Basic Transplantation

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
JASN November 2004, 15 (11) 2893-2901; DOI: https://doi.org/10.1097/01.ASN.0000142425.23036.AC

Abstract

Binding of the P-, L-, and E-selectins to sialyl Lewisx (sLex) retards circulating leukocytes, thereby facilitating their attachment to the blood vessels of allografts. Whether the selectin inhibitor bimosiamose (BIMO; C46H54O16 · 0.25 H2O [867.4 molecular weight]) inhibits the rejection process of kidney allografts in a rat model was examined. Rat recipients acutely rejected kidney allografts at a mean survival time of 8.8 ± 0.75 d. 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 ± 34.5 d (P < 0.01), respectively. Combination of BIMO with cyclosporine produced synergistic interactions, as documented by the combination index (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) and FTY720 (CI = 0.22). While the mechanism of immunosuppression was being analyzed, decreased infiltration of CD4+, CD8+, and macrophages on day 7 after grafting was observed. Multiple cytokines were also expressed, including IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IL-18, TNF-α, and IFN-γ 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.

Whereas nearly all cells in an adult human are sessile, the majority of leukocytes provide a mobile defense of the individual’s integrity (1). Precursors of leukocytes continuously undergo differentiation in the bone marrow and the thymus, whereas mature leukocytes continuously traffic between the spleen and the lymph nodes. Efficient deployment to an inflammatory site requires polarization, mobilization, and migration of leukocytes during processes 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 toward organ allografts (3,4⇓).

Adhesive interactions with the vascular endothelium initiate the migration of leukocytes to sites of inflammation. In this cascade of events (rolling, attachment, spreading, and transendothelial migration), the selectin family (P-, E-, and L-selectins) is largely involved in rolling and attachment (5). Rolling is initiated by the interaction of selectins with sialyl Lewisx (sLex) ligand (6); all selectins have an NH2-terminal, lectin-like domain binding to sLex and other related ligands in a Ca2+-dependent manner (7). Two selectins are expressed on endothelial cells, namely, P-selectins, which are stored in granules and rapidly translocated to the cell surface, and E-selectins, which are induced by inflammatory cytokines (8). Within minutes after reperfusion, endothelial cells express P-selectins that mediate leukocyte 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-like glycoprotein expressed on leukocytes, platelets, and CD34+ cells (9). Functional PSGL-1 is modified with sialylation and fucosylation of O-linked sugars, as well as sulfation of tyrosine residues on the N-terminus (10). Engagement of PSGL-1 by P-selectins slows the movement of leukocytes and sparks a cascade of signaling pathways, leading to the expression of multiple adhesion molecules (11). During rolling, the leukocytes start expressing integrins triggered by cytokines and chemokines present on the surface of the endothelial cells (12). All of these processes lead to firm adhesion and migration of leukocytes through the vessel wall and into the inflammatory site. Similar events have been shown in signal transduction pathways (13) that orchestrate leukocyte trafficking (14), organogenesis (15), hematopoiesis (16), and vascular remodeling (17). Thus, initial selectin-mediated rolling of leukocytes along with the expression of adhesion molecules and the production of cytokines and chemokines may determine the pattern and severity of the I/R injury and of allograft rejection (13).

A new, potent selectin antagonist, bimosiamose (BIMO; 1.6-bis [3-(3-carboxymethylphenyl)-4-(2-α-D-mannopyranosyloxy)phenyl]hexane; Encysive Pharmaceuticals, Bellaire, TX), is a synthetic sLex glycomimetic with potent inhibitory effects on all three selectins (18). In vitro studies examined the competitive inhibitory effects of BIMO on the binding of beads coated with human E-, P-, or L-selectin/IgG fusion protein to HL60 cells expressing natural sLex (19). Although the inactive control was ineffective, a 50% inhibitory effect (IC50) was achieved by BIMO at 500 μM for E-selectin, 70 μM for P-selectin, and 560 μM for L-selectin. An in situ perfusion with BIMO before the I/R injury inflicted by bilateral artery clamping prevented mortality and improved kidney function (20). The damage caused by hemorrhagic shock was diminished after pretreatment with BIMO (21). Furthermore, BIMO blockade of selectin/PSGL-1 interaction inhibited the PSGL-1–induced expression of adhesion molecules (19). The involvement of selectins in the pathogenesis of renal I/R injury was confirmed by results obtained with another selectin inhibitor, glycyrrhizin (22). Perfusion of kidneys with glycyrrhizin before artery cross-clamping attenuated I/R injury when evaluated 72 h later. These results suggested that selectin inhibitors might help to protect organ allografts.

The results of the present experiments revealed that BIMO blocked intragraft 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.

Materials and Methods

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 environment and 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 the standards prescribed by the “Guide for the Care and Use of Laboratory Animals” (National Research Council, Washington, DC).

Experimental Design

In the I/R study, LEW kidneys perfused with 2 or 4 mg of BIMO in 2 ml of saline or UW solution (ViaSpan; Barr Laboratories, Inc., Pomona, NY) were exposed to 30 min of cold ischemia and 30 min of anastomosis time during transplantation to nephrectomized LEW recipients. At 24 h, GFR were measured using the iohexol method (23), and serum creatinine and blood urea nitrogen levels were measured by conventional methods.

For the allograft survival study, ACI recipients of LEW kidney allografts were treated with 0, 2.5, 5, 10, or 20 mg/kg BIMO delivered intravenously by 7-d osmotic pumps (Alza, Mountain View, CA). Kidney allografts from recipients that were treated with 20 mg/kg BIMO were examined for the expression of cytokines—IL-1α, IL-1β, TNF-β, IL-3, IL-4, IL-5, IL-6, IL-10, TNF-α, IL-2, IFN-γ, macrophage inhibition factor (MIF), and PSGL-1—and chemokines fractalkine (CX3CL1), macrophage inflammatory protein (MIP)-3β (CCL19), MIP-3α (CCL20), and macrophage chemoattractant protein (MCP)-1 (CCL2) by ribonuclease protection assay (RPA). Some recipients were treated with 2.5, 5, 10, or 20 mg/kg BIMO with 1.5, 2.5, or 5 mg/kg CsA; 0.125 or 0.25 mg/kg FTY720; or 0.8 mg/kg SRL by gavage; other recipients were treated with monotherapy of 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 were pretreated intraperitoneally on days −2, −1, and 0 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 observers independently assessed the degree of vasculopathy, glomerular changes, 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 of kidneys were sectioned on a cryostat and stained by immunoperoxidase using rat anti-rat/mouse monoclonal antibodies (BD Pharmingen, San Diego, CA) and an Envision kit (Dako, Carpinteria, CA) directed against CD4+, CD8+, CD25+, macrophages, and polymorphonuclears (PMN). Labeled cells within 10 consecutive high-power fields were 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 intervals were analyzed for iohexol concentrations. GFR values (ml/min) were calculated by the formula (U × V)/P, where U is urinary iohexol concentration (mg/ml), V is urine output (ml/24 h), and P is plasma iohexol concentration (mg/ml) (24). The results were presented as mean ± SD, and statistical significance was 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 of survival), fu is the fraction unaffected (1–fa), D is the administered drug dose, Dm is the dose required for 50-d survival (the median effect), and m is the slope coefficient. Logarithmic conversion of the median effect equation linearizes the relationship: log(fa/fu) = m log(D) − m log(Dm), with correlation coefficient (r) >0.75. The two-drug interaction was assessed by a combination index (CI) analysis, which uses the dose of each drug alone and the doses of each drug in combination necessary to achieve the same days of survival: Embedded Image

where D1C and D2C are the doses of drugs when used in combination and D1A and D2A are the corresponding doses of drugs used alone. CI values <1 reflect synergistic, 1 additive, and >1 antagonistic interactions.

RPA

The RiboQuant method (BD Pharmingen, San Diego, CA) with multiprobe DNA templates were used with the T7 RNA polymerase-directed synthesis of high specific activity α32P-labeled antisense RNA probes for hybridization with specific mRNA encoding various cytokines and chemokines. The total RNA was isolated by the RNA-Bee method (Tel-Test Inc., Friendswood, TX) and stored in RNase-free water at −80°C (27). RPA was performed as 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 injection of 0, 250, 500, or 750 mg/kg BIMO. Similarly, mice (n = 5/sex/dose) received an intravenous injection of 1250, 1625, or 2000 mg/kg BIMO. Animals were then observed for clinical signs for 14 d after injection before being killed and necropsy was performed. In repeat 14-d dosing experiments, rats (n = 10/sex/dose) were treated with daily intravenous injections of 0, 60, 120, or 300 mg/kg BIMO, and dogs (n = 3/sex/dose) received intravenous injections for 14 d with 0, 60, 140, or 190 mg/kg BIMO. During both studies, the clinical signs, body weight, and food consumption were recorded. Blood samples were collected on days 1 and 14 for analysis of drug levels, hematology, and clinical chemistry. Upon completion of the dosing period, animals were killed and subjected to detailed necropsy, including organ weight evaluation and histopathologic evaluation of the tissues.

Statistical Analyses

The t test was used to assess the equality of the mean values between treatment groups, and ANOVA was used to calculate mean values with SD. P < 0.05 was considered to be significant.

Results

Effect of BIMO on I/R Injury of Kidney Transplants

To evaluate the in vivo effects on I/R injury, we perfused kidneys ex vivo with 2 or 4 mg of BIMO suspended in 2 ml of saline before ischemia and isografting (Figure 1A). When the GFR was measured 24 h later, the 4-mg BIMO dose produced protection against the injury; these results were confirmed by serum creatinine and blood urea nitrogen levels (Figure 1A). In addition, graft perfusion with 2 mg of BIMO accompanied by a single perioperative intravenous injection of 50 mg/kg BIMO into the recipient tended to show a similar degree of protection (Figure 1A). Because of the documented effects 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 delivered by graft perfusion alone immediately after harvesting and when delivered by intravenous infusion before reperfusion.

Figure1

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 examined in the Lew to ACI combination. Untreated recipients rejected kidney allografts at a mean survival time of 8.8 ± 0.8 d (Figure 2A). Treatment of donors with 25, 50, or 100 mg/kg either three (days −2, −1, and 0) or five times (days −4, −3, −2, −1, and 0) extended kidney allograft survivals (Figure 2A). Similarly, a 7-d intravenous continuous infusion of 2.5, 5, 10, or 20 mg/kg BIMO prolonged survivals 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 recipients that were treated with 5 or 10 mg/kg BIMO received an intravenous injection immediately after the operation with 50 mg/kg BIMO and received a transplant of kidney allografts that were perfused with 1 mg/2 ml BIMO, the survivals were significantly extended to 38.2 ± 3.8 d (P < 0.04) and 55.7 ± 8.1 d (P = 0.01), respectively (data not shown), compared with recipients that were treated by intravenous pump (Figure 2B). These results showed that BIMO therapy has potent immunosuppressive effects in vivo.

Figure2

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 from recipients that were treated with the highest dose of BIMO (20 mg/kg) revealed significant differences from those from untreated controls (Figure 2C). Hematoxylin and eosin staining of kidney transplants showed reduced infiltration on day 3 after grafting in the BIMO group compared with the untreated rejector group. On day 7 after grafting, kidney allografts from the BIMO group showed significantly reduced infiltration of mononuclear cells and none of the kidney damage that was observed in the rejector group (Figure 2C). Immunostaining analysis confirmed that BIMO therapy significantly reduced infiltration of kidney allografts (day 7 after grafting) with CD4+, CD8+, macrophages, PMN, and CD25+ cells (P < 0.0001; Figure 2D). These results document that BIMO blocks the process of allograft rejection by reducing infiltration with leukocytes. To examine further the mechanism of immunosuppression, we evaluated the expression of cytokines and chemokines. As shown by RPA, kidney transplants from untreated rejectors displayed increased mRNA levels for IL-1β, IL-3, IL-6, IL-10, TNF-α, and IFN-γ on postgrafting days 3 and 5, which decreased on day 7 (Figure 3). These findings correlated with the kinetics of allograft destruction. In contrast, kidney allografts from BIMO-treated recipients showed reduced levels of cytokine mRNA at all of these times (Figure 3). BIMO also inhibited the expression of PSGL-1 (Figure 4A), CX3CL1, CCL19, CCL20, and CCL2 (Figure 4B) mRNA. These results indicate that selectin inhibition of mononuclear cell infiltration correlates with reduced production of multiple cytokines and chemokines.

Figure3

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-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IL-18, TNF-α, and IFN-γ 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.

Figure4

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 immunosuppressive drugs that display distinct mechanisms of action: CsA blocks a calcineurin-dependent T cell receptor pathway and, consequently, cytokine production (28); SRL blocks the mammalian target of rapamycin after cytokine/cytokine receptor interaction (29); and FTY720 affects chemokine/chemokine receptor–dependent homing of lymphocytes in lymphoid organs (30). BIMO was combined with these agents to test the effects on kidney allograft survival (Figure 5). Recipients of kidney allografts were treated with a 7-d BIMO regimen alone or in combination with CsA, SRL, or FTY720. A median effect analysis was used to calculate CI values that describe the type of interactions between BIMO and other drugs. It is interesting that each of the three immunosuppressive drugs produced synergistic effects in combination with BIMO. The CI values were 0.05 to 0.77 for CsA (Figure 5A), 0.3 to 1.0 for SRL (Figure 5B), and 0.22 to 0.70 for FTY720 (Figure 5C). These results document that BIMO interacts synergistically with immunosuppressive drugs with distinct mechanisms of action.

Figure5

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 is its apparent lack of toxic effects. Preclinical studies performed in accordance with good laboratory practices showed that BIMO was well tolerated in rodents and dogs after intravenous administration. Single doses of 500 or 1250 mg/kg BIMO delivered intravenously to rats and mice, respectively, did not cause mortality. In a 14-d course, doses of 60 mg/kg BIMO delivered by intravenous injection to rats had no observable effects on hematology and clinical chemistry parameters and produced no histologic changes. Similarly, dogs that were treated intravenously for 14 d with 60 mg/kg per day BIMO had no hematologic, biochemical, or histopathologic changes. Thus, BIMO may provide significant benefits without producing toxic effects. Further experiments are planned to explore the potential adverse effects of treatment with BIMO combined with CsA, SRL, or FTY720.

Discussion

This study examined the effect of selectin inhibitor BIMO on both I/R injury and allograft rejection. Our results show that inhibition of selectins by BIMO blocks intragraft infiltration and activation of leukocytes during an I/R injury and inhibits alloimmune responses. These inhibitory effects correlate with a dramatic decrease in infiltration with CD4+, CD8+, macrophages, and PMN, as well as expression of cytokines and chemokines at the graft site. We also demonstrated that BIMO acts synergistically with immunosuppressive agents that display various mechanisms of action, extending allograft survival. These results show that BIMO is a beneficial and versatile complementary agent that can spare drug doses and thereby mitigate toxicity. Preclinical experiments documented that the clinical formulation of BIMO has an excellent toxicology profile: a single dose as high as 1 g/kg was necessary to produce LD50, whereas a threefold higher dose than that used in our study of 60 mg/kg delivered intravenously for 14 d caused no observable toxicities or any chromosomal aberrations.

Continuous intravenous delivery of BIMO (5 to 20 mg/kg) extended kidney allograft survival in a dose-dependent manner. Identical doses of 10 mg/kg BIMO delivered intravenously by pump for 7 d or by daily bolus injections produced similar survivals of 37.0 ± 13.6 and 33.4 ± 11 d, respectively. The most dramatic change that accompanied BIMO therapy was a marked decrease in graft mononuclear cell infiltration, which was associated with reduced expression of cytokines and chemokines. Because both selectins and chemokines are actively involved in directing the destination of T cells, deficient production of cytokines reflects changes in overall inflammatory processes after selectin blockade (31).

Naïve T cells express CCR7 that are sensitive to secondary lymphoid chemokines on endothelial cells and stromal cells in T cell areas of secondary lymphoid organs (32). For example, expression of CCR7 and L-selectin determines development of central (CCR7high and L-selectinhigh) and peripheral (CCR7low and L-selectinlow) memory T cells (32). Activated T cells downregulate CCR7 and upregulate CXCR5 and CCR4, enabling their T-B cell collaboration. Polarization of T cells into IL-2/IFN-γ–producing Th1 and IL-4/IL-10–producing Th2 also is regulated by selectins and chemokine receptors. In particular, Th1 expresses P-selectinhigh (33), as well as CCR5, CXCR3, and CCR1, whereas Th2 expresses P-selectinlow and high levels of CCR3 and CCR4. Expression of P- and E-selectins correlated with generation of Leishmania-specific Th1, which was dependent on the presence of IL-12 (34). Mice deficient in P- and E-selectins developed significantly less inflammatory response. Thus, blockade of all selectins affects multiple, interconnected events, resulting in inhibited T cell activation and decreased production of cytokines and chemokines.

The complex interdependence of cytokines, chemokines, and adhesion molecules with intracellular signaling was displayed by the application of an inhibitor of the Rho/ROCK pathway Y-27632 (35). Continuous treatment with Y-27632 significantly prolonged the survival of heart allografts. Immunohistochemistry showed that Y-27632 therapy reduced intragraft expression of intercellular adhesion molecule and vascular cell adhesion molecule-1 on day 7 after grafting. A recent report also revealed a new mechanism of 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 ischemic preservation. Again, immunostaining confirmed that PARP inhibition in heart transplants correlated with reduced expression of P-selectin and intercellular adhesion molecule-1 (36). Thus, blockade of selectins may interfere with multiple signaling pathways involved in graft rejection and I/R injury.

Our results show that selectin inhibition by BIMO dramatically reduced intragraft expression of the chemokines CX3CL1, CCL19, CCL20, and CCL2. As chemokines and their receptors are involved in several different functions of leukocytes—including migration, cell–cell interactions, and intracellular signaling—their inhibition (or activation) may affect I/R injury as well as acute and chronic rejection (37). Mounting evidence shows that chemokines and their receptors are involved in I/R events. For example, deficiency of CCR1 on PMN protected kidneys and livers against I/R injury (38). CX3CL1 is upregulated on vascular endothelial cells, thereby promoting direct leukocyte adhesion and transmigration during inflammation or I/R injury (39). Both CX3CL1 and its receptor CX3CR1 are involved in the rejection process (40). Targeting of CXCR3 by specific monoclonal antibodies in normal mice or utilization of CXCR3−/− recipients delayed acute allograft rejection (41). CX3CR1−/− mice also displayed significantly reduced leukocyte infiltration of heart allografts (42). Furthermore, CCR1−/− mice permanently accepted class II MHC- and class I/II MHC-mismatched heart allografts without or with concomitant treatment with low doses of CsA, respectively (43). Although CXCL10−/− recipients acutely rejected heart allografts, normal mice displayed prolonged survivals of CXCL10−/− heart allografts with significantly reduced infiltration of allografts and intragraft production of cytokines (44).

Our in vivo results showed that BIMO acted synergistically to prolong 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 standard therapeutic drugs, thereby damping their toxicities. CsA, a calcineurin inhibitor blocking signal 1 in the T cell activation pathway, causes nephrotoxicity that may be alleviated by using synergistic interaction with SRL (45). Although SRL—an agent that blocks mammalian target of rapamycin in the signal 3 cytokine/cytokine receptor pathway—is not nephrotoxic, it causes disorders of lipid metabolism (46). Each of these immunosuppressive drugs may be at least partially replaced by BIMO, maintaining immunosuppression and potentially mitigating their toxicities. However, the most promising synergistic interaction of BIMO is with FTY720, because this drug modulates lymphocyte trafficking responsible for homing of lymphocytes in spleen and lymph nodes (47). Permanent sequestration of lymphocytes in secondary lymphoid organs prevents allograft rejection (48). Although the exact mechanism of FTY720 action is not yet completely explained, synergism with a selectin inhibitor may provide a potent immunosuppressive combination. Furthermore, FTY720 pretreatment reduced warm hepatic I/R injury by inhibiting T lymphocyte infiltration and minimized I/R injury in kidneys (49).

In conclusion, selection inhibition by BIMO produces multiple benefits by protecting against I/R injury, thereby improving kidney function, and by inhibiting allograft rejection. Furthermore, BIMO acts alone as well as synergistically with other immunosuppressants displaying diverse mechanisms of action. Remarkably, toxicology profiles revealed that BIMO causes no observable toxic reactions, proffering a unique and versatile agent to improve standard immunosuppressive protocols. However, to confirm these results, further analyses of the potential toxic effects of treatment with BIMO combined with CsA, SRL, or FTY720 must be performed.

Acknowledgments

This study was supported in part by a grant from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK 38016-15) and AI061052.

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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, Barry D. Kahan
JASN Nov 2004, 15 (11) 2893-2901; DOI: 10.1097/01.ASN.0000142425.23036.AC
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