Resolvin E1 Reduces Proinflammatory Markers in Human Pancreatic Islets in vitro

Abstract

Background: In clinical islet transplantation, inflammatory responses initiated by the transplanted islets and by the host immune system cause acute and chronic graft loss. The resolution of acute inflammation is an active process mediated by specific signals and mediators such as resolvin E1 (RvE1). We investigated the effect of RvE1 on i) the inflammatory status of human pancreatic islets, ii) islet viability and apoptosis, and iii) the instant blood-mediated inflammatory reaction (IBMIR) in vitro.

Methods: Pro-inflammatory cytokines and tissue factor (TF) in isolated human islets were determined by real-time RT-qPCR (mRNA levels), CBA and Gyrolab bioaffy (protein levels) after lipopolysaccaride (LPS) stimulation. Islet viability was measured using insulin secretion in a dynamic model, ADP/ATP ratio and total ATP content. Apoptosis was measured using commercial kits after stimulation with proinflammatory cytokines. To assess effect on IBMIR, human islets were mixed with non-anticoagulated, RvE1 or vehicle pretreated ABO-compatible blood in heparin-coated tubing loops.

Results: Treatment of human islets with RvE1 (500 nM) for 24 h reduced LPS-induced increase in mRNA and protein levels of selected pro-inflammatory markers (IL-8, MCP-1, and TF). RvE1 lowered the ADP/ATP ratio, but had no effect on insulin secretion. RvE1 reduced the apoptotic effect of proinflammatory cytokines. Additionally, RvE1 reduced platelet consumption and TAT complex formation during the first 5 min after islet-blood contact.

Conclusions: RvE1 suppresses proinflammatory markers and lowers the ADP/ATP ratio in human islets in vitro. RvE1 demonstrates anti-apoptotic effects in a proinflammatory milieu. Additionally, RvE1 has modest dampening effects on IBMIR. We conclude that RvE1 may have potential in clinical islet transplantation.

islet - transplantation - resolvin - cytokine - IBMIR

References

• 1 Arita M, Bianchini F, Aliberti J. et al . Stereochemical assignment, antiinflammatory properties, and receptor for the omega-3 lipid mediator resolvin E1.  J Exp Med. 2005;  201 713-722
• 2 Arita M, Ohira T, Sun YP. et al . Resolvin E1 selectively interacts with leukotriene B4 receptor BLT1 and ChemR23 to regulate inflammation.  J Immunol. 2007;  178 3912-3917
• 3 Arita M, Yoshida M, Hong S. et al . Resolvin E1, an endogenous lipid mediator derived from omega-3 eicosapentaenoic acid, protects against 2,4,6-trinitrobenzene sulfonic acid-induced colitis.  Proc Natl Acad Sci USA. 2005;  102 7671-7676
• 4 Bannenberg G, Moussignac RL, Gronert K. et al . Lipoxins and novel 15-epi-lipoxin analogs display potent anti-inflammatory actions after oral administration.  Br J Pharmacol. 2004;  143 43-52
• 5 Bannenberg GL, Chiang N, Ariel A. et al . Molecular circuits of resolution: formation and actions of resolvins and protectins.  J Immunol. 2005;  174 4345-4355
• 6 Barshes NR, Wyllie S, Goss JA. Inflammation-mediated dysfunction and apoptosis in pancreatic islet transplantation: implications for intrahepatic grafts.  J Leukoc Biol. 2005;  77 587-597
• 7 Bennet W, Sundberg B, Groth CG. et al . Incompatibility between human blood and isolated islets of Langerhans: a finding with implications for clinical intraportal islet transplantation?.  Diabetes. 1999;  48 1907-1914
• 8 Bradbury DA, Simmons TD, Slater KJ. et al . Measurement of the ADP:ATP ratio in human leukaemic cell lines can be used as an indicator of cell viability, necrosis and apoptosis.  J Immunol Methods. 2000;  240 79-92
• 9 Campbell EL, Louis NA, Tomassetti SE. et al . Resolvin E1 promotes mucosal surface clearance of neutrophils: a new paradigm for inflammatory resolution.  FASEB J. 2007;  21 3162-3170
• 10 Dona M, Fredman G, Schwab JM. et al . Resolvin E1, an EPA-derived mediator in whole blood, selectively counterregulates leukocytes and platelets.  Blood. 2008;  112 848-855
• 11 Eich T, Eriksson O, Lundgren T. Visualization of early engraftment in clinical islet transplantation by positron-emission tomography.  N Engl J Med. 2007;  356 2754-2755
• 12 Eich T, Eriksson O, Sundin A. et al . Positron emission tomography: a real-time tool to quantify early islet engraftment in a preclinical large animal model.  Transplantation. 2007;  84 893-898
• 13 Goto M, Eich TM, Felldin M. et al . Refinement of the automated method for human islet isolation and presentation of a closed system for in vitro islet culture.  Transplantation. 2004;  78 1367-1375
• 14 Goto M, Holgersson J, Kumagai-Braesch M. et al . The ADP/ATP ratio: A novel predictive assay for quality assessment of isolated pancreatic islets.  Am J Transplant. 2006;  6 2483-2487
• 15 Heit JJ, Apelqvist AA, Gu X. et al . Calcineurin/NFAT signalling regulates pancreatic beta-cell growth and function.  Nature. 2006;  443 345-349
• 16 Herrera BS, Ohira T, Gao L. et al . An endogenous regulator of inflammation, resolvin E1, modulates osteoclast differentiation and bone resorption.  Br J Pharmacol. 2008;  155 1214-1223
• 17 Hjelmesaeth J, Hagen LT, Asberg A. et al . The impact of short-term ciclosporin A treatment on insulin secretion and insulin sensitivity in man.  Nephrol Dial Transplant. 2007;  22 1743-1749
• 18 Johansson H, Lukinius A, Moberg L. et al . Tissue factor produced by the endocrine cells of the islets of Langerhans is associated with a negative outcome of clinical islet transplantation.  Diabetes. 2005;  54 1755-1762
• 19 Johansson U, Olsson A, Gabrielsson S. et al . Inflammatory mediators expressed in human islets of Langerhans: implications for islet transplantation.  Biochem Biophys Res Commun. 2003;  308 474-479
• 20 Johnson JD, Ao Z, Ao P. et al . Different effects of FK506, rapamycin, and mycophenolate mofetil on glucose-stimulated insulin release and apoptosis in human islets.  Cell Transplant. 2009;  in press
• 21 Kim JH, Park SG, Lee HN. et al . ATP measurement predicts porcine islet transplantation outcome in nude mice.  Transplantation. 2009;  87 166-169
• 22 Kono H, Rock KL. How dying cells alert the immune system to danger.  Nat Rev Immunol. 2008;  8 279-289
• 23 Kurt-Jones EA, Popova L, Kwinn L. et al . Pattern recognition receptors TLR4 and CD14 mediate response to respiratory syncytial virus.  Nat Immunol. 2000;  1 398-401
• 24 Lenzen S, Drinkgern J, Tiedge M. Low antioxidant enzyme gene expression in pancreatic islets compared with various other mouse tissues.  Free Radic Biol Med. 1996;  20 463-466
• 25 Lewis EC, Mizrahi M, Toledano M. et al . Alpha1-Antitrypsin monotherapy induces immune tolerance during islet allograft transplantation in mice.  Proc Natl Acad Sci U S A. 2008;  105 16236-16241
• 26 Lund T, Fosby B, Korsgren O. et al . Glucocorticoids reduce pro-inflammatory cytokines and tissue factor in vitro and improve function of transplanted human islets in vivo.  Transpl Int. 2008;  21 669-678
• 27 Marzorati S, Melzi R, Nano R. et al . In vitro modulation of monocyte chemoattractant protein-1 release in human pancreatic islets.  Transplant Proc. 2004;  36 607-608
• 28 Mita A, Ricordi C, Miki A. et al . Anti-proinflammatory effects of sirolimus on human islet preparations.  Transplantation. 2008;  86 46-53
• 29 Moberg L, Johansson H, Lukinius A. et al . Production of tissue factor by pancreatic islet cells as a trigger of detrimental thrombotic reactions in clinical islet transplantation.  Lancet. 2002;  360 2039-2045
• 30 Moberg L, Olsson A, Berne C. et al . Nicotinamide inhibits tissue factor expression in isolated human pancreatic islets: implications for clinical islet transplantation.  Transplantation. 2003;  76 1285-1288
• 31 Paraskevas S, Maysinger D, Wang R. et al . Cell loss in isolated human islets occurs by apoptosis.  Pancreas. 2000;  20 270-276
• 32 Piemonti L, Leone BE, Nano R. et al . Human pancreatic islets produce and secrete MCP-1/CCL2: relevance in human islet transplantation.  Diabetes. 2002;  51 55-65
• 33 Rhode A, Pauza ME, Barral AM. et al . Islet-specific expression of CXCL10 causes spontaneous islet infiltration and accelerates diabetes development.  J Immunol. 2005;  175 3516-3524
• 34 Ruth MR, Proctor SD, Field CJ. Effects of feeding fish oil on mesenteric lymph node cytokine responses in obese leptin receptor-deficient JCR:LA-cp rats.  Int J Obes (Lond). 2009;  33 96-103
• 35 Samuelsson B, Dahlen SE, Lindgren JA. et al . Leukotrienes and lipoxins: structures, biosynthesis, and biological effects.  Science. 1987;  237 1171-1176
• 36 Schroppel B, Zhang N, Chen P. et al . Differential expression of chemokines and chemokine receptors in murine islet allografts: the role of CCR2 and CCR5 signaling pathways.  J Am Soc Nephrol. 2004;  15 1853-1861
• 37 Schulthess FT, Paroni F, Sauter NS. et al . CXCL10 impairs beta cell function and viability in diabetes through TLR4 signaling.  Cell Metab. 2009;  9 125-139
• 38 Serhan CN. Lipoxins and aspirin-triggered 15-epi-lipoxin biosynthesis: an update and role in anti-inflammation and pro-resolution.  Prostaglandins Other Lipid Mediat. 2002;  68-69 433-455
• 39 Serhan CN. Resolution phase of inflammation: novel endogenous anti-inflammatory and proresolving lipid mediators and pathways.  Annu Rev Immunol. 2007;  25 101-137
• 40 Serhan CN, Clish CB, Brannon J. et al . Novel functional sets of lipid-derived mediators with antiinflammatory actions generated from omega-3 fatty acids via cyclooxygenase 2-nonsteroidal antiinflammatory drugs and transcellular processing.  J Exp Med. 2000;  192 1197-1204
• 41 Serhan CN, Hong S, Gronert K. et al . Resolvins: a family of bioactive products of omega-3 fatty acid transformation circuits initiated by aspirin treatment that counter proinflammation signals.  J Exp Med. 2002;  196 1025-1037
• 42 Serhan CN, Yang R, Martinod K. et al . Maresins: novel macrophage mediators with potent antiinflammatory and proresolving actions.  J Exp Med. 2009;  206 15-23
• 43 Shapiro AM, Gallant HL, Hao EG. et al . The portal immunosuppressive storm: relevance to islet transplantation?.  Ther Drug Monit. 2005;  27 35-37
• 44 Shapiro AM, Ricordi C, Hering BJ. et al . International trial of the Edmonton protocol for islet transplantation.  N Engl J Med. 2006;  355 1318-1330
• 45 Steer SA, Scarim AL, Chambers KT. et al . Interleukin-1 stimulates beta-cell necrosis and release of the immunological adjuvant HMGB1.  PLoS Med. 2006;  3 e17
• 46 Vassiliou EK, Kesler OM, Tadros JH. et al . Bone marrow-derived dendritic cells generated in the presence of resolvin E1 induce apoptosis of activated CD4+ T cells.  J Immunol. 2008;  181 4534-4544
• 47 Yokomizo T, Izumi T, Chang K. et al . A G-protein-coupled receptor for leukotriene B4 that mediates chemotaxis.  Nature. 1997;  387 620-624

Supplementary Data Online

[Fig. 6], [7], [Table 2.]

Fig. 6 Dose-response experiments. Dose-response of RvE1 on key inflammatory mediators in human islets, showing best effect at 500 nM IL-8 (A), MCP-1 (B) and TF (C) were used to assess the effect of increasing doses of RvE1 on human islets. Glucocorticoids (MP, methylprednisolone, 1 μg/ml) were used as a positive control, while lipopolysaccharide (LPS, 1 μg/ml) was used as a negative control. Substance exposure was 24 h. Cycle times for the highest-expressing group are shown in the corresponding bar. n=6, purity ranging from 50–95%. *p<0.05, significantly different from vehicle treated. Data represent the mean±SEM.

Fig. 7 Calculation of dynamic insulin secretion (example) Pictures of hand-picked islets was analyzed with Cellimage^® (Andrew Friberg, Uppsala, Sweden), mean islet size calculated, and insulin secretion normalized to mU/ml insulin per 100 islet equivalents (IEQ). This example represent untreated, control islets.

Table 1 Donor and islet preparation characteristics.

Donor pancreas	Age (years)	Sex	Cold ischaemia time (h)	BMI weight (kg/m2)	Pancreas weight (g)	Islet Equivalents	Purity of fractions (%)
1	46	female	12.3	24	93	83 600	50 and 65
2	54	male	3.5	29	133	220 000	85 and 90
3	55	female	13	22	95	388 000	95
4	56	female	6.3	27	122	335 000	90 and 95
5	77	female	12.5	21	90	195 000	50 and 75
6	34	male	11.5	26	105	283 000	55 and 70
7	54	male	7.5	25	110	143 000	70 and 80
8	32	male	6	25	118	94 000	60 and 90
Donor pancreas	Insulin/DNA content (ng)	Insulin Stimulation Index ratio	ADP/ATP (pmol/μg DNA)	TF content	MCP-1(pmol/μg DNA)	IL-8 (pmol/μg DNA)
1	7.1	6.1	0.2	0.025	0.004	0.013
2	6.9	3.4	0.19	0.009	0.011	0.038
3	1.5	9.3	0.07	0.027	0.006	0.014
4	5.4	12	<0.05	0.022	0.002	0.008
5	6.1	7.1	<0.05	0.049	0.012	0.058
6	5.9	1.4	<0.05	0.039	0.011	0.057
7	4.6	1.1	0.11 0	107	–	0.055
8	3.3	4	<0.05	0.04	–	0.017

Effect of RvE1 on human islet gene expression in a TaqMan low density array

The table shows expression levels based on the individual ΔCt-values and is denoted as high, medium and low. No expression is denoted (-). Islets were treated with or without RvE1 (500 nM) for 24 h, with addition of lipopolysaccharide (LPS, 1 μg/ml) after 18 h. The genes tended to be upregulated or downregulated in RvE1 treated islets correspond to a p value below 0.15. n=4, purity ranging from 65–95%. Data represent the mean±SEM. For method, see below.

TaqMan low density array

In the current study, we applied a TaqMan LDA card to examine the expression of 48 genes in human islets after exposure to RvE1 (500 nM) for 24 h and compared the results to vehicle treated islets (controls). Aliquots (n=2) of islets from 2 independent donors in quadruplicates where used. The 48 genes belonged to several pathways involved in inflammation, coagulation and apoptosis. 1 μg of RNA was reverse transcribed using the High Capacity Archive Kit (Applied Biosystems) in a final volume of 50 μL. For real-time PCR analysis we created a TaqMan LDA card based on a 7900 HT Micro Fluidic card (Applied Biosystems, Foster City, CA). Each gene was present as duplicates in the analysis. GAPDH was used as a reference gene. The total volume of each PCR reaction was 1 μL containing an equivalent of 2ng RNA. The data acquired were analyzed with the Sequence Detector software (version 1.6.3, Applied Biosystems) and the relative gene expression levels were calculated using the 2-ΔΔCT method (Livak & Schmittgen 2001). To provide a mRNA expression profile of RvE1 exposed islets and controls, we displayed the expression levels as high, medium and low, based on ΔCt values (Ct (target gene)-Ct (GAPDH)) ([Table 2] ). The groups were made by dividing the range between the lowest and highest ΔCt into three groups for high, medium and low expression. Genes with a ΔCt above 15 cycles were defined as absent. For the calculation of differential expression in RvE1 exposed islets compared to controls, the ΔCt-value for RvE1 was divided with ΔCt-value for controls (values >1: downregulation, and <1: upregulation). Trends obtained were used for targeted real-time qRT-PCR analysis.

Reference

Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method . Methods 2001; 25 : 402 – 408.

Table 2 Expression of genes on the Taqman^®Low Density Array.

Gene	Assay ID	Mean expression level	ΔCt RvE1/ΔCt Control
			Mean	p-value
NFKB1	Hs00765730_m1	medium	1.30	0.52
CCL2	Hs00234140_m1	medium	1.04	0.39
SMAD3	Hs00232222_m1	medium	1.00	0.87
DIABLO	Hs00219876_m1	medium	1.04	0.27
SLC30A10	Hs00218883_m1	low	0.98	0.12
CCR2	Hs00174150_m1	–	–	–
INS	Hs02741908_m1	high	0.98	0.22
CD40	Hs00386848_m1	medium	1.02	0.65
CASP8	Hs00154256_m1	low	0.98	0.33
BAD	Hs00188930_m1	low	1.05	0.27
TNF	Hs00174128_m1	low	1.04	0.08
VEGFA	Hs00900054_m1	medium	0.98	0.38
TLR4	Hs00152939_m1	medium	1.00	1.00
IL15	Hs00542562_m1	low	1.09	0.44
CCL3	Hs00234142_m1	low	1.02	0.39
FOXP3	Hs00203958_m1	low	0.99	0.86
JAK1	Hs00233820_m1	low	1.03	0.67
CXCL10	Hs00171042_m1	low	1.07	0.20
ANGPT1	Hs00181613_m1	low	0.96	0.41
IL8	Hs00174103_m1	high	1.23	0.10
STAT1	Hs00234829_m1	medium	1.00	0.99
CCR5	Hs00152917_m1	low	1.09	0.04
BAX	Hs00180269_m1	medium	1.04	0.51
EGFR	Hs00193306_m1	medium	1.08	0.35
SSTR5	Hs00265647_s1	low	1.02	0.62
CASP3	Hs00234385_m1	low	1.03	0.13
CCL5	Hs00174575_m1	medium	1.04	0.10
CXCL12	Hs00930455_m1	low	1.00	0.92
IL1B	Hs00174097_m1	medium	1.02	0.31
GCG	Hs00174967_m1	high	0.98	0.39
INSR	Hs00169631_m1	medium	1.04	0.35
C5	Hs00156197_m1	low	0.98	0.48
IL4	Hs00174122_m1	–	–	–
IL18	Hs00155517_m1	low	1.05	0.03
IFNG	Hs00174143_m1	–	–	–
TXNIP	Hs00197750_m1	medium	1.03	0.21
ACTB	Hs99999903_m1	medium	0.82	0.28
IL6	Hs00174131_m1	low	1.00	0.83
FAS	Hs00163653_m1	low	1.01	0.81
TGFB1	Hs99999918_m1	medium	0.95	0.03
SLC39A1	Hs00205358_m1	medium	0.98	0.64
MAPK1	Hs00177066_m1	medium	0.99	0.78
CASP9	Hs00154261_m1	low	1.03	0.08
CCL4	Hs99999148_m1	medium	1.04	0.29
BCL2	Hs00153350_m1	low	0.97	0.61
IL10	Hs00174086_m1	low	1.16	0.53
ICAM1	Hs00164932_m1	medium	1.02	0.33

Correspondence