Role of Donor-Derived Monocyte Chemoattractant Protein-1 in Murine Islet Transplantation
Bernd Schröppel*,,
Nan Zhang,,
Peng Chen*,
Dongmei Chen,
Jonathan S. Bromberg, and
Barbara Murphy*,
* Division of Nephrology, Recanati/Miller Transplantation Institute, and Department of Gene and Cell Medicine, Mount Sinai School of Medicine, New York, New York
Address correspondence to: Dr. Bernd Schröppel, Division of Nephrology, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1243, New York, NY 10029-6574. Phone: 212-241-8004; Fax: 212-987-0389; E-mail: bernd.schroppel{at}mssm.edu
Monocyte chemoattractant protein-1 (CCL2/MCP-1) is a proinflammatorychemokine produced by several cell types, including pancreaticislets. High levels of donor-derived CCL2 have been associatedwith poor islet allograft outcome in patients with type 1 diabetes;however, the causal relationship is unknown. The constitutiveand inducible expression of chemokines and their receptors bypancreatic islets in vitro were investigated, specifically therole of donor-derived CCL2 in marginal mass murine islet transplantation.The results showed that inflammatory cytokine stimulation ofislets induced de novo expression of CCL2, CCL5/RANTES, CXCL9/MIG,and CXCL10/IP-10 and increased expression of CXCL2/macrophage-inflammatoryprotein-2. CCL2 mRNA and protein were highly expressed within2 d in cultures. Transplantation of islets with high levelsof CCL2 into syngeneic recipients led to a significantly greaterinflux of CCR2+ cells and higher expression of monocyte/macrophage-associatedinflammatory cytokines compared with low CCL2-donor islets.The level of pretransplantation CCL2 inversely correlated (P< 0.0001) with isograft function. In contrast, in CCR2–/–recipients, this correlation was not present. A direct toxiceffect of CCL2 on islets was excluded by assessing cell viabilityand insulin release in vitro. In conclusion, CCL2 secreted byislets plays an important role in the immediate islet graftfunction. Strategies to decrease islet-derived CCL2 releasemay increase the success of islet transplantation.
In the immediate period after islet transplantation, the marginalislet mass is further compromised by cell damage caused byischemia-reperfusion and the inflammatory response. In additionto adhesion molecules, such as selectins and integrins, thecomplex process of extravasation and influx of leukocyte subsetsinto the site of tissue injury is mediated, to a significantextent, by the expression of specific chemokines and chemokinereceptors (1). Monocyte chemoattractant protein-1 (CCL2/MCP-1)is a proinflammatory chemokine produced by monocytes/macrophagesand lymphocytes in addition to many other cell types, includingendothelial cells, mesangial cells, renal tubular cells, andfibroblasts, in response to cytokines, oxidized LDL, and LPS(2–4). Several studies have now shown that CCL2 is alsostrongly expressed by human and rodent pancreatic islets undervarious conditions (5–11). The biologic actions of CCL2are mainly mediated through the interaction with the CC chemokinereceptor 2 (CCR2), the other ligands for which include CCL8/MCP-2,CCL7/MCP-3, and CCL12/MCP-5. CCR2 receptors are found on thesurface of inflammatory mononuclear cells, endothelial cells,and fibroblasts (12). Through binding to the CCR2 receptor,its ligands signal monocytes, natural killer, and activatedand memory T cells to migrate to sites of injury as part ofthe inflammatory response (13,14).
We hypothesized that the local generation of chemokines by isletcells may be important in the initiation and regulation of inflammatoryprocesses during insulitis and islet graft destruction. Thishypothesis is supported by recent data that demonstrated thathigh levels of CCL2 released by human islet cells may play animportant role in the clinical outcome of islet allografts inpatients with type 1 diabetes, because high levels of donor-derivedCCL2 secretion were associated with poor islet graft outcome(6). Despite recent publications on CCL2 secretion by pancreaticislets (5–11), no mechanistic data outlining the roleof donor-derived CCL2 are available. In the present study, wespecifically investigated the role of donor-derived CCL2 inmurine islet transplantation.
Mice and Diabetic Model
Animals were treated in strict compliance with regulations establishedby the Institutional Animal Care and Use Committee. Mice wereborn and housed under specific pathogen-free conditions. Therecipients were rendered diabetic by a single intraperitonealinjection of 180 mg/kg streptozotocin (Sigma, St. Louis, MO)and considered diabetic when the tail-vein blood glucose concentrationas determined by OneTouch glucometer (Lifescan, Milpitas, CA)was >300 mg/dl for 2 consecutive days. Inbred C57BL/6 (H-2b)mice (Jackson Laboratories, Bar Harbor, ME) were used at 10to 12 wk of age (25 to 30 g). The CCR2 gene knockout mice (backgroundC57BL/6; F10) were provided by W.A. Kuziel (Department of Microbiology,Institute for Cellular and Molecular Biology, University ofTexas, Austin, TX), and deletion was confirmed by genotyping(15).
Islet Isolation, Culture, and Transplantation
Islet isolation and their culture and transplantation were previouslydescribed in detail (5). Briefly, 3 ml of cold Hank’sbuffer/collagenase P solution (1.5 mg/ml; Roche Diagnostics,Mannheim, Germany) was infused into the pancreatic duct in situ,and the removed pancreas was digested at 37°C for 20 min.Islets were purified on a discontinuous Ficoll gradient (Sigma).For marginal mass syngeneic transplantation, 200 handpickedislets were transplanted immediately after isolation or after48 h of culture in serum-free medium beneath the renal capsule,and tail-vein glucose was measured daily. This represents halfthe number of islets usually transplanted. Mice were anesthetizedwith ketamine. Posttransplantation glucose reduction was calculatedas percentage of the pretransplantation value. As an indirectmeasure of cell viability in vitro, proliferation was assessedby a modified methyl-tetrazolium (MTT) dye procedure (16). TheMTT staining (absorbance at 600 nm) for -TC3 cells was foundto increase linearly with the cell number from 5 x 103 to 1.2x 105 cells loaded per well in a 96-well plate (data not shown).All experiments were done in quintuplicate. Reagents were routinelytested for the presence of endotoxin using the Limulus AmebocyteLysate kit (Bio-Whittaker, Walkersville, MD) and contained <0.1endotoxin unit/ml.
Islet and -TC3 Stimulation
The mouse pancreatic insulinoma -TC3 line (gift from Dr. S.Efrat, Albert Einstein College of Medicine, Bronx, NY), whichproduces both proinsulin I and II and efficiently processeseach into mature insulin in a manner comparable to normal cellsin isolated islets, were cultured as described previously (17).Cells (500 islets/ml and 5 x 106-TC3 cells/ml) were stimulatedat 37°C for 24 h in 1 ml of fresh serum-free medium in thepresence or absence of a combination of recombinant murine IL-1(R&D Systems, Minneapolis, MN; 2 ng/ml; specific activity1.1 x 106 units/mg), TNF- (BD PharMingen, San Diego, CA; 20ng/ml; specific activity 1.0 x 109 units/mg), and IFN- (BD PharMingen;20 ng/ml; specific activity 1.0 x 108 units/mg).
RNA Isolation and cDNA Synthesis
Total RNA was extracted from 5 x 106-TC3 cells, 500 islets,or islet grafts with 1 ml of phenol/guanidine isothiocyanatethat contained Trizol solution (Life Technologies, BRL, GrandIsland, NY). The integrity of total RNA was determined by denaturingagarose gel electrophoresis and ethidium bromide staining. ForcDNA synthesis, 1 µg of total RNA was primed with oligo(dT)Superscript Moloney murine leukemia virus reverse transcriptase(Life Technologies BRL) as described previously (5).
Quantitative Real-Time PCR
Kinetic PCR was performed on a LightCycler (Roche Diagnostics)by using FastStart QuantiTect SYBR Green PCR kit (Qiagen, Valencia,CA) as a double-strand DNA-specific binding dye using the followingprimer pairs or as described previously (5,18). CD64 sense 5'-GGGAAGACACCGCTACACAT-3',antisense 5'-GGAGATGACA CGGATGCTCT-3'; CD68 sense 5'-CCAATTCAGGGTGGAAGAAA-3',antisense 5'-ATGGGTACCGTCACAACCTC-3'; IL-1 sense 5'-CAACCAACAAGTGATATTCTCCATG-3',antisense 5'-GATCCACACTCTCCAGCTGCA; TNF- sense 5'-CATCTTCTCAAAATTCGAGTGACAA-3',antisense: 5'-TGGGAGTAGACA AGGTACAACCC –3'; macrophage-inflammatoryprotein 1 (MIP-1) sense 5'-ATGAAGGTCTCCACCACTGC-3', antisense5'-GATGAATTGGCGTGGAATCT-3'. All runs included a control forgenomic DNA contamination, where the reverse transcriptase wasomitted in the sample. The PCR reactions were cycled 40 timesafter initial denaturation (95°C, 15 min) with the followingparameters: denaturation at 94°C for 15s, annealing at 56°Cfor 20 s, and extension at 72°C for 15 s with temperaturetransition rates of 20°C/s. Control reactions for productidentification consisted of analyzing the melting peaks (°C)and determining the length of the PCR products on agarose gel.
cDNA Array
The relative mRNA expression of 67 chemokines and chemokinereceptors and four different housekeeping genes was analyzedwith a nonradioactive GEArray Q series (SuperArray, Bethesda,MD), according to the manufacturer’s protocol. Two microgramsof total RNA was reverse-transcribed into cDNA with Moloneymurine leukemia virus reverse transcriptase (Life TechnologiesBRL). Biotin-labeled cDNA probes were hybridized to chemokineand chemokine receptor gene-specific cDNA fragments that werespotted on the GEArray membranes. The image was analyzed bySuperArray software. The relative expression level of each geneis determined by comparing the signal intensity of each genein the array after normalization to the signal of the housekeepinggene cyclophilin A. Arbitrary units were calculated with thefollowing formula: (chemokine signal – background signal)/(cyclophilinA signal – background signal). Results of <5% wereconsidered negative. For the cDNA array expression analysis,a twofold increase in gene expression was considered to be significantfor genes with basal expression values significantly over backgroundvalues, which was defined as >5% of the housekeeping gene.
In Situ Hybridization In situ hybridization was performed as described previously(5). Negative controls included hybridization performed on replicatetissue sections using the sense riboprobe.
Glucose-Stimulated Insulin Secretion
Groups of 30 isolated islets or 3 x 104 cells were culturedwith complete CMRL 1066 medium (10% FBS, 100 U/ml penicillin,0.1 mg/ml streptomycin, and 2 mM l-glutamine) that contained5.5 or 16.7 mM glucose in 24-well tissue culture plates (Corning,Corning, NY). Supernatants were analyzed for insulin contentusing a rodent-specific insulin ELISA kit (Crystal Chem, Chicago,IL).
CCL2 Protein Assay
Aliquots of various pancreatic islet preparations (50 islets/ml;n = 13) were cultured in 24-well plates, and supernatants wereharvested at different time points and stored at –80°Cuntil assayed. CCL2 was measured with a sandwich ELISA kit fromR&D Systems according to the manufacturer’s instructions.Absorbance was measured at 450 nm.
Statistical Analyses
All experiments were repeated at least three times. Groups werecompared using two-sided t test for continuous variables. Valuesare expressed as the mean ± SEM. No corrections weremade for multiple comparisons. Linear regression and correlationswere calculated using InStat 2.01 software.
Differential Expression of Chemokines and Their Receptors in Cells and Isolated Islets under Basal and Stimulated Conditions
We first characterized the constitutive and stimulated expressionof chemokines and chemokine receptors of islets in vitro. Asislets are composed of different residential cell populations,an established cell line (-TC3) was also studied to delineatethe contribution of this cell type. Immediately after the isolationand purification procedure, islets expressed the transcriptsof specific chemokine genes, namely CCL6, CCL7, CCL17, CCL19,CCL21, CCL28, CXCL2, CXCL5, CXCL11, and CXCL15. The expressionof CCL2 was induced after 24 h in culture. In addition, mostof the chemokines identified immediately after isolation werefurther upregulated after 24 h in culture. The same chemokinemRNA expression pattern was observed in -TC3 in long-term culture.Transcripts for the chemokine receptors CCR7 and CXCR5 werefound in both -TC3 and isolated islets (Table 1).
Table 1. Chemokine and chemokine receptor gene expression in murine -TC3 cells and islets immediately after isolation (0 h) and after 24 h in culture, without the addition of cytokines, detected by cDNA arraya
One mechanism by which invading mononuclear cells destroy cellsis by local production of cytokines such as TNF-, IL-1, andIFN-. To study whether proinflammatory conditions result inthe induction of chemokines in islets, we exposed cells in cultureto a combination of TNF-, IL-1, and IFN- for 24 h at a concentrationused previously (10,11). Cytokine stimulation of islets ledto an induction of CCL2, CCL5/RANTES (regulated on activationnormal T cell expressed and secreted), CXCL2/MIP-2, CXCL9/MIG,and CXCL10/IP-10. With the exception of CXCL2, all genes werealso induced in the cell line (-TC3), suggesting a source otherthan cells within the islets for CXCL2 (Table 2). These findingswere confirmed by QPCR (data not shown).
Table 2. IL-1, IFN-, and TNF- induced chemokine gene expression in islets and cultured -TC3 cells detected by cDNA arraya
To determine whether CCL2 mRNA expression was associated withprotein synthesis and release, we determined CCL2 levels inculture medium by ELISA and found them to correlate well withthe pattern of mRNA expression. Islets that were cultured upto 2 d had a progressive increase in CCL2 production (Figure 1).These in vitro data suggest that islets and cells constitutivelyexpressed the mRNA for several chemokines and chemokine receptors,and exposure to proinflammatory cytokines upregulated additionalpotent chemokines, with the potential to attract cells expressingCCR2, CCR5, and CXCR3, such as activated T cells and monocytes/macrophages.
Figure 1. Effect of islet cell culture on CCL2 secretion. CCL2 secretion of 13 different islet preparations immediately after isolation (0 h), 1 and 2 d of culture. CCL2 concentration is given as pg/ml supernatant and pg/islet.
High Levels of Pretransplantation CCL2 Effectively Recruit CCR2+ Monocytes/Macrophages and T Cells
To assess whether donor-derived CCL2 has the potential to attractCCR2+ cells in vivo, we analyzed intragraft mRNA expressionin isografts by QPCR and localized it with in situ hybridization.By using the isografts, we were able to determine recruitmentindependent of allogeneic stimulus. High levels of pretransplantationCCL2 by islets resulted in greater intragraft CCR2 mRNA expression(Figure 2). In situ hybridization localized a significant CCR2+mononuclear cellular infiltrate surrounding the islet cells(Figure 3). Molecular markers for leukocytes suggested increasedrecruitment of monocytes/macrophages (CD64, CD68) and T cells(CD3) into the CCL2-expressing donor grafts (Figure 2), withearly recruitment of monocytes/macrophages on day 1 followedby late recruitment of T cells on day 4.
Figure 2. Isograft CCL2, CCR2, monocyte/macrophage marker CD64 and CD68, and T cell marker CD3 mRNA expression. Grafts were analyzed by QPCR 1 and 4 d after transplantation of cultured or freshly isolated islets (*P < 0.05 freshly isolated versus cultured islets).
Figure 3. CCR2 was localized by in situ hybridization using an antisense riboprobe, demonstrating mRNA expression (in situ signal indicated by the deposition of silver grains) by infiltrating mononuclear cells. As negative controls, serial sections were hybridized with the CCR2 sense probe, demonstrating a very small number of nonspecifically deposited silver grains. Original magnification x400.
Intragraft Cytokine mRNA Expression Pattern
Expression of seven cytokines was analyzed on days 1 and 4 posttransplantationof cultured and uncultured islets to assess their local production.There was no detectable expression of IL-2 and IFN- (data notshown). Both cytokines are mainly produced by T cells and naturalkiller cells after their activation, suggesting that significantT cell activation does not occur within 4 d posttransplantation.In addition, there was no difference of IL-10 expression inthe two groups and time point’s analyzed (data not shown).
In contrast, there was evidence for production of monocyte/macrophage-derivedcytokines. Cultured islets showed higher expression of IL-1,TNF-, TGF-1, and MIP-1 when compared with freshly isolated isletsafter transplantation (Figure 4). MIP-1 is a product of monocytesand macrophages and primary stimulator of other proinflammatorycytokines such as IL-1, IL-6, and TNF-. This is consistent withthe pattern of cytokine production seen in cultured transplantedislets. These data suggest that mainly monocyte/macrophage-derivedcytokines and not T cell-derived cytokines are likely mediatorsfor the impaired islet graft function.
Figure 4. Isograft cytokine mRNA expression. Grafts were analyzed by QPCR 1 and 4 d after transplantation of cultured or freshly isolated islets (*P < 0.05 freshly isolated versus cultured islets).
Effect of Islet-Derived CCL2 on Islet Isograft Function
We next examined whether the islet cell culture or the infiltratingmononuclear cells are detrimental to islet grafts using a marginalmass transplantation model in streptozotocin-induced diabeticsyngeneic C57BL/6 mice. In this model, the degree of glucosereduction, expressed as percentage reduction of the pretransplantationglucose concentration, reflects islet graft function in vivo.The mean glucose reduction over 4 d posttransplantation wassignificantly lower when islets were transplanted after 2 dof culture compared with freshly isolated islets grafts (–40± 15.9 versus –55 ± 7.7%; P = 0.038). Wethen examined whether pretransplantation CCL2 levels rangingfrom 0.46 pg/islet (22.9 pg/ml) to 4.12 pg/islet (206 pg/ml)correlated with islet graft function. We found that the glucosereduction 1 d posttransplantation inversely correlated withthe level of CCL2 secretion of islets (P < 0.0001; Figure 5).This effect was independent of the time interval analyzed(mean posttransplantation glucose reduction day 1 to 2 r = –0.923(95% confidence interval [CI], –0.98 to –0.74),day 1 to 3 r = –0.921 (95% CI, –0.98 to –0.74),and day 1 to 4 r = –0.915 (95% CI, –0.98 to –0.72).Isografts that were transplanted to CCR2–/– recipientsdemonstrated reduction of glucose independent of the level ofCCL2 production by islets (Figure 5). Although the CCR2–/–animals were significantly protected from this effect, damageto the islets could not be completely accounted for by MCP-1/CCR2interaction because a minimal decrease in percentage of glucosereduction was seen in the absence of the receptor, suggestinga role for other cytokines or chemokines.
Figure 5. Effect of islet-derived CCL2 on islet isograft function after marginal mass transplantation of streptozotocin-induced diabetic mice. (A) Linear regression of pretransplant CCL2 concentration with glucose reduction 1 d posttransplantation. Linear coefficient (r) and its 95% confidence interval and P-value are shown. High pretransplant CCL2 significantly correlated with poor islet graft function in wild type recipients. This correlation was absent when the CCR2 pathway was disrupted by transplanting CCR2–/– mice. (B) Recipients divided into low and high donor-derived CCL2 and the effect on glucose reduction. CCL2 cutoff value was the 50th percentile (82 pg/ml).
CCL2 Is not Cell Toxic and Does not Impair Insulin Release In Vitro
We and others have shown that islet cell expression of CCR2mRNA can be induced under various conditions (5,19). Using themore sensitive QPCR assay (data not shown), we demonstratedthat islet cells constitutively expressed small amounts of CCR2mRNA. Therefore, to determine whether the detrimental effectof high levels of CCL2 secretion was due to direct toxicity,we measured in vitro glucose-stimulated insulin release and cell viability, in the presence of recombinant CCL2 at concentrationssimilar to those produced by cultured islets (Figure 1). Isletcell insulin release at basal (5.5 mM) or high glucose (16.7mM) conditions was unaffected by rCCL2 at a range of concentrations(100 to 1000 pg/ml; Figure 6). -TC3 cells that were incubatedwith rCCL2 concentrations ranging from 100 to 105 pg/ml werealso evaluated for cell viability using the MTT method. We foundthat rCCL2 had no direct affect on -TC3 cell proliferation orviability (Figure 7). These results strongly suggest that CCL2itself had no deleterious effect on cell viability or endocrinefunction.
Figure 6. Effect of CCL2 on islet and -TC3 insulin secretion. Cells were cultured for 12 h with 0 (vehicle control), 100, and 1000 pg/ml rCCL2 and then exposed to 5.5 mM or 16.7mM glucose for 1 h. Supernatants were analyzed for insulin content with ELISA. Data are shown as mean insulin secretion ± SEM.
Figure 7. Effect of CCL2 on -TC3 cell viability was measured by MTT metabolism. Data are % of control mean ± SEM of three experiments performed in quintuplicate. -TC3 were plated in 96-well plates and cultured for 48 hours in the presence of rCCL2 at various concentrations.
Highly differentiated pancreatic cells have prominent chemokine-producingcapabilities and, therefore, the potential to induce or alterinflammatory as well as noninflammatory processes (5–11).Murine islet cells are not passive bystanders of their own destructionbut have the potential to secrete CCL2, and this study is thefirst to show that this is detrimental for immediate posttransplantationgraft function. The important role of the CCR2 pathway in conjunctionwith low-dose immunosuppression has been demonstrated in a murineallogeneic islet transplantation model (18,20). However, fewdata are available to support the role of donor-derived chemokinesin transplantation (21).
Our data suggest that islets have the capacity to behave ina similar manner to cells of the immune system, recruiting leukocytesfrom the blood stream, resulting in the amplification of themononuclear cell infiltration within the islet itself. Thissupports the data of a recent study in islet transplant patientswith type 1 diabetes that found a correlation with high basaldonor islet CCL2 production before transplantation with a poorclinical outcome (6). Data in the literature are conflictingas to whether islets’ expressing CCL2 constitutively maybe due to the differences in species and technique sensitivities.The data that islets express and secrete CCL2 as soon as theyare cultured are consistent with an inflammatory function forCCL2 in this setting, and migration studies with isolated monocyteshave shown that CCL2 secreted by islets is biologically active(6,7). Our data that in vitro culture of islets augmented CCL2secretion are in agreement with previous data on human isletscultured up to 6 (6) or 11 days after isolation (22). Our invivo experiments showed that the inflammatory mediator CCL2exerts a biologic effect by providing a gradient to attractCCR2+ cells to the site of inflammation. Moreover, the amountof CCL2 produced negatively correlated with islet graft function,a correlation absent when the CCR2 pathway was disrupted.
Our data suggest that the primary cause of transplanted isletdysfunction is antigen-nonspecific inflammation at the graftsite mediated by the monocyte/macrophage-associated inflammatoryproducts IL-1, TNF-, and MIP-1. Recent data also documentedsignificant expression of macrophage-associated inflammatorygenes, including IL-1, IL-6, TNF-, and CCL2, in islets froma brain-dead donor (23), and these islet grafts were found tohave a decreased functionality in vivo and in vitro (24). Together,the data suggest that donor-derived CCL2 leads to monocyte/macrophageinfiltration, further cytokine secretion, and activation ofthe innate and adaptive immune response to cause graft dysfunction.
Of note, transgenic mice with targeted CCL2 expression for cells develop intense insulitis, with a predominance of macrophagesbut no diabetes (25). This suggests that the local productionof CCL2 is not sufficient to cause tissue destruction but mightcontribute to the recruitment of a first wave of mononuclearcells, and the final result depends on the presence of otherchemokines. Our data with early monocyte/macrophage infiltrationfollowed by T cells would be consistent with that hypothesis.In addition, in our marginal mass model, which resembles clinicalislet transplantation, mild insulitis might affect islet graftfunction. Our results exclude, however, that CCL2 is directly cell toxic, as it did not affect cell viability or the releaseof insulin, confirming previous studies with human islets (6).
Our data have several potential clinical implications. First,they provide evidence that islet cell culture affects isletgraft function in vivo. Transplantation of islets immediatelyafter isolation was done in the original description of theEdmonton protocol (26) and may explain the high success ratenot reached in other sites despite the same immunosuppressiveprotocol (27). At this point, however, it is unknown whetherislet cell culture has a negative impact on graft function inimmunosuppressed recipients. Second, measurement of pretransplantationCCL2 level may be a useful tool to preselect islets to increaseengraftment success rates. Third, strategies to prevent in vitroCCL2 secretion might offer new approaches in clinical islettransplantation. Of note, NF-B was found to play an importantrole for CCL2 expression in cells and might be a potentialtarget for pretransplantation, ex vivo gene therapy (9). Inaddition, it was previously shown that the in vitro CCL2 expressionin human islets can be decreased by cyclosporine and the vitaminB derivative nicotinamide (28).
Using a cDNA array approach, we also identified the inductionof potent chemokines CCL5, CXCL9, CXCL10, and CXCL2 after treatmentwith cytokines. It is interesting that CXCL9 and CXCL10 arenot only chemoattractant but also angiostatic and, therefore,of possible importance during islet transplantation, in whichrevascularization of the islets is required for successful engraftment(29). However, the role of these chemokines in the recruitmentof inflammatory cells and angiogenesis is unclear and needsto be studied further. We have also shown that islets as wellas -TC3 cells expressed the chemokine receptor CCR7, along withboth its ligands CCL19/SLC and CCL21/MIP-3. Expression of bothCCL19 and CCR7 was also found in glomerular mesangial cells,where this pathway was important for cell survival, suggestinga homeostatic or regulatory role for chemokines expressed bynonlymphoid tissue (30).
In conclusion, high pretransplantation CCL2 concentration hasa negative impact on immediate islet graft function as a resultof increased inflammatory cell recruitment. Therefore, strategiesto decrease CCL2 release by islet preparations or blocking theCCL2/CCR2 pathway in the recipient might increase the successof islet engraftment and long-term insulin independence in humanislet transplantation.
Acknowledgment
B.M. was supported by National Institutes of Health researchgrant RO1 AI 49289-01.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Received for publication September 8, 2004.
Accepted for publication November 10, 2004.
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Abstract
Introduction
cell damage caused by ischemia-reperfusion and the inflammatory response. In addition to adhesion molecules, such as selectins and integrins, the complex process of extravasation and influx of leukocyte subsets into the site of tissue injury is mediated, to a significant extent, by the expression of specific chemokines and chemokine receptors (1). Monocyte chemoattractant protein-1 (CCL2/MCP-1) is a proinflammatory chemokine produced by monocytes/macrophages and lymphocytes in addition to many other cell types, including endothelial cells, mesangial cells, renal tubular cells, and fibroblasts, in response to cytokines, oxidized LDL, and LPS (2–4). Several studies have now shown that CCL2 is also strongly expressed by human and rodent pancreatic islets under various conditions (5–11). The biologic actions of CCL2 are mainly mediated through the interaction with the CC chemokine receptor 2 (CCR2), the other ligands for which include CCL8/MCP-2, CCL7/MCP-3, and CCL12/MCP-5. CCR2 receptors are found on the surface of inflammatory mononuclear cells, endothelial cells, and fibroblasts (12). Through binding to the CCR2 receptor, its ligands signal monocytes, natural killer, and activated and memory T cells to migrate to sites of injury as part of the inflammatory response (13,14). 
(BD PharMingen, San Diego, CA; 20 ng/ml; specific activity 1.0 x 109 units/mg), and IFN-
(BD PharMingen; 20 ng/ml; specific activity 1.0 x 108 units/mg). 
B was found to play an important role for CCL2 expression in 
