Selectin Inhibitor Bimosiamose Prolongs Survival of Kidney Allografts by Reduction in Intragraft Production of Cytokines and Chemokines
Robert Langer*,
Mouer Wang*,
Stanislaw M. Stepkowski*,
Wayne W. Hancock,
Rongxiang Han,
Ping Li,
Lily Feng,
Robert A. Kirken,
Kurt L. Berens¶,
Brian Dupre¶,
Hemangshu Podder*,
Richard A.F. Dixon¶ and
Barry D. Kahan*
*Division of Immunology and Organ Transplantation, Department of Surgery, the University of Texas Medical School, Houston, Texas; Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; Department of Nephrology, Baylor College of Medicine, Houston, Texas; Department of Integrative Biology and Pharmacology, the University of Texas Medical School, Houston, Texas; and ¶Encysive Pharmaceuticals Inc., Bellaire, Texas
Correspondence to Dr. Stanislaw M. Stepkowski, Division of Immunology and Organ Transplantation, The University of Texas Medical School–Houston, 6431 Fannin, Suite 6.240, Houston, TX 77030. Phone: 713-500-7372; Fax: 713-500-0784; E-mail: Stanislaw.Stepkowski{at}uth.tmc.edu
Binding of the P-, L-, and E-selectins to sialyl Lewisx (sLex)retards circulating leukocytes, thereby facilitating their attachmentto the blood vessels of allografts. Whether the selectin inhibitorbimosiamose (BIMO; C46H54O16 · 0.25 H2O [867.4 molecularweight]) inhibits the rejection process of kidney allograftsin a rat model was examined. Rat recipients acutely rejectedkidney allografts at a mean survival time of 8.8 ± 0.75d. An intravenous 7-d infusion by osmotic pump of 2.5, 5, 10,or 20 mg/kg BIMO extended kidney allograft survival to 11.5± 2.2 d (P < 0.03), 25.4 ± 11.4 d (P < 0.006),37.4 ± 13.6 d (P < 0.001), and 39.8 ± 34.5d (P < 0.01), respectively. Combination of BIMO with cyclosporineproduced synergistic interactions, as documented by the combinationindex (CI) values of 0.34 to 0.43 (CI <1 is synergistic;CI = 1 is additive; and CI >1 is antagonistic). Similarly,BIMO interacted synergistically with sirolimus (CI = 0.64) andFTY720 (CI = 0.22). While the mechanism of immunosuppressionwas being analyzed, decreased infiltration of CD4+, CD8+, andmacrophages on day 7 after grafting was observed. Multiple cytokineswere also expressed, including IL-1, IL-1, IL-2, IL-4, IL-6,IL-10, IL-12, IL-18, TNF-, and IFN- in kidney allografts ondays 3, 5, and 7 after grafting, as measured by a ribonucleaseprotection assay. Furthermore, at similar time points, BIMOtreatment reduced intragraft expression of P-selectin glycoproteinligand-1, CX3CL1, CCL19, CCL20, and CCL2. Thus, BIMO blocksallograft rejection by reduction of intragraft expression ofcytokines and chemokines.
Whereas nearly all cells in an adult human are sessile, themajority of leukocytes provide a mobile defense of the individual’sintegrity (1). Precursors of leukocytes continuously undergodifferentiation in the bone marrow and the thymus, whereas matureleukocytes continuously traffic between the spleen and the lymphnodes. Efficient deployment to an inflammatory site requirespolarization, mobilization, and migration of leukocytes duringprocesses that are controlled by adhesion molecules and chemokines(2). These molecules participate in the process of ischemia/reperfusion(I/R), as well as in acute and chronic rejection responses towardorgan allografts (3,4).
Adhesive interactions with the vascular endothelium initiatethe migration of leukocytes to sites of inflammation. In thiscascade of events (rolling, attachment, spreading, and transendothelialmigration), the selectin family (P-, E-, and L-selectins) islargely involved in rolling and attachment (5). Rolling is initiatedby the interaction of selectins with sialyl Lewisx (sLex) ligand(6); all selectins have an NH2-terminal, lectin-like domainbinding to sLex and other related ligands in a Ca2+-dependentmanner (7). Two selectins are expressed on endothelial cells,namely, P-selectins, which are stored in granules and rapidlytranslocated to the cell surface, and E-selectins, which areinduced by inflammatory cytokines (8). Within minutes afterreperfusion, endothelial cells express P-selectins that mediateleukocyte rolling over the vascular lining.
The main ligand for P-selectin is P-selectin glycoprotein ligand-1(PSGL-1), which is a disulfide-bonded homodimeric mucin-likeglycoprotein expressed on leukocytes, platelets, and CD34+ cells(9). Functional PSGL-1 is modified with sialylation and fucosylationof O-linked sugars, as well as sulfation of tyrosine residueson the N-terminus (10). Engagement of PSGL-1 by P-selectinsslows the movement of leukocytes and sparks a cascade of signalingpathways, leading to the expression of multiple adhesion molecules(11). During rolling, the leukocytes start expressing integrinstriggered by cytokines and chemokines present on the surfaceof the endothelial cells (12). All of these processes lead tofirm adhesion and migration of leukocytes through the vesselwall and into the inflammatory site. Similar events have beenshown in signal transduction pathways (13) that orchestrateleukocyte trafficking (14), organogenesis (15), hematopoiesis(16), and vascular remodeling (17). Thus, initial selectin-mediatedrolling of leukocytes along with the expression of adhesionmolecules and the production of cytokines and chemokines maydetermine the pattern and severity of the I/R injury and ofallograft rejection (13).
A new, potent selectin antagonist, bimosiamose (BIMO; 1.6-bis[3-(3-carboxymethylphenyl)-4-(2--D-mannopyranosyloxy)phenyl]hexane;Encysive Pharmaceuticals, Bellaire, TX), is a synthetic sLexglycomimetic with potent inhibitory effects on all three selectins(18). In vitro studies examined the competitive inhibitory effectsof BIMO on the binding of beads coated with human E-, P-, orL-selectin/IgG fusion protein to HL60 cells expressing naturalsLex (19). Although the inactive control was ineffective, a50% inhibitory effect (IC50) was achieved by BIMO at 500 µMfor E-selectin, 70 µM for P-selectin, and 560 µMfor L-selectin. An in situ perfusion with BIMO before the I/Rinjury inflicted by bilateral artery clamping prevented mortalityand improved kidney function (20). The damage caused by hemorrhagicshock was diminished after pretreatment with BIMO (21). Furthermore,BIMO blockade of selectin/PSGL-1 interaction inhibited the PSGL-1–inducedexpression of adhesion molecules (19). The involvement of selectinsin the pathogenesis of renal I/R injury was confirmed by resultsobtained with another selectin inhibitor, glycyrrhizin (22).Perfusion of kidneys with glycyrrhizin before artery cross-clampingattenuated I/R injury when evaluated 72 h later. These resultssuggested that selectin inhibitors might help to protect organallografts.
The results of the present experiments revealed that BIMO blockedintragraft production of multiple cytokines and chemokines,consequently inhibiting I/R injury and kidney allograft rejection.BIMO also acted synergistically in combination with cyclosporine(CsA; Sandimmune; Novartis Research, East Hanover, NJ), sirolimus(SRL; Rapamune; Wyeth Research, Philadelphia, PA), and FTY720(Novartis) to prolong the survival of kidney allografts.
Animals
Male ACI (RT1a), Lewis (Lew; RT1l), and Wistar Furth (WF; RT1u)rats (160 to 200 g) from Harlan Sprague Dawley (Indianapolis,IN) were housed in a temperature- and light-controlled environmentand fed ad libitum with regular or low-salt diet (0.05% sodium;Teklad Premier, Madison, WI) with free access to tap water.All experiments were performed with strict adherence to thestandards prescribed by the "Guide for the Care and Use of LaboratoryAnimals" (National Research Council, Washington, DC).
Experimental Design
In the I/R study, LEW kidneys perfused with 2 or 4 mg of BIMOin 2 ml of saline or UW solution (ViaSpan; Barr Laboratories,Inc., Pomona, NY) were exposed to 30 min of cold ischemia and30 min of anastomosis time during transplantation to nephrectomizedLEW recipients. At 24 h, GFR were measured using the iohexolmethod (23), and serum creatinine and blood urea nitrogen levelswere measured by conventional methods.
For the allograft survival study, ACI recipients of LEW kidneyallografts were treated with 0, 2.5, 5, 10, or 20 mg/kg BIMOdelivered intravenously by 7-d osmotic pumps (Alza, MountainView, CA). Kidney allografts from recipients that were treatedwith 20 mg/kg BIMO were examined for the expression of cytokines—IL-1,IL-1, TNF-, IL-3, IL-4, IL-5, IL-6, IL-10, TNF-, IL-2, IFN-,macrophage inhibition factor (MIF), and PSGL-1—and chemokinesfractalkine (CX3CL1), macrophage inflammatory protein (MIP)-3(CCL19), MIP-3 (CCL20), and macrophage chemoattractant protein(MCP)-1 (CCL2) by ribonuclease protection assay (RPA). Somerecipients were treated with 2.5, 5, 10, or 20 mg/kg BIMO with1.5, 2.5, or 5 mg/kg CsA; 0.125 or 0.25 mg/kg FTY720; or 0.8mg/kg SRL by gavage; other recipients were treated with monotherapyof 2.5, 5, 10, or 20 mg/kg CsA; 0.125, 0.25, or 0.5 mg/kg FTY720;or 0.4, 0.8, or 1.6 mg/kg SRL. In some experiments, donors werepretreated intraperitoneally on days –2, –1, and0 or –4, –3, –2, –1, and 0 with 25,50, or 100 mg/kg BIMO.
Pathology and Immunopathology
Kidneys that were fixed in 10% formalin were paraffin embedded;sectioned (3 µm); and stained with hematoxylin and eosin,periodic acid-Schiff, or Masson’s Trichrome. Two observersindependently assessed the degree of vasculopathy, glomerularchanges, and tubulointerstitial damage in multiple kidney sections.Tubular and glomerular changes were separately graded as 0 =none; 1+ = <5%; 2+ = 5 to 25%; 3+ = 26 to 50%; and 4+ = >50%.A similar vascular scale included 0, none; 1+, minimal; 2+,mild; 3+, moderate; and 4+, severe. Snap-frozen portions ofkidneys were sectioned on a cryostat and stained by immunoperoxidaseusing rat anti-rat/mouse monoclonal antibodies (BD Pharmingen,San Diego, CA) and an Envision kit (Dako, Carpinteria, CA) directedagainst CD4+, CD8+, CD25+, macrophages, and polymorphonuclears(PMN). Labeled cells within 10 consecutive high-power fieldswere counted, using sections from three renal allografts/group,and results (mean ± SD) were compared by a t test.
Renal Function
Renal function was evaluated by iohexol (Omnipaque, 300 mg/ml;Nycomed, Inc., Princeton, NJ), as recommended by Inman et al.(23). Urine and blood samples collected at 20-min intervalswere analyzed for iohexol concentrations. GFR values (ml/min)were calculated by the formula (U x V)/P, where U is urinaryiohexol concentration (mg/ml), V is urine output (ml/24 h),and P is plasma iohexol concentration (mg/ml) (24). The resultswere presented as mean ± SD, and statistical significancewas assessed using a t test.
Analysis of Drug Interaction
The median effect equation was used to assess two-drug interactions(25,26). The dose-effect relationship is described by the equation(fa/fu) = (D/Dm)m, where fa is the fraction affected (days ofsurvival), fu is the fraction unaffected (1–fa), D isthe administered drug dose, Dm is the dose required for 50-dsurvival (the median effect), and m is the slope coefficient.Logarithmic conversion of the median effect equation linearizesthe relationship: log(fa/fu) = m log(D) – m log(Dm), withcorrelation coefficient (r) >0.75. The two-drug interactionwas assessed by a combination index (CI) analysis, which usesthe dose of each drug alone and the doses of each drug in combinationnecessary to achieve the same days of survival:
where D1C and D2C are the doses of drugs when used in combinationand D1A and D2A are the corresponding doses of drugs used alone.CI values <1 reflect synergistic, 1 additive, and >1 antagonisticinteractions.
RPA
The RiboQuant method (BD Pharmingen, San Diego, CA) with multiprobeDNA templates were used with the T7 RNA polymerase-directedsynthesis of high specific activity 32P-labeled antisense RNAprobes for hybridization with specific mRNA encoding variouscytokines and chemokines. The total RNA was isolated by theRNA-Bee method (Tel-Test Inc., Friendswood, TX) and stored inRNase-free water at –80°C (27). RPA was performedas recommended in the manufacturer’s instructions.
Toxicology
Toxicology studies were performed by Inveresk Research. Rats(n = 5/sex/dose) were treated with a single intravenous injectionof 0, 250, 500, or 750 mg/kg BIMO. Similarly, mice (n = 5/sex/dose)received an intravenous injection of 1250, 1625, or 2000 mg/kgBIMO. Animals were then observed for clinical signs for 14 dafter injection before being killed and necropsy was performed.In repeat 14-d dosing experiments, rats (n = 10/sex/dose) weretreated with daily intravenous injections of 0, 60, 120, or300 mg/kg BIMO, and dogs (n = 3/sex/dose) received intravenousinjections for 14 d with 0, 60, 140, or 190 mg/kg BIMO. Duringboth studies, the clinical signs, body weight, and food consumptionwere recorded. Blood samples were collected on days 1 and 14for analysis of drug levels, hematology, and clinical chemistry.Upon completion of the dosing period, animals were killed andsubjected to detailed necropsy, including organ weight evaluationand histopathologic evaluation of the tissues.
Statistical Analyses
The t test was used to assess the equality of the mean valuesbetween treatment groups, and ANOVA was used to calculate meanvalues with SD. P < 0.05 was considered to be significant.
Effect of BIMO on I/R Injury of Kidney Transplants
To evaluate the in vivo effects on I/R injury, we perfused kidneysex vivo with 2 or 4 mg of BIMO suspended in 2 ml of saline beforeischemia and isografting (Figure 1A). When the GFR was measured24 h later, the 4-mg BIMO dose produced protection against theinjury; these results were confirmed by serum creatinine andblood urea nitrogen levels (Figure 1A). In addition, graft perfusionwith 2 mg of BIMO accompanied by a single perioperative intravenousinjection of 50 mg/kg BIMO into the recipient tended to showa similar degree of protection (Figure 1A). Because of the documentedeffects of UW solution to protect organs better than saline,we used 1 or 2 mg of BIMO suspended in 2 ml of UW (Figure 1B).The addition of BIMO to UW further enhanced kidney function.Thus, selectin inhibition mitigates I/R injuries when deliveredby graft perfusion alone immediately after harvesting and whendelivered by intravenous infusion before reperfusion.
Figure 1. Graft perfusion with bimosiamose (BIMO) inhibits ischemia/reperfusion (I/R) injury. (A) ACI kidneys were perfused ex vivo immediately after harvesting with 2 ml of saline that contained 2 or 4 mg of BIMO and transplanted to ACI recipients. In the last group, kidneys that were perfused with 2 ml of saline with 2 mg of BIMO were transplanted to recipients that received a single injection (day 0) of 50 mg/kg BIMO. (B) ACI kidneys that were perfused ex vivo with 2 ml of UW solution that contained 2 or 4 mg of BIMO were transplanted to ACI recipients. Within 24 h after grafting, GFR values were measured using an iohexol method. In addition, blood samples were used to measure serum creatinine and blood urea nitrogen levels. The statistically significant P values were calculated (n = 3–5) by a t test.
Effect of BIMO on Allograft Rejection
The effects of BIMO on kidney allograft rejection were examinedin the Lew to ACI combination. Untreated recipients rejectedkidney allografts at a mean survival time of 8.8 ± 0.8d (Figure 2A). Treatment of donors with 25, 50, or 100 mg/kgeither three (days –2, –1, and 0) or five times(days –4, –3, –2, –1, and 0) extendedkidney allograft survivals (Figure 2A). Similarly, a 7-d intravenouscontinuous infusion of 2.5, 5, 10, or 20 mg/kg BIMO prolongedsurvivals to 11.6 ± 2.2 d (P < 0.02), 25.4 ±11.4 d (P < 0.006), 37.4 ± 13.6 d (P < 0.002),and 41.8 ± 34.1 d (P < 0.04; Figure 2B). When recipientsthat were treated with 5 or 10 mg/kg BIMO received an intravenousinjection immediately after the operation with 50 mg/kg BIMOand received a transplant of kidney allografts that were perfusedwith 1 mg/2 ml BIMO, the survivals were significantly extendedto 38.2 ± 3.8 d (P < 0.04) and 55.7 ± 8.1 d(P = 0.01), respectively (data not shown), compared with recipientsthat were treated by intravenous pump (Figure 2B). These resultsshowed that BIMO therapy has potent immunosuppressive effectsin vivo.
Figure 2. Treatment of recipients with BIMO alone prolongs kidney allograft survival. (A) ACI recipients received a transplant of LEW kidneys that were harvested from donors that were received an intraperitoneal injection of 25, 50, or 100 mg/kg BIMO either three times (days –2, –1, and 0) or five times (–4, –3, –2, –1, and 0). (B) ACI recipients of LEW kidney allografts were treated with 2.5, 5, 10, or 20 mg/kg BIMO delivered by an intravenous 7-d osmotic pump. The results in A and B are presented as mean survival time ± SD with statistically significant P values calculated (n = 5–6) by a t test. (C) Histologic examination was performed on kidney allografts from recipients that were untreated or treated with 20 mg/kg BIMO for 3 and 7 d after grafting. (D) Immunostaining was performed on kidney allografts from untreated recipients (rejector) or recipients that were treated with 20 mg/kg BIMO for 7 d after grafting. Sections of snap-frozen kidney portions were stained by immunoperoxidase by rat anti-rat/mouse monoclonal antibodies directed to CD4+, CD8+, CD25+, macrophages, and polymorphonuclears using an Envision kit. Results represent labeled cells in 10 consecutive high-power fields from three kidney allografts per group and were compared by a t test (mean ± SD). For more details, see the Materials and Methods section.
Histologic examination that was performed on allografts fromrecipients that were treated with the highest dose of BIMO (20mg/kg) revealed significant differences from those from untreatedcontrols (Figure 2C). Hematoxylin and eosin staining of kidneytransplants showed reduced infiltration on day 3 after graftingin the BIMO group compared with the untreated rejector group.On day 7 after grafting, kidney allografts from the BIMO groupshowed significantly reduced infiltration of mononuclear cellsand none of the kidney damage that was observed in the rejectorgroup (Figure 2C). Immunostaining analysis confirmed that BIMOtherapy significantly reduced infiltration of kidney allografts(day 7 after grafting) with CD4+, CD8+, macrophages, PMN, andCD25+ cells (P < 0.0001; Figure 2D). These results documentthat BIMO blocks the process of allograft rejection by reducinginfiltration with leukocytes. To examine further the mechanismof immunosuppression, we evaluated the expression of cytokinesand chemokines. As shown by RPA, kidney transplants from untreatedrejectors displayed increased mRNA levels for IL-1, IL-3, IL-6,IL-10, TNF-, and IFN- on postgrafting days 3 and 5, which decreasedon day 7 (Figure 3). These findings correlated with the kineticsof allograft destruction. In contrast, kidney allografts fromBIMO-treated recipients showed reduced levels of cytokine mRNAat all of these times (Figure 3). BIMO also inhibited the expressionof PSGL-1 (Figure 4A), CX3CL1, CCL19, CCL20, and CCL2 (Figure 4B)mRNA. These results indicate that selectin inhibition ofmononuclear cell infiltration correlates with reduced productionof multiple cytokines and chemokines.
Figure 3. BIMO blocks intragraft production of different cytokines. Kidney allografts that were harvested on days 1, 3, 5, and 7 after grafting from untreated recipients (rejectors) or recipients that were treated with 20 mg/kg BIMO for 7 d were examined for expression of IL-1, IL-1, IL-2, IL-4, IL-6, IL-10, IL-12, IL-18, TNF-, and IFN- mRNA by a ribonuclease protection assay (RPA). The differences in the expression of cytokine mRNA were confirmed by similar expression of the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase and L32. The experiment was repeated three times with almost identical results for days 1, 3, and 5 and repeated seven times for day 7.
Figure 4. BIMO inhibits intragraft expression of PSGL-1, CX3CL1, CCL19, CCL20, and CCL2 mRNA. LEW kidney allografts were transplanted to untreated (control) ACI recipients or those that were treated with 20 mg/kg BIMO. (A) Expression of PSGL-1 mRNA was examined on days 0, 3, and 7 after grafting. (B) Expression of CX3CL1, CCL19, CCL20, and CCL2 mRNA was examined on days 1, 3, 5, and 7 after grafting. The experiments performed using an RPA method were repeated three times with almost identical results. For more details, see the Materials and Methods section.
Synergy of BIMO with Immunosuppressive Drugs
We also examined the interaction of BIMO with three immunosuppressivedrugs that display distinct mechanisms of action: CsA blocksa calcineurin-dependent T cell receptor pathway and, consequently,cytokine production (28); SRL blocks the mammalian target ofrapamycin after cytokine/cytokine receptor interaction (29);and FTY720 affects chemokine/chemokine receptor–dependenthoming of lymphocytes in lymphoid organs (30). BIMO was combinedwith these agents to test the effects on kidney allograft survival(Figure 5). Recipients of kidney allografts were treated witha 7-d BIMO regimen alone or in combination with CsA, SRL, orFTY720. A median effect analysis was used to calculate CI valuesthat describe the type of interactions between BIMO and otherdrugs. It is interesting that each of the three immunosuppressivedrugs produced synergistic effects in combination with BIMO.The CI values were 0.05 to 0.77 for CsA (Figure 5A), 0.3 to1.0 for SRL (Figure 5B), and 0.22 to 0.70 for FTY720 (Figure 5C).These results document that BIMO interacts synergisticallywith immunosuppressive drugs with distinct mechanisms of action.
Figure 5. BIMO acts synergistically with cyclosporine (CsA), sirolimus (SRL), or FTY720 to prolong survival of kidney allografts. ACI recipients of LEW kidney allografts were untreated (control) or treated by an intravenous 7-d osmotic pump with 2.5, 5, or 10 mg/kg BIMO alone (see also results in Figure 2B) or in combination with 3-d oral gavage of 2.5 or 5 mg/kg CsA; 0.4, 0.8, or 1.6 mg/kg SRL; or 0.125, 0.25, or 0.5 FTY720. The results are presented as mean ± SD with statistically significant P values calculated (n = 5–6) by a t test. Drug interaction was compared by the median effect analysis and calculation of the combination index (CI) values (CI <1 shows synergistic; CI = 1, additive; and CI >1, antagonistic interactions). For more details, see the Materials and Methods section.
Toxicity Study of BIMO
We propose that the major advantage of treatment with BIMO isits apparent lack of toxic effects. Preclinical studies performedin accordance with good laboratory practices showed that BIMOwas well tolerated in rodents and dogs after intravenous administration.Single doses of 500 or 1250 mg/kg BIMO delivered intravenouslyto rats and mice, respectively, did not cause mortality. Ina 14-d course, doses of 60 mg/kg BIMO delivered by intravenousinjection to rats had no observable effects on hematology andclinical chemistry parameters and produced no histologic changes.Similarly, dogs that were treated intravenously for 14 d with60 mg/kg per day BIMO had no hematologic, biochemical, or histopathologicchanges. Thus, BIMO may provide significant benefits withoutproducing toxic effects. Further experiments are planned toexplore the potential adverse effects of treatment with BIMOcombined with CsA, SRL, or FTY720.
This study examined the effect of selectin inhibitor BIMO onboth I/R injury and allograft rejection. Our results show thatinhibition of selectins by BIMO blocks intragraft infiltrationand activation of leukocytes during an I/R injury and inhibitsalloimmune responses. These inhibitory effects correlate witha dramatic decrease in infiltration with CD4+, CD8+, macrophages,and PMN, as well as expression of cytokines and chemokines atthe graft site. We also demonstrated that BIMO acts synergisticallywith immunosuppressive agents that display various mechanismsof action, extending allograft survival. These results showthat BIMO is a beneficial and versatile complementary agentthat can spare drug doses and thereby mitigate toxicity. Preclinicalexperiments documented that the clinical formulation of BIMOhas an excellent toxicology profile: a single dose as high as1 g/kg was necessary to produce LD50, whereas a threefold higherdose than that used in our study of 60 mg/kg delivered intravenouslyfor 14 d caused no observable toxicities or any chromosomalaberrations.
Continuous intravenous delivery of BIMO (5 to 20 mg/kg) extendedkidney allograft survival in a dose-dependent manner. Identicaldoses of 10 mg/kg BIMO delivered intravenously by pump for 7d or by daily bolus injections produced similar survivals of37.0 ± 13.6 and 33.4 ± 11 d, respectively. Themost dramatic change that accompanied BIMO therapy was a markeddecrease in graft mononuclear cell infiltration, which was associatedwith reduced expression of cytokines and chemokines. Becauseboth selectins and chemokines are actively involved in directingthe destination of T cells, deficient production of cytokinesreflects changes in overall inflammatory processes after selectinblockade (31).
Naïve T cells express CCR7 that are sensitive to secondarylymphoid chemokines on endothelial cells and stromal cells inT cell areas of secondary lymphoid organs (32). For example,expression of CCR7 and L-selectin determines development ofcentral (CCR7high and L-selectinhigh) and peripheral (CCR7lowand L-selectinlow) memory T cells (32). Activated T cells downregulateCCR7 and upregulate CXCR5 and CCR4, enabling their T-B cellcollaboration. Polarization of T cells into IL-2/IFN-–producingTh1 and IL-4/IL-10–producing Th2 also is regulated byselectins and chemokine receptors. In particular, Th1 expressesP-selectinhigh (33), as well as CCR5, CXCR3, and CCR1, whereasTh2 expresses P-selectinlow and high levels of CCR3 and CCR4.Expression of P- and E-selectins correlated with generationof Leishmania-specific Th1, which was dependent on the presenceof IL-12 (34). Mice deficient in P- and E-selectins developedsignificantly less inflammatory response. Thus, blockade ofall selectins affects multiple, interconnected events, resultingin inhibited T cell activation and decreased production of cytokinesand chemokines.
The complex interdependence of cytokines, chemokines, and adhesionmolecules with intracellular signaling was displayed by theapplication of an inhibitor of the Rho/ROCK pathway Y-27632(35). Continuous treatment with Y-27632 significantly prolongedthe survival of heart allografts. Immunohistochemistry showedthat Y-27632 therapy reduced intragraft expression of intercellularadhesion molecule and vascular cell adhesion molecule-1 on day7 after grafting. A recent report also revealed a new mechanismof I/R injury through the poly-ADP-ribose polymerase (PARP)pathway (36). The PARP inhibitor PJ34 improved myocardial contractility,coronary blood flow, and endothelial function after ischemicpreservation. Again, immunostaining confirmed that PARP inhibitionin heart transplants correlated with reduced expression of P-selectinand intercellular adhesion molecule-1 (36). Thus, blockade ofselectins may interfere with multiple signaling pathways involvedin graft rejection and I/R injury.
Our results show that selectin inhibition by BIMO dramaticallyreduced intragraft expression of the chemokines CX3CL1, CCL19,CCL20, and CCL2. As chemokines and their receptors are involvedin several different functions of leukocytes—includingmigration, cell–cell interactions, and intracellular signaling—theirinhibition (or activation) may affect I/R injury as well asacute and chronic rejection (37). Mounting evidence shows thatchemokines and their receptors are involved in I/R events. Forexample, deficiency of CCR1 on PMN protected kidneys and liversagainst I/R injury (38). CX3CL1 is upregulated on vascular endothelialcells, thereby promoting direct leukocyte adhesion and transmigrationduring inflammation or I/R injury (39). Both CX3CL1 and itsreceptor CX3CR1 are involved in the rejection process (40).Targeting of CXCR3 by specific monoclonal antibodies in normalmice or utilization of CXCR3–/– recipients delayedacute allograft rejection (41). CX3CR1–/– mice alsodisplayed significantly reduced leukocyte infiltration of heartallografts (42). Furthermore, CCR1–/– mice permanentlyaccepted class II MHC- and class I/II MHC-mismatched heart allograftswithout or with concomitant treatment with low doses of CsA,respectively (43). Although CXCL10–/– recipientsacutely rejected heart allografts, normal mice displayed prolongedsurvivals of CXCL10–/– heart allografts with significantlyreduced infiltration of allografts and intragraft productionof cytokines (44).
Our in vivo results showed that BIMO acted synergistically toprolong allograft survival when combined with CsA, SRL, or FTY720.Because each of these drugs has a distinct mechanism of action,selectin inhibition may allow reduction in daily doses of standardtherapeutic drugs, thereby damping their toxicities. CsA, acalcineurin inhibitor blocking signal 1 in the T cell activationpathway, causes nephrotoxicity that may be alleviated by usingsynergistic interaction with SRL (45). Although SRL—anagent that blocks mammalian target of rapamycin in the signal3 cytokine/cytokine receptor pathway—is not nephrotoxic,it causes disorders of lipid metabolism (46). Each of theseimmunosuppressive drugs may be at least partially replaced byBIMO, maintaining immunosuppression and potentially mitigatingtheir toxicities. However, the most promising synergistic interactionof BIMO is with FTY720, because this drug modulates lymphocytetrafficking responsible for homing of lymphocytes in spleenand lymph nodes (47). Permanent sequestration of lymphocytesin secondary lymphoid organs prevents allograft rejection (48).Although the exact mechanism of FTY720 action is not yet completelyexplained, synergism with a selectin inhibitor may provide apotent immunosuppressive combination. Furthermore, FTY720 pretreatmentreduced warm hepatic I/R injury by inhibiting T lymphocyte infiltrationand minimized I/R injury in kidneys (49).
In conclusion, selection inhibition by BIMO produces multiplebenefits by protecting against I/R injury, thereby improvingkidney function, and by inhibiting allograft rejection. Furthermore,BIMO acts alone as well as synergistically with other immunosuppressantsdisplaying diverse mechanisms of action. Remarkably, toxicologyprofiles revealed that BIMO causes no observable toxic reactions,proffering a unique and versatile agent to improve standardimmunosuppressive protocols. However, to confirm these results,further analyses of the potential toxic effects of treatmentwith BIMO combined with CsA, SRL, or FTY720 must be performed.
Acknowledgments
This study was supported in part by a grant from the NationalInstitute of Diabetes and Digestive and Kidney Diseases (NIDDK38016-15) and AI061052.
Mellado M, Rodriguez-Frade JM, Manes S, Martinez -AC. Chemokine signaling and functional responses: The role of receptor dimerization and TK pathway activation. Annu Rev Immunol 19: 397–421, 2001[CrossRef][Medline]
Burne-Taney MJ, Rabb H: The role of adhesion molecules and T cells in ischemic renal injury. Curr Opin Nephrol Hypertens 12: 85–90, 2003[CrossRef][Medline]
Weis M, von Scheidt W: Cardiac allograft vasculopathy: A review. Circulation 96: 2069–2077, 1997[Abstract/Free Full Text]
Butcher EC: Leukocyte-endothelial cell recognition: Three (or more) steps to specificity and diversity. Cell 67: 1033–1036, 1991[CrossRef][Medline]
Kannagi R: Regulatory roles of carbohydrate ligands for selectins in the homing of lymphocytes. Curr Opin Struct Biol 12: 599–608, 2002[CrossRef][Medline]
Frenette PS, Mayadas TN, Rayburn H, Hynes RO, Wagner DD: Susceptibility to infection and altered hematopoiesis in mice deficient in both P- and E-selectins. Cell 84: 563–574, 1996[CrossRef][Medline]
Tracey JB, Rinder HM: Characterization of the P-selectin ligand on human hematopoietic progenitors. Exp Hematol 24: 1494–1500, 1996[Medline]
Moore KL: Structure and function of P-selectin glycoprotein ligand-1. Leuk Lymphoma 29: 1–15, 1998[Medline]
Burch EE, Patil VR, Camphausen RT, Kiani MF, Goetz DJ: The N-terminal peptide of PSGL-1 can mediate adhesion to trauma-activated endothelium via P-selectin in vivo. Blood 100: 531–538, 2002[Abstract/Free Full Text]
Springer TA. Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration. Annu Rev Physiol 57: 1995
Baggiolini M, Dewald B, Moser B: Human chemokines: An update. Annu Rev Immunol 15: 675–705, 1997[CrossRef][Medline]
Cyster JG: Chemokines and cell migration in secondary lymphoid organs. Science 286: 2098–2102, 1999[Abstract/Free Full Text]
Ma Q, Jones D, Borghesani PR, Segal RA, Nagasawa T, Kishimoto T, Bronson RT, Springer TA: Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4- and SDF-1-deficient mice. Proc Natl Acad Sci U S A 95: 9448–9453, 1998[Abstract/Free Full Text]
Kim CH, Broxmeyer HE: Chemokines: Signal lamps for trafficking of T and B cells for development and effector function. J Leukoc Biol 65: 6–15, 1999[Abstract]
Zhou Y, Genbacev O, Fisher SJ: The human placenta remodels the uterus by using a combination of molecules that govern vasculogenesis or leukocyte extravasation. Ann N Y Acad Sci 995: 73–83, 2003[Medline]
Kogan TP, Dupre B, Bui H, McAbee KL, Kassir JM, Scott IL, Hu X, Vanderslice P, Beck PJ, Dixon RA: Novel synthetic inhibitors of selectin-mediated cell adhesion: Synthesis of 1,6-bis[3-(3-carboxymethylphenyl)-4-(2-alpha-D-mannopyranosyloxy)phenyl]hexane (TBC1269). J Med Chem 41: 1099–1111, 1998[CrossRef][Medline]
Davenpeck KL, Berens KL, Dixon RA, Dupre B, Bochner BS: Inhibition of adhesion of human neutrophils and eosinophils to P-selectin by the sialyl Lewis antagonist TBC1269: Preferential activity against neutrophil adhesion in vitro. J Allergy Clin Immunol 105: 769–775, 2000[CrossRef][Medline]
Nemoto T, Burne MJ, Daniels F, O’Donnell MP, Crosson J, Berens K, Issekutz A, Kasiske BL, Keane WF, Rabb H: Small molecule selectin ligand inhibition improves outcome in ischemic acute renal failure. Kidney Int 60: 2205–2214, 2001[CrossRef][Medline]
Ramos-Kelly JR, Toledo-Pereyra LH, Jordan J, Rivera-Chavez F, Rohs T, Holevar M, Dixon RA, Yun E, Ward PA: Multiple selectin blockade with a small molecule inhibitor downregulates liver chemokine expression and neutrophil infiltration after hemorrhagic shock. J Trauma 49: 92–100, 2000[Medline]
Subramanian S, Bowyer MW, Egan JC, Knolmayer TJ: Attenuation of renal ischemia-reperfusion injury with selectin inhibition in a rabbit model. Am J Surg 178: 573–576, 1999[CrossRef][Medline]
Inman SR, Plott WK, Richardson BF, Lewis RM: Iohexol clearance is an accurate and reliable measure of glomerular filtration rate in rats [Abstract]. J Am Soc Nephrol 8: 347A, 1997
Gaspari F, Perico N, Ruggenenti P, Mosconi L, Amuchastegui CS, Guerini E, Daina E, Remuzzi G: Plasma clearance of nonradioactive iohexol as a measure of glomerular filtration rate. J Am Soc Nephrol 6: 257–263, 1995[Abstract]
Chou TC, Kahan BD: A rigorous approach to the diagnosis of immunosuppressive synergy combination therapies. In: Modern Immunosuppressives: Milestones in Drug Therapy, edited by Schuurman H-J, Switzerland, Birkhauser, 2001 pp 183–200
Chou T-C, Talalay P: Quantitative analysis of dose-effect relationships: The combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 22: 27–55, 1984[CrossRef][Medline]
Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156–159, 1987[Medline]
Ho S, Clipstone N, Timmermann L, Northrop J, Graef I, Fiorentino D, Nourse J, Crabtree GR: The mechanism of action of cyclosporin A and FK506. Clin Immunol Immunopathol 80: S40–S45, 1996[CrossRef][Medline]
Huang S, Bjornsti MA, Houghton PJ: Rapamycins: Mechanism of action and cellular resistance. Cancer Biol Ther 2: 222–232, 2003[Medline]
Henning G, Ohl L, Junt T, Reiterer P, Brinkmann V, Nakano H, Hohenberger W, Lipp M, Forster R: CC chemokine receptor 7-dependent and -independent pathways for lymphocyte homing: Modulation by FTY720. J Exp Med 194: 1875–1881, 2001[Abstract/Free Full Text]
Vestweber D, Blanks JE: Mechanisms that regulate the function of the selectins and their ligands. Physiol Rev 79: 181–213, 1999[Abstract/Free Full Text]
Bjorkdahl O, Barber KA, Brett SJ, Daly MG, Plumpton C, Elshourbagy NA, Tite JP, Thomsen LL: Characterization of CC-chemokine receptor 7 expression on murine T cells in lymphoid tissues. Immunology 110: 170–179, 2003[CrossRef][Medline]
Haddad W, Cooper CJ, Zhang Z, Brown JB, Zhu Y, Issekutz A, Fuss I, Lee HO, Kansas GS, Barrett TA: P-selectin and P-selectin glycoprotein ligand 1 are major determinants for Th1 cell recruitment to nonlymphoid effector sites in the intestinal lamina propria. J Exp Med 198: 369–377, 2003[Abstract/Free Full Text]
Zaph C, Scott P: Th1 cell-mediated resistance to cutaneous infection with Leishmania major is independent of P- and E-selectins. J Immunol 171: 4726–4732, 2003[Abstract/Free Full Text]
Ohki S, Iizuka K, Ishikawa S, Kano M, Dobashi K, Yoshii A, Shimizu Y, Mori M, Morishita Y: A highly selective inhibitor of Rho-associated coiled-coil forming protein kinase, Y-27632, prolongs cardiac allograft survival of the BALB/c-to-C3H/He mouse model. J Heart Lung Transplant 20: 956–963, 2001[CrossRef][Medline]
Hancock WW: Chemokines and transplant immunobiology. J Am Soc Nephrol 13: 821–824, 2002[Free Full Text]
Pratschke J, Topham PS, Gao W, Paz D, Tilney NL, Gerard CJ, Hancock WW: New model of cold ischemia/reperfusion injury: CCR1 knockout mice are protected against renal injury [Abstract 961]. Presented at the XVIII Annual Scientific Meeting of the American Society of Transplantation, Chicago, IL, May 19–21, 1999
Soriano SG, Amaravadi LS, Wang YF, Zhou H, Yu GX, Tonra JR, Fairchild-Huntress V, Fang Q, Dunmore JH, Huszar D, Pan Y: Mice deficient in fractalkine are less susceptible to cerebral ischemia-reperfusion injury. J Neuroimmunol 125: 59–65, 2002[CrossRef][Medline]
Robinson LA, Nataraj C, Thomas DW, Howell DN, Griffiths R, Bautch V, Patel DD, Feng L, Coffman TM: A role for fractalkine and its receptor (CX(3)CR1) in cardiac allograft rejection. J Immunol 165: 6067–6072, 2000[Abstract/Free Full Text]
Hancock WW, Lu B, Gao W, Csizmadia V, Faia K, King JA, Smiley ST, Ling M, Gerard NP, Gerard C: Requirement of the chemokine receptor CXCR3 for acute allograft rejection. J Exp Med 192: 1515–1520, 2000[Abstract/Free Full Text]
Haskell CA, Hancock WW, Salant DJ, Gao W, Csizmadia V, Peters W, Faia K, Fituri O, Rottman JB, Charo IF: Targeted deletion of CX(3)CR1 reveals a role for fractalkine in cardiac allograft rejection. J Clin Invest 108: 679–688, 2001[CrossRef][Medline]
Gao W, Topham PS, King JA, Smiley ST, Csizmadia V, Lu B, Gerard CJ, Hancock WW: Targeting of the chemokine receptor CCR1 suppresses development of acute and chronic cardiac allograft rejection. J Clin Invest 105: 35–44, 2000[Medline]
Hancock WW, Gao W, Csizmadia V, Faia KL, Shemmeri N, Luster AD: Donor-derived IP-10 initiates development of acute allograft rejection. J Exp Med 193: 975–980, 2001[Abstract/Free Full Text]
Dunn J, Grevel J, Napoli K, Lewis RM, Van Buren CT, Kahan BD: The impact of steady-state cyclosporine concentrations on renal allograft outcome. Transplantation 49: 30–34, 1990[Medline]
Hoogeveen RC, Ballantyne CM, Pownall HJ, Opekun AR, Hachey DL, Jaffe JS, Oppermann S, Kahan BD, Morrisett JD: Effect of sirolimus on the metabolism of apoB100-containing lipoproteins in renal transplant patients. Transplantation 72: 1244–1250, 2001[CrossRef][Medline]
Henning G, Ohl L, Junt T, Reiterer P, Brinkmann V, Nakano H, Hohenberger W, Lipp M, Forster R: CC chemokine receptor 7-dependent and -independent pathways for lymphocyte homing: Modulation by FTY720. J Exp Med 194: 1875–1881, 2001
Wang ME, Tejpal N, Qu X, Yu J, Okamoto M, Stepkowski SM, Kahan BD: Immunosuppressive effects of FTY720 alone or in combination with cyclosporine or sirolimus. Transplantation 65: 899–905, 1998[CrossRef][Medline]
Anselmo DM, Amersi FF, Shen XD, Gao F, Katori M, Lassman C, Ke B, Coito AJ, Ma J, Brinkmann V, Busuttil RW, Kupiec-Weglinski JW, Farmer DG: FTY720 pretreatment reduces warm hepatic ischemia reperfusion injury through inhibition of T-lymphocyte infiltration. Am J Transplant 2: 843–849, 2002[CrossRef][Medline]
Received for publication November 21, 2003.
Accepted for publication July 30, 2004.
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Abstract
Introduction
, IL-1
, IL-2, IL-4, IL-6, IL-10, IL-12, IL-18, TNF-
in kidney allografts on days 3, 5, and 7 after grafting, as measured by a ribonuclease protection assay. Furthermore, at similar time points, BIMO treatment reduced intragraft expression of P-selectin glycoprotein ligand-1, CX3CL1, CCL19, CCL20, and CCL2. Thus, BIMO blocks allograft rejection by reduction of intragraft expression of cytokines and chemokines. 
