR. The renal vessels of uninephrectomized LEW rats were occluded for 45 minutes to ascertain the effects of warm ischemia, a condition becoming more clinically relevant with the use of non-heartbeating organ donors. For parallel studies of cold ischemia, the kidney was perfused in situ with 10 mL iced UW solution. In the treatment group, sPSGL (5 pg in 1 mL) was perfused after UW cooling to block P-selectin; 50 pg of this material was then injected IV into the animal after 3 hours of reperfusion to inhibit E-selectin. Kidneys (n = 3/time point) were removed at 1,3,5, and 7 days for immunohistology and molecular biology. PMN were stained with an anti-rat PMN FITC-conjugated gamma globulin and were counted under the fluorescence microscope. For immunohistology, the specimens were stained with ED-l for macrophages, W3/25 for CD4’ cells, OX-8 for CD8+ cells and OX-3 for MHC class II. Expression of adhesion molecules, cytokines, and growth factors were examined by RT-PCR.’ The specific primers (E-selectin, ICAM-1, IL-2, IL-2R, IFNg, IL-l, IL-6, TGFP, TNFLu,and p-actin) were used as described.3 RESULTS
Significant changes occurred within hours of warm ischemia/reperfusion, similar but slightly less intense patterns after cold perfusion. The early adhesion molecule, Eselectin, was rapidly upregulated (3 X /3-actin control) by 6 hours, associated with peak infiltration of PMN’s. ICAMwas highly expressed (3 X p-actin) between 1 and 3 days. Lymphocytes (primarily CD4+) and macrophages (ED-l’) entered the ischemic organs by day 2, peaking at day 5; this infiltration pattern paralleled upregulated MHC class II antigen expression. By RT-PCR; T cell products, IL-2, TNFa, IFNy, remained highly expressed after day 2; IL-2R 0041-l 315/97/$17.00 PII SO041 -1345(96)00577-5
expression did not change. Macrophages and their products, IL-6, IL-l, TGFP, were upregulated. Expression of lymphocytes and their products relating to upregulation of class II may increase the immunogenicity of the injured organ, which may explain the clinical correlation between ischemia and acute and possibly chronic rejection.’ Parallel studies were performed using a soluble recombinant form of PSGL-l(sPSGL) which binds and blocks both P- and E-selectins. Expression of E-selectin remained at baseline in the treated groups throughout the follow-up period. In addition, leukocytes (PMN’s, lymphocytes and macrophages) never infiltrated the injured organs; class II expression did not increase. T cell and macrophage products were markedly inhibited, although IL-2R expression was unaffected. DISCUSSION
It seems increasingly clear that primary graft dysfunction secondary to ischemia/reperfusion injury may decrease both short- and long-term function.’ As knowledge about the molecular events occurring in the kidney after this nonspecific insult is relatively limited, we have examined the early kinetics of inflammation activity in a rat model4 In general, adhesion molecules, cells and their products are highly expressed after both warm and cold ischemia. Expression of the early adhesion molecules, P- and E-selectin seems to trigger adhesion of host leukocyte population to graft endothelial cells.4 In the present model, a temporal relationship of E-selectin and subsequent cell migration into the organ on day 3 to 5 is apparent. Soluble PSGL-1 perfused into the ischemic graft and given to the animal during the re-perfusion phase, inhibited all acute (within 7 days) inflammatory events occurring in the kidney. The data imply that minimal cellular and cytokine activity occurs once E-selectin and initial PMN adhesion to graft endothelium, presumably a triggering mechanism for later events, are blocked. This therapeutic strategy may provide a bio-
From the Surgical Research Laboratory, Harvard Medical School and Department of Surgery, Brigham and Women’s Hospital, Boston, Massachusetts, USA. Address reprint requests to N.L. Tilney, MD, 75 Francis St, Boston, MA 02115.
0 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
1324
Transplantation
Proceedings,
29, 1324-l 325 (1997)
EARLY CHANGES
IN ISCHEMIA/REPERFUSION
logically inert organ to the recipient subsequent host reactivity.
INJURY
and may diminish
1325
2. Platzer C, Ode-Hakim 58:264, 1994
S, Reike P, et al: Transplantation
3. Nadeau KC, Azuma H, Tilney NL: Proc Nat1 Acad Sci USA REFERENCES
92:8729, 1995
1. Almond PS, Matas A, Gillingham K, et al: Transplantation 551752, 1993
4. Goes N, Urmson 59:565, 1995
J, Ramassar
V, et al: Transplantation
Early Cellular and Molecular Changes in Ischemia/Reperfusion Injury: Inhibition by a Selectin Antagonist, P-Selectin Glycoprotein Ligand-1 M. Takada, K.C. Nadeau, G.D. Shaw, and N.L. Tilney
I
SCHEMIA/reperfusion injury associated with organ transplantation may influence initial graft function, the development of acute rejection and late chronic changes.’ In this study, initial events surrounding both warm and cold ischemia/reperfusion have been examined. As the very early induced adhesion molecule, E-selectin (CD62E), is upregulated quickly and seems to trigger subsequent cellular events, we have assessed the inhibitory role of soluble P-selectin glycoprotein ligand (sPSGL) in the process. MATERIALS
AND
METHODS
The early (0 to 7 days) events surrounding this injury were examined serially by immunohistology and RT-PCR. The renal vessels of uninephrectomized LEW rats were occluded for 45 minutes to ascertain the effects of warm ischemia, a condition becoming more clinically relevant with the use of non-heartbeating organ donors. For parallel studies of cold ischemia, the kidney was perfused in situ with 10 mL iced UW solution. In the treatment group, sPSGL (5 pg in 1 mL) was perfused after UW cooling to block P-selectin; 50 pg of this material was then injected IV into the animal after 3 hours of reperfusion to inhibit E-selectin. Kidneys (n = 3/time point) were removed at 1,3,5, and 7 days for immunohistology and molecular biology. PMN were stained with an anti-rat PMN FITC-conjugated gamma globulin and were counted under the fluorescence microscope. For immunohistology, the specimens were stained with ED-l for macrophages, W3/25 for CD4’ cells, OX-8 for CD8+ cells and OX-3 for MHC class II. Expression of adhesion molecules, cytokines, and growth factors were examined by RT-PCR.’ The specific primers (E-selectin, ICAM-1, IL-2, IL-2R, IFNg, IL-l, IL-6, TGFP, TNFLu,and p-actin) were used as described.3 RESULTS
Significant changes occurred within hours of warm ischemia/reperfusion, similar but slightly less intense patterns after cold perfusion. The early adhesion molecule, Eselectin, was rapidly upregulated (3 X /3-actin control) by 6 hours, associated with peak infiltration of PMN’s. ICAMwas highly expressed (3 X p-actin) between 1 and 3 days. Lymphocytes (primarily CD4+) and macrophages (ED-l’) entered the ischemic organs by day 2, peaking at day 5; this infiltration pattern paralleled upregulated MHC class II antigen expression. By RT-PCR; T cell products, IL-2, TNFa, IFNy, remained highly expressed after day 2; IL-2R 0041-l 315/97/$17.00 PII SO041 -1345(96)00577-5
expression did not change. Macrophages and their products, IL-6, IL-l, TGFP, were upregulated. Expression of lymphocytes and their products relating to upregulation of class II may increase the immunogenicity of the injured organ, which may explain the clinical correlation between ischemia and acute and possibly chronic rejection.’ Parallel studies were performed using a soluble recombinant form of PSGL-l(sPSGL) which binds and blocks both P- and E-selectins. Expression of E-selectin remained at baseline in the treated groups throughout the follow-up period. In addition, leukocytes (PMN’s, lymphocytes and macrophages) never infiltrated the injured organs; class II expression did not increase. T cell and macrophage products were markedly inhibited, although IL-2R expression was unaffected. DISCUSSION
It seems increasingly clear that primary graft dysfunction secondary to ischemia/reperfusion injury may decrease both short- and long-term function.’ As knowledge about the molecular events occurring in the kidney after this nonspecific insult is relatively limited, we have examined the early kinetics of inflammation activity in a rat model4 In general, adhesion molecules, cells and their products are highly expressed after both warm and cold ischemia. Expression of the early adhesion molecules, P- and E-selectin seems to trigger adhesion of host leukocyte population to graft endothelial cells.4 In the present model, a temporal relationship of E-selectin and subsequent cell migration into the organ on day 3 to 5 is apparent. Soluble PSGL-1 perfused into the ischemic graft and given to the animal during the re-perfusion phase, inhibited all acute (within 7 days) inflammatory events occurring in the kidney. The data imply that minimal cellular and cytokine activity occurs once E-selectin and initial PMN adhesion to graft endothelium, presumably a triggering mechanism for later events, are blocked. This therapeutic strategy may provide a bio-
From the Surgical Research Laboratory, Harvard Medical School and Department of Surgery, Brigham and Women’s Hospital, Boston, Massachusetts, USA. Address reprint requests to N.L. Tilney, MD, 75 Francis St, Boston, MA 02115.
0 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
1324
Transplantation
Proceedings,
29, 1324-l 325 (1997)
EARLY CHANGES
IN ISCHEMIA/REPERFUSION
logically inert organ to the recipient subsequent host reactivity.
INJURY
and may diminish
1325
2. Platzer C, Ode-Hakim 58:264, 1994
S, Reike P, et al: Transplantation
3. Nadeau KC, Azuma H, Tilney NL: Proc Nat1 Acad Sci USA REFERENCES
92:8729, 1995
1. Almond PS, Matas A, Gillingham K, et al: Transplantation 551752, 1993
4. Goes N, Urmson 59:565, 1995
J, Ramassar
V, et al: Transplantation