Exp Clin Endocrinol Diabetes 2010; 118(4): 237-244
DOI: 10.1055/s-0029-1241825
Article

© J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart · New York

Resolvin E1 Reduces Proinflammatory Markers in Human Pancreatic Islets in vitro

T. Lund1 , 2 , S. M. Mangsbo3 , H. Scholz2 , P. Gjorstrup4 , T. H. Tötterman3 , O. Korsgren3 , A. Foss1 , 2
  • 1Division of Surgery, Section for Transplantation, Oslo University Hospital, Oslo, Norway
  • 2Institute for Surgical Research, Oslo University Hospital, Oslo, Norway
  • 3Division of Clinical Immunology, Uppsala University Hospital, Uppsala, Sweden
  • 4Resolvyx Pharmaceuticals, Bedford, USA
Further Information

Publication History

received 28.07.2009 first decision 28.07.2009

accepted 14.10.2009

Publication Date:
29 January 2010 (online)

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.

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Supplementary Data Online

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

Zoom Image

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.

Zoom Image

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

T. LundMD 

Surgical Clinic Transplant Unit/Institute for Surgical Research

Oslo University Hospital

N-0027 Oslo

Norway

Phone: +47/23/07 35 18

Fax: +47/23/07 36 30

Email: tormod.lund@rr-research.no