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Published as doi: 10.1096/fj.06-7910com.
 (The FASEB Journal. 2007;21:2840-2848.)
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Toll-like receptor 4 suppression leads to islet allograft survival

Alyssa Goldberg*, Margherita Parolini{dagger}, Beek Y. Chin*, Eva Czismadia*, Leo E. Otterbein*, Fritz H. Bach* and Hongjun Wang*,1

* Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA; and

{dagger} Universita di Milano-Bicocca, Milan, Italy

1Correspondence: Beth Israel Deaconess Medical Center, Harvard Medical School, 99 Brookline Ave., Boston, MA 02215, USA. E-mail: hwang3{at}bidmc.harvard.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Carbon monoxide (CO) exposure of an islet donor frequently leads to islet allograft long-term survival and tolerance in recipients. We show here that CO confers its protective effects at least in part by suppressing Toll-like receptor 4 (TLR4) up-regulation in pancreatic ß cells. TLR4 is normally up-regulated in islets during the isolation procedure; donor treatment with CO suppresses TLR4 expression in isolated islets as well as in transplanted grafts. TLR4 up-regulation allows initiation of inflammation, which leads to islet allograft rejection; islet grafts from TLR4-deficient mice survive indefinitely in BALB/c recipients and show significantly less inflammation at various days after transplantation compared with grafts from a control donor. Isolated islets preinfected with a TLR4 dominant negative virus before transplantation demonstrated prolonged survival in recipients. Despite the salutary effects of TLR4 suppression, HO-1 expression is still needed in the recipient for islet survival: TLR4-deficient islets were rejected promptly after being transplanted into recipients in which HO-1 activity was blocked. In addition, incubation of an insulinoma cell line, ßTC3, with an anti-TLR4 antibody protects those cells from cytokine-induced apoptosis. Our data suggest that TLR4 induction in ß cells is involved in ß cell death and graft rejection after transplantation. CO exposure protects islets from rejection by blocking TLR4 up-regulation.—Goldberg, A., Parolini, M., Chin, B. Y., Czismadia, E., Otterbein, L. E., Bach, F. H., Wang, H. Toll-like receptor 4 suppression leads to islet allograft survival.


Key Words: islet transplantation • carbon monoxide • inflammation • type 1 diabetes


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HEME OXYGENASES ARE THE RATE-LIMITING enzymes that degrade heme into equal molar amounts of CO, free iron, and biliverdin. Biliverdin is rapidly converted to bilirubin and free iron up-regulates the expression of ferritin (1) . Three isoforms of HO exist: HO-1 is highly inducible while HO-2 and HO-3 are constitutively expressed (2 , 3) . HO-1 has been identified as a ubiquitous stress protein and can be induced in many cell types by various stimuli such as heme, inflammatory cytokines, endotoxin, heavy metals, hormones, and heat shock (4 5 6 7 8) . HO-1 induced in stress conditions exerts anti-inflammatory effects and modulates apoptosis and cell proliferation (9 , 10) . Expression of HO-1 in vivo suppresses the inflammatory responses in endotoxic shock (11 12 13) , hyperoxia (14) , acute pleurisy (15) , allo- and xenotransplantation (16 , 17) , and ischemia reperfusion injury (18) and thereby provides salutary effects in these conditions. Induction of HO-1 in a transplanted organ can be critical to the survival of that graft after transplantation (19) and has been shown to improve islet function in a minimal marginal mass model (20) .

Survival of islet allografts after transplantation has been based largely on suppressing the T lymphocyte immune response in the recipient that is responsible for rejection of the graft. Induction of HO-1 or treating the islets with CO while still in the donor can also protect those islets from immune rejection after they are transplanted (21 22 23) . CO treatment of the donor only (the "donor effect") suppresses the proinflammatory response in the islets after transplantation, which may account for islet survival. However, the mechanisms of such protection are not understood.

Toll-like receptors (TLRs) are transmembrane receptors that are critical for the response to microbial pathogens (24) . TLRs initiate an innate immune response after recognition of pathogen-associated molecular patterns (25) . Activation of TLRs triggers an inflammatory response that is mediated by macrophages, neutrophils, and complement (25 26 27 28) . The TLR signaling pathway ultimately results in the activation of NF-{kappa}B, which leads to the transcription and production of inflammatory cytokines (29) . Currently, 13 human Toll-like receptor proteins have been identified (30) . Among them, TLR2 and TLR4 are well known as the receptors for lipopolysaccharide (LPS), a product of the outer membrane of Gram-negative bacteria. Recent evidence suggests that not only exogenous but also endogenous ligands produced during stress or cell damage, such as heat shock protein 60 (31 , 32) , the EDA domain of fibronectin (33) , saturated fatty acids (34) , and even the transplant process (35) , can activate TLR4. Indeed, endogenous TLR4 ligands representing the danger signal (36) may initiate an immune response in the absence of infection. TLR4 is not only expressed on macrophages, but also on cells of many tissues without a recognized immune function, notably the heart (37) , vasculature (38 , 39) , liver (40) , and others (41) . Evidence is accumulating that TLR4 plays an important role in the pathogenesis of atherosclerosis (42) , chronic cardiac allograft rejection (43) , and liver and heart ischemia/reperfusion injury (40) . Several groups have also demonstrated that TLR4 activation is directly involved in the rejection of transplanted organs (43 44 45) . Although activation of innate immune cells itself is not sufficient for acute graft rejection without the participation of T cells, activation of TLRs and the ensuing inflammation might be important for development of the alloimmune response to the transplanted organ (43) . To date, there are limited data on TLR4 expression in islets (46) , and the role of TLR4 in islet allograft rejection has yet to be elucidated. The present study was designed to evaluate these parameters and to test whether CO exposure to the islet donors protects islet allografts from rejection by preventing TLR4 up-regulation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
C57BL/10ScNJ (TLR4–/–), C57BL/10, C57BL/6, BALB/c, DBA/1, and DBA/2 mice at 6–8 wk of age were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Although inbred mouse strains C57BL/6 and C57BL/10 have genotypic differences and different susceptibilities to infection, they are similar in phenotype and physiology, and are often treated as alternative strains that can be substituted in experiments (47) . For a detailed comparison of these two strains, see http://www.informatics.jax.org/external/festing/mouse/docs/C57BL.shtml. We used C57BL/10 mice as controls when we transplanted C57BL/10ScNJ islets to BALB/c recipients and C57BL/6 in all other studies. No difference was observed when transplanting C57BL/6 or C57BL/10 islets to BALB/c recipients. The animal protocol was approved by the Animal Care Committee of the Beth Israel Deaconess Medical Center.

Islet isolation and transplantation
Islets were isolated as described by Pileggi et al. (4) . Polymyxin B (10 U/ml) was added to all media to avoid any effects of LPS during islet isolation. Islet purity was assessed by dithizone (Sigma-Aldrich, St. Louis, MO, USA) staining after isolation. An algorithm was used to calculate the 150 µm diameter islet equivalent number (IEQ) (48 , 49) . Islet cell viability was assessed using fluorescence staining with acridine orange and propidium iodide (Sigma) (50) . Our isolation protocol usually yields 90–95% of viable cells before transplantation. Recipients were rendered diabetic using streptozotocin (STZ, 225 mg/kg, i.p.; Sigma). Five days after STZ administration, mice with two consecutive blood glucose levels exceeding 350 mg/dl were used as recipients. Islets (500–600 IEQ) were transplanted under the kidney capsule of the recipients. Blood glucose levels of the recipients were measured twice weekly with a glucometer (Roche, Basel, Switzerland) after islet transplantation. Animals with a blood glucose level of <200 mg/dl were considered normoglycemic. Grafts were deemed rejected when two consecutive glucose levels were >300 mg/dl after a period of primary graft function.

Tolerance test
The kidneys under which the initial islets were transplanted were removed from some animals that had islets surviving long-term, after which islets syngeneic with the original donor (C57BL/10) were transplanted under the other kidney with no further treatment. If those second transplanted islets also survived for >100 days, the recipients were considered tolerant. Antigen-specific tolerance was assessed by transplanting islets from a third-party strain (DBA/1) that does not share either class I or class II antigens with the original donor.

Adenoviral infection
TLR4 dominant negative (TLR4-dn) adenovirus, TLR4-expressing adenovirus (TLR4-wt), as well as a control adenovirus containing ß-gal obtained from the University of Pittsburgh Vector Core Facility were used in the study. Freshly isolated islets were washed with serum-free RPMI 1640 medium, then cultured in the same medium that contains virus (multiplicity of infection, MOI: 10:1) for 1 h before transfer to a complete medium (CMRI-1640 plus 10% FBS, 2 mmol/L L-glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin). Islets were cultured in the complete medium for 24 h in 5% CO2 at 37°C before transplantation.

Real-time RT-PCR analysis
Islet grafts were harvested 1, 3, and 7 days after transplantation. Total RNA was extracted using Qiagene RNA kit (Qiagen Inc., Chatworth, CA, USA). DNase treatment was performed according to the manufacturer's suggestion (Qiagen) to prevent contamination by genomic DNA during real-time RT-PCR. Real-time RT-PCR was performed to quantify the amount of target gene in each sample at the mRNA level using the ABI PRISM® 7700 Sequence Detection Systems as described (21) . Expression of the following genes was analyzed in freshly isolated islets and islet grafts after transplantation: TNF-{alpha}, inducible nitric oxide synthase (iNOS), monocyte chemoattractant protein-1 (MCP-1), granzyme B, and Fas (CD95). GAPDH expression were quantified in each sample and used as endogenous control.

Western blot
Membrane and cytosolic portions of cell lysates from pancreas or isolated islets were separated with a Membrane Protein Extraction kit (Biovision, Oxon, UK) as recommended. Protein samples (40 µg from cytosolic and 20 µg from membrane portion) were separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were incubated with goat anti-TLR4 antibody (L-14, Santa Cruz, CA, USA), mouse anti-GAPDH antibody (cytosol marker, Sigma), and rabbit antipan cadherin antibody (plasma membrane marker; Cambridge, MA, USA), respectively, and followed by peroxidase-labeled secondary antibodies. Signals were visualized using an ECL detection kit (Amersham Pharmacia Biotech, Little Chalfont, UK).

CO and zinc protoporphyrin-IX (ZnPP) treatment
CO exposure to the donor was performed in a chamber containing 250 parts per million CO for 2 h before islet harvest. ZnPP (Frontier Scientific, Logan, UT, USA) was dissolved in 0.1M sodium hydroxide and the pH was adjusted to 7.4 with hydrochloride acid. ZnPP at 20 mg/kg was given to the recipient on days –1, 1, 3, 5, 7 days after transplantation to block HO-1 activity in the recipient.

Apoptosis assay, detection of I{kappa}B{alpha} degradation, and NF-{kappa}B activation
ßTC3 cells were seeded into 6-well plates at a concentration of 1 x 106/well. Cells were incubated with the anti-TLR4 antibody (rat monoclonal IgG2a, MTS510, Santa Cruz Biotech., Santa Cruz, CA, USA) at 1, 5, and 25 µg/ml as well as normal rat IgG2a control antibody (25 µg/ml) in DMEM with 10% FBS for 30 min at 37°C. Cell death induced by recombinant murine IL-1ß (100 U/ml) plus recombinant rat INF-{gamma} (1000 U/ml) (R&D Systems, Minneapolis, MN, USA) was quantified by flow cytometry using propidium iodide staining at 24 h after cytokine stimulation.

In a separate set of experiments, ßTC3 cells were incubated with the anti-TLR4 antibody (25 µg/ml) or rat IgG2a control antibody for 30 min at 37°C and stimulated with IL-1ß (100 U/ml) plus INF-{gamma} (1000 U/ml). Cells were collected at 0, 15, 30, and 60 min after cytokine stimulation to assess I{kappa}B{alpha} expression and NF-{kappa}B activation. I{kappa}B{alpha} expression was detected by Western blot with an anti-I{kappa}B{alpha} antibody (Santa Cruz). I{kappa}B{alpha} was quantified by the ratio of I{kappa}B{alpha} expression divided by the amount of ß-actin of individual samples as analyzed by ImageJ software. To assess NF-{kappa}B activity, nuclear extractions of cells were separated from the cytoplasmic fraction using the NE-PERTM Nuclear and Cytoplasmic Extraction Reagents (Pierce, Rockford, IL, USA) as recommended. P65 and P50 activities were assayed at 3 µg/well using the TansAM NF-{kappa}B Family kit (Active Motif, Carlsbad, CA, USA) as suggested.

Immunohistochemistry
Isolated islets were snap-frozen in liquid nitrogen for immunohistological staining. Tissue sections of 5 µm were stained with the anti-TLR4 (L-14, Santa Cruz), anti-insulin antibodies, and IgG controls for both antibodies. Secondary antibodies were FITC-labeled anti-goat and Alex anti-mouse antibodies (Vector Labs, Burlingame, CA, USA). Counterstaining with DAPI was performed after dehydration and slides were covered with mounting medium for observation.

Statistical analyses
Kaplan-Meier survival curves were performed by using Statview software, and the statistical differences were assessed by the Log-rank test. Values of P < 0.05 were considered significant. Survival data are expressed as mean survival time ± standard deviation (MST±SD). Differences between cytokine expressions were compared for statistical significance by the Mann-Whitney U Test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CO exposure of the islet donor suppresses TLR4 up-regulation ex vivo in isolated islets and in vivo in grafts at various days after transplantation
We studied TLR4 expression in DBA/2 islets/ß cells within the pancreas and after isolation by staining with an anti-TLR4 and an anti-insulin antibody. No visible TLR4 expression was detected on the membrane of islet cells in the pancreas (data not shown). However, significant TLR4 induction was detected on the membrane of insulin-producing ß cells after these cells were isolated from the pancreas, a process that took ~4 h (Fig. 1 A). We interpret this as showing that the stress of isolation leads to membrane expression of TLR4, since polymyxin B (10 U/ml) was added to all media to avoid any effects of LPS during islet isolation. CO treatment of donors led to much less TLR4 expression in isolated islets (Fig. 1A ). This result was confirmed by Western blots studying TLR4 expression in cytosolic and membrane fractions of the pancreas and the isolated islets (Fig. 1B ). In addition, TLR4 expression in islet grafts at various days after transplantation was quantified at the mRNA level by real-time RT-PCR; expression was significantly suppressed in grafts from CO-treated donors compared with grafts from controls at 1 and 3 days after transplantation (Fig. 1C ).


Figure 1
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  		Figure 1. TLR4 activation in murine islet ß cells. A) TLR4 expression in fresh isolated islets from control and CO-treated donors. TLR4 was detected on the surface of insulin-producing ß cells ~4 h after these cells were isolated from the pancreas (a, b, c). Significantly less TLR4 expression was detected in ß cells isolated from CO-treated donors (d, e, f). Islets from control donors were stained with IgG controls for both anti-TLR4 and anti-insulin antibodies (g, h, i). Data are representative of three independent experiments. B) TLR4 expression was detected mostly in the cytosolic fraction (indicated by GAPDH expression) of the pancreas and in the membrane fraction (indicated by cadherin expression) of isolated islets as analyzed by Western blot. Islets from CO-treated donor have much less TLR4 expression on the cell membrane than those from the control donors. C) Expression of TLR4 in the islet allografts after transplantation was quantified at the mRNA level by real-time RT-PCR. TLR4 expression was significantly suppressed in grafts from CO-treated donors compared with those from control donors 1 and 3 days after transplantation. *P < 0.05 vs. control. Values are means of 3–4 grafts per group.

The majority of TLR4-deficient islet allografts survive long-term (>100 days) after transplantation into BALB/c recipients while islets from congenic-resistant wild-type mice are rejected promptly
We tested whether TLR4-deficient islets would survive longer than control islets after transplantation. TLR4-deficient islets were obtained using TLR4 knockout mice or by infecting islets with TLR4 dn-virus. Islets (500–600 IEQ) isolated from TLR4-deficient mice (C57BL/10ScNJ) or their wild-type C57BL/10 controls were transplanted into BALB/c recipients rendered diabetic by stroptozotocin injection. As shown in Fig. 2 , islet grafts from C57BL/10 controls were rejected in 19.0 ± 1.73 days (n=5). In striking contrast, four of six grafts from TLR4–/– mice survived indefinitely in the recipients while the other two were rejected at 18 and 49 days (P=0.0074 vs. control).


Figure 2
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  		Figure 2. Survival of islets from TLR4–/– mice and islets infected with TLR4-dn virus in recipients after transplantation. C57BL/10 islets were rejected in 19.0 ± 1.7 days in BALB/c recipients (x, n=5). Four of 6 grafts from TLR4–/– mice survived indefinitely (>100 days) in the recipients whereas the other two were rejected at 18 and 49 days ({circ}, n=6, P=0.007 vs. control as analyzed by log rank test). Three of four C57BL/6 islet grafts infected with TLR4 dominant-negative (TLR4-dn) survived for >100 days, with one rejecting on day 20 (•, n=4).

We tested whether blocking TLR4 with a recombinant dominant-negative adenovirus in isolated islets in vitro would prolong islet graft survival in the C57BL/6 to BALB/c combination. C57BL/6 islets were infected with TLR4 dominant-negative (TLR4-dn) or wild-type (TLR4-wt) adenoviruses, as well as a control virus containing ß-gal, for 1 h in serum-free medium. Islets were cultured in CMRI-1640 complete medium in 5% CO2 at 37°C for 24 h after viral infection before being transplanted into diabetic recipients. Islet grafts infected with control virus or TLR4-wt virus was rejected in 20.0 ± 1.6 and 20.4 ± 2.6 days, respectively (data not shown). On the contrary, three of four grafts infected with the TLR4-dn virus survived for >100 days, whereas one rejected on day 20 (P=0.002 vs. control, and P=0.0022 vs. grafts infected with TLR4-wt virus; Fig. 2 ). Our data also suggest that inducing islet long-term survival leads to antigen-specific tolerance. This was tested in those recipients carrying long-term surviving islets by transplanting second islet grafts without further treatment either from the same donor strain (C57BL/10) or a third party strain (DBA/1) after removing the long-term surviving islet grafts from the first transplant. One graft from a third party strain was rejected in 18 days whereas the two grafts from the same donor strain survived long-term (>100 days).

Islets grafts from TLR4–/– donors show less inflammation at various days after transplantation than grafts from wild-type donors
Islet isolation and transplantation up-regulates a series of proinflammatory cytokines, chemokines, and proapoptotic genes that presumably facilitate rejection of the graft. We quantified proinflammatory and proapoptotic genes (including TNF-{alpha}, iNOS, MCP-1, and granzyme B) and death receptor Fas (CD95) at the mRNA level by real-time RT-PCR in islet grafts from TLR4–/– donors or their wild-type controls 1, 3, and 7 days after transplantation. Grafts from TLR4-deficient donor mice showed significantly less expression of TNF-{alpha}, iNOS, MCP-1, and Fas at most of the time points measured compared with grafts from wild-type control animals (Fig. 3 A–D). No difference in granzyme B expression was observed between the two groups (Fig. 3E ).


Figure 3
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  		Figure 3. Expression of proinflammatory and proapoptotic genes in islet grafts at various days after transplantation. Islet grafts from TLR4–/– donors as well as control donors were harvested 1, 3, and 7 days after transplantation to measure gene expression by real-time RT-PCR. Grafts from TLR4–/– donors (black bars) showed much less expression of TNF-{alpha} (A), iNOS (B), MCP-1 (C), and Fas (D) at most of the time points measured compared with grafts from C57BL/6 controls (gray bars). No significant difference was seen on the expression level of granzyme B between grafts from TLR4–/– donors and wild-type donors (E). #P < 0.01 vs. control and *P < 0.05 vs. control. Values are means of 3–4 grafts per group.

Blocking HO-1 activity in the recipient leads to rapid rejection of TLR4-deficient islet allografts after transplantation
To test whether HO-1 expression in the recipient contributes to the survival of TLR4–/– islets after transplantation, we transplanted TLR4–/– islets to diabetic BALB/c recipients in which HO-1 activity was blocked by its inhibitor, ZnPP. As shown in Fig. 4 , TLR–/– islets transplanted into recipients in which HO-1 activity was blocked were rejected at 17.0 ± 2.2 days (n=5), with no prolongation of survival observed compared with TLR4–/– islets transplanted into untreated diabetic recipients or C57BL/10 islets transplanted into BALB/c recipients in which HO-1 was blocked by ZnPP (Fig. 4) . These results indicate that despite the salutary effects of TLR4 suppression, HO-1 expression in the recipient is essential for the survival of TLR4–/– islets after transplantation.


Figure 4
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  		Figure 4. Survival of TLR4–/– islets in BALB/c recipients in which HO-1 was blocked by ZnPP. Islets from C57BL/10 or C57BL/10ScNJ mice were transplanted into BALB/c recipients in which HO-1 activity was blocked or not by ZnPP. Grafts from TLR4–/– donors (C57BL/10ScNJ) were rejected promptly after being transplanted into recipients in which HO-1 activity was blocked by ZnPP ({circ}, n=5, P>0.05 vs. control).

Blocking TLR4 with an anti-TLR4 antibody prolongs BABL/c islet allograft survival in C57BL/6 recipients
We tested whether blocking TLR4 in isolated islets in vitro with an anti-TLR4 antibody would prevent islet allograft from immune rejection after transplantation. Freshly isolated islets precultured with the anti-TLR4 antibody (25 µg/ml) or normal rat IgG2a control were transplanted into diabetic recipients. As shown in Fig. 5 , islets cultured with control rat IgG2a were rejected in 18.0 ± 3.1 days (n=5). Preincubation of islets with the anti-TLR4 antibody significantly prolonged islet allograft survival for up to 30.6 ± 12.6 days (n=5, P<0.05 vs. control).


Figure 5
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  		Figure 5. Survival of C57BL/6 islets incubated with the anti-TLR4 antibody in BALB/c recipients. C57BL/6 islets precultured with the anti-TLR4 antibody or control IgG were transplanted into BALB/c recipients. Grafts precultured with anti-TLR4 antibody ({circ}, n=5) survived significantly longer in BALB/c recipients than islets precultured with normal rat IgG2a (x, n=5, P<0.05 vs. control).

Blocking TLR4 with the anti-TLR4 antibody protects islet cells from cytokine-induced apoptosis by inhibiting I{kappa}B{alpha} degradation
Recombinant murine IL-1ß and rat INF-{gamma} induce islet ß cell death. To test whether incubating cells with anti-TLR4 antibody can protect those cells from apoptosis, we preincubated ßTC3 cells with different concentrations of anti-TLR4 antibody (1, 5, and 25 µg/ml) as well as normal rat IgG2a control antibody (25 µg/ml) before cytokine stimulation. Our data indicate that significantly fewer islets (8.9%±0.9, 3.6%±0.8) incubated with the anti-TLR4 antibody (5 and 25 µg/ml) underwent apoptosis compared with islet cells incubated with the rat IgG2a control (14.5%±1.3). Cells cultured only with rat IgG2a without cytokines showed 1.8% ± 0.3 cell death (Fig. 6 A, B).


Figure 6
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  		Figure 6. Analysis of cytokine-induced apoptosis in ßTC3 cells by flow cytometry. Cell apoptosis induced by IL-1ß and INF-{gamma} was quantified by flow cytometry using propidium iodide staining (A). Significantly fewer islets incubated with the anti-TLR4 antibody (5 and 25 µg/ml) underwent apoptosis than islet cells incubated with the rat IgG2a control (B). The gray bar indicates cells without stimulation. The lined bars indicate cells incubated with anti-TLR4 antibody; black bars represent cells pretreated with IgG control before stimulation. Values are representatives of 3 independent experiments. #P < 0.01 vs. control.

To elucidate a potential mechanism that would explain why blocking TLR4 in ßTC3 cells protects those cells from cytokine-induced apoptosis, we evaluated whether degradation of I{kappa}B{alpha}, the inhibitor of NF-{kappa}B, was affected. Degradation of I{kappa}B{alpha} leads to NF-{kappa}B activation and the production of proinflammatory cytokines. Cells pretreated with the anti-TLR4 antibody or control IgG2a were stimulated with IL-1ß (100 U/ml) plus INF-{gamma} (1000 U/ml) and harvested 0, 15, 30, and 60 min after cytokine incubation. As shown in Fig. 7 A, INF-{gamma} and IL-ß induced the rapid degradation of I{kappa}B{alpha} in cells pretreated with rat IgG2a control antibody; I{kappa}B{alpha} was completely absent 15 and 30 min after cytokine treatment, and the expression level went back to normal 1 h later. On the contrary, significantly less I{kappa}B{alpha} degradation was observed when cells were preincubated with the anti-TLR4 antibody (Fig. 7A, B ), suggesting that blocking TLR4 in ßTC3 protects those cells from cytokine-induced apoptosis by inhibiting NF-{kappa}B activation.


Figure 7
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  		Figure 7. Analysis of I{kappa}B{alpha} degradation and NF-{kappa}B activation in ßTC3 cells after stimulation with IL-1ß and INF-{gamma}. Expression of I{kappa}B{alpha} was detected by Western blot using an anti-I{kappa}B{alpha} antibody. IL-1ß and INF-{gamma} stimulation leads to a rapid degradation of I{kappa}B{alpha} in control cells. No significant I{kappa}B{alpha} degradation was observed in cells preincubated with the anti-TLR4 antibody (A). I{kappa}B{alpha} expression level was quantified using ImageJ software. The relative quantity of I{kappa}B{alpha} (densities of I{kappa}B{alpha} divided by densities of ß-actin of each sample) was compared between the two groups (B). Significantly less P50 (at 15 and 30 min, C) and P65 (at 30 min, D) was observed in the nuclear extracts of cells pretreated with the anti-TLR4 antibody than in cells pretreated with rat IgG control antibody after cytokine stimulation. Data shown are representative of at least 3 independent experiments. *P < 0.05 vs. control as analyzed by Mann-Whitney U test.

These results were confirmed by measuring NF-{kappa}B activation in nuclear extracts from cells pretreated with rat IgG control or the anti-TLR4 antibodies after cytokine stimulation. As evident in Fig. 7C, D , P65 and P50 activities were significantly lower in cells pretreated with the anti-TLR4 antibody compared to those treated with the IgG controls.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have observed, as have others, that the induced expression of HO-1 in recipients of allogeneic islets results in prolonged survival of the islets and tolerance in the recipient to donor islet antigens (20 , 51) . We have extended these observations by evaluating the potential value of inducing HO-1 in, or administering CO or bilirubin to, the donor only, the recipient only, or both (21 , 23) . Our results show that any one of the above treatments is salutary and results in prolonged, and often long-term, islet graft survival. The combination of treating donor and recipient appears to provide somewhat better results. Perhaps most surprising was the observation that donor treatment alone led to long-term (>100 days) survival of islets in untreated recipients and antigen-specific tolerance in those recipients.

In an effort to understand this last finding, we examined the inflammation in islets after transplantation. Without any treatment, there is extensive expression of proinflammatory cytokines in the transplanted islets as early as 1 day after transplantation, as measured by RT-PCR. However, if HO-1 is induced in the donor or the donor is treated with CO or bilirubin, there is marked suppression of the proinflammatory response in the islets after transplantation even though the recipient is untreated. We hypothesized at that time that the low level of inflammation failed to support a strong immune rejection response against the islets. However, we had no mechanism to explain the suppression of inflammation by donor treatment.

Our present data provide one explanation: exposing donors to CO results in the inhibition of TLR4 up-regulation during the isolation procedure. The lack of TLR4 expression could explain why there is much less inflammation in the islets after transplantation. We suggest this based on the danger hypothesis: if there is less inflammation, the immune response will also be weakened (36) . We evaluated the role of TLR4 by demonstrating that the islet harvest process itself leads to rapid induction and expression of TLR4. By using TLR4–/– mouse donors, we were able to show that islets from these mice were not rejected when transplanted to an allogeneic recipient. These experiments provide the strongest evidence that TLR4 expression is a key element in leading to islet damage and rejection and that the suppression of the TLR4 response likely contributes to the effects of CO treatment. It is worth noting that the TLR4–/– mice were described to have a spontaneous interleukin 12 receptor ß (IL12Rß) mutation (52) . TLR4–/– mice from the Jackson Laboratory that we used, however, carry only the wild-type IL12Rß allele. This eliminates the possibility that deficiency of IL12Rß contributed to islet allograft survival in our transplantation model.

While the above experiments are key to testing our hypothesis that TLR4 expression plays a key role, direct or indirect, in islet allograft rejection, we were interested in whether a similar effect could be achieved with procedures that might be useful clinically. We thus employed a TLR4 dominant negative mutant expressed in an adenovirus as well as an antibody directed against TLR4 to assess whether treatment with these reagents was efficacious. The use of a dominant negative mutant adenovirus appeared to be as useful in terms of achieving long-term survival as the TLR4–/– islets. Use of the anti-TLR4 antibody was far less effective, but islets treated with the antibody showed significantly prolonged, although not long-term, survival.

In our study, culturing ßTC3 cells with the anti-TLR4 antibody in vitro protects those cells from cytokine-induced apoptosis. Proinflammatory cytokines produced by cells of the innate immune response that infiltrate transplanted islets have important implications for islet survival and function after transplantation (53 , 54) . Even under the best metabolic control, islets are subject to early dysfunction after transplantation (55 , 56) , caused in part by the intra-islet release of proinflammatory cytokines such as TNF-{alpha}, INF-{gamma}, and IL-1ß that are generated by islet resident macrophages (57 , 58) . The deleterious effects of these cytokines relate in large measure to activation of NF-{kappa}B in the ß cells of the islet, which leads to up-regulation of iNOS and consequent production of nitric oxide (NO) in the cells. NO has been shown to be directly injurious to islets by inhibiting insulin secretion and inducing islet/ß cell apoptosis (59 , 60) . We showed in our experiments that blocking TLR4 in ßTC3 cells before stimulating these cells with IL-1ß and INF-{gamma} protects those cells from apoptosis. Similar mechanisms may exist in isolated islets as well as in ßTC3 cells that can explain our transplantation data: suppression of TLR4 in islets blocked NF-{kappa}B activation and proinflammatory cytokine production, which would facilitate the survival of islet allografts after transplantation.

An important question with regard to these findings is, For how long after transplantation must the TLR4 receptor be blocked from being expressed in order to obtain the long-term survival of the transplanted islets? We posit that TLR4 will have to be suppressed for only a week, or maybe even less, to get past the time when the islets are disrupted by their preparation and transplantation and perhaps to establish antigen-specific suppression.

Our data show that blocking HO-1 activity in the recipient leads to rapid rejection of TLR4-deficient islet allografts after transplantation. We interpret these data to show that presumably an effective anti-inflammatory response is still needed in the recipient even though we have suppressed the inflammatory response by donor treatment.

These data may lead to clinical application. Certainly treatment of the islets with either CO or bilirubin, or both, could be achieved either by treatment of the donor or by treatment of the islets immediately after their removal and during their isolation. While we have not tested whether the latter approach would be as efficacious as treatment of the donor, our data in other systems suggest that such a protocol is worth testing in experimental animals prior to possible use in humans.


   ACKNOWLEDGMENTS
 
This work was supported in part by JDRF 5–2005-989 (H.W.); Riva Foundation 01–2006 and NIH HL077721 (F.H.B.), and the Julie Henry Fund of the Division of Transplantation, Department of Surgery, BIDMC, Harvard Medical School. This is publication #823 from our laboratories.

Received for publication December 8, 2006. Accepted for publication March 26, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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