Inflammation in atherosclerosis

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Research Paper

J Vasc Res 2005;42:266–271 DOI: 10.1159/000085721

Lipopolysaccharide-Induced Cytokine

and Chemokine Expression in Human

Carotid Lesions

Ken Jatta

a

Dick Wågsäter

a

Lars Norgren

b

Björn Stenberg

b

Allan Sirsjö

a

Division of Biomedicine, Department of Caring Sciences, University of Örebro, and b

Department of Surgery, Örebro University Hospital, Örebro , Sweden

sis, and that low levels of circulating LPS may affect the levels of pro-infl ammatory cytokines much more in ath-erosclerotic vessels than in normal vessels and may con-tribute to the development of the atherosclerotic le-sion.

Introduction

The pathophysiology of atherosclerosis involves a trig-gered infl ammatory process and activation of immuno-competent cells. Characteristics for the atherogenic in-fl ammatory process include monocyte, T-cell and mast cell recruitment from the bloodstream into the wall of the artery [1, 2] . Several epidemiological and animal studies have highlighted the importance of pro-infl ammatory cy-tokines in atherogenesis [3, 4] .

Infectious agents as potential participants in the in-ﬂ ammatory process of atherosclerosis are gaining accep-tance in experimental science. Several epidemiological studies have postulated an association between infectious agents, like gram-negative bacteria, and chronic cardio-vascular disease such as atherosclerosis [5, 6] . This has been demonstrated in animal models where the adminis-tration of lipopolysaccharide (LPS), a major pathogenic component of gram-negative bacteria cell wall, is found

Key Words

IL-1 , -6, -8, -10 TNF- MCP-1 IP-10 RANTES MIG Atherosclerosis

Abstract

The release of cytokines and chemokines from activated immune-competent cells plays a crucial role in determin-ing the pathology of the atherogenic progress. We inves-tigated the effect of bacterial lipopolysaccharide (LPS) on cytokine/chemokine expression in carotid lesions and normal renal arteries. The lesions or renal arteries were incubated for 6 h at 37 ° C in serum-free media treated with or without LPS. After LPS treatment, increased pro-tein levels of IL-1 , IL-6, IL-8, IL-10, TNF- and MCP-1 were observed in the culture medium from the lesions measured with cytometric bead array. We were able to detect the induction of IL-1 , IL-6, IL-8, IL-10, TNF- and MCP-1 mRNA in the lesions after stimulation with LPS using real-time PCR. In renal arteries, LPS also induces mRNA expression of all chemokines and cytokines vestigated with the exception of IL-6. However, LPS in-duces signifi cantly higher levels of TNF- , IL-1 and IL-10 mRNA in lesions compared to renal arteries. The results suggest that infectious agents are capable of enhancing the production of cytokines/chemokines in an already ongoing infl ammatory process such as in

Received: November 9, 2004 Accepted after revision: March 20, 2005 Published online: May 11, 2005

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to increase the atherosclerotic lesion size [5] . However, the molecular mechanisms in which LPS exerts its action in the development of atherosclerosis are yet to be ex-plored. Hence, we set to investigate the effect of the bac-terial cell wall component (LPS) in an ongoing inﬂ amma-tory process as in atherosclerosis.

Materials and Methods

Patient Samples

Carotid specimens from 12 patients were taken from plaques removed during carotid endarterectomy. All patients were oper-ated on due to symptomatic carotid artery stenosis greater than 70%. The arterial specimens were washed immediately in phos-phate-buffered saline to remove peripheral blood cells. All speci-mens contained advanced atherosclerotic lesions. Each arterial specimen was then divided into two sections, approximately 700 mg each. The tissue sections were then incubated separately

for 6 h, at 37 ° C in 2 ml Dulbecco’s modiﬁ ed Eagle’s medium/

Ham’s F12 medium (Gibco, Rockville, Md., USA) containing 2% albumin (Pharmacia) with or without 100 ng LPS. Thereafter, the conditioned media were collected, aliquoted and stored in different

vials than the tissue samples at –70 ° C until analysis. Six patients

scheduled for nephrectomy were included, and biopsies from the renal artery were obtained preoperatively and placed in Dulbecco’s modiﬁ ed Eagle’s medium/Ham’s F12 medium (Gibco) enriched with human albumin 30 mg/ml (Biovitrum AB, Stockholm, Swe-den). The renal arteries were divided into two equal pieces and incubated with 100 ng/ml LPS from Escherichia coli, O55:B5

(Sig-ma Chemical, St. Louis, Mo., USA) or left unstimulated at 37 ° C

for 6 h. The renal arteries are histologically free of atherosclero-sis.

Total RNA Preparation

Artery sections were frozen in liquid nitrogen and disrupted in a Mikro-Dismembrator II (B. Braun), and RNeasy Fibrous Tissue Mini Kit (Qiagen, USA) was used to isolate RNA according to the manufacturer’s instructions. To ensure proper results, the integrity and quantity of the isolated RNA were evaluated with the Agilent 2100 bioanalyzer using the RNA 6000 Nano Assay kit (Agilent

Technologies). Recovered RNA was stored at –70 ° C until use.

Reverse Transcription and PCR Ampliﬁ cation

One microgram of total RNA was diluted in Rnase-free H 2 O to

20 l for the synthesis of cDNA by reverse transcription using

Su-perScript™ II Rnase H – _{reverse transcriptase (Invitrogen)}

accord-ing to the manufacturer’s instructions. The ﬁ nal cDNA product was

diluted to the double volume with H 2 O and stored at –20 ° C until

use. PCRs were performed in a 96-well plate using a PCR mix

(22 l) consisting of 2 l cDNA sample, 12.5 l TaqMan®

Uni-versal Master Mix (2 ! ), and 1.25 l of primers and probes for

IL-8, IL-10 and MCP-1 (designed and premixed by Applied

Bio-systems, Foster City, Calif., USA, to a concentration of 18 M for

each primer and 5 M for the probe) diluted in Rnase-free water.

For the ampliﬁ cation of IL-1 , IL-6 and TNF- genes, the

follow-ing set of primers and probes were designed by Primer Express and obtained from Invitrogen and Applied Biosystems, respectively:

IL-1 F-5 -CTG ATG GCC CTA AAC AGA TGA AG-3 , IL-1

R-5 -GGT CGG AGA TTC GTA GCA GCT GGA T-3 and probe

5 TTC CAG GAC CTG GAC CTC TGC CCT C3 ; IL6 F5

-CGG GAA CGA AAG AGA AGC TCT A-3 , IL-6 R-5 -CGC TTG

TGG AGA AGG AGT TCA-3 and probe 5 -TCC CCT CCA GGA

GCC CAG CT-3 ; TNF- F-5 -AGG CGG TGC TTG TTC CTC

A-3 ; TNF- R-5 -GTT CGA GAA GAT GAT CTG ACT GCC-3

and probe 5 -CCA GAG GGA AGA GTT CCC CAG GGA C-3 .

Gene expressions were normalized to -actin gene expression with

the primers -actin F-5 -CTG GCT GCT GAC CGA GG-3 ,

-ac-tin R-5 -GAA GGT CTC AAA CAT GAT CTG GGT-3 and the

-actin probe 5 -CCT GAA CCC CAA GGC CAA CCG-3 . The

probes were labelled with FAM as reporter dye and TAMRA as quencher dye. The ABI Prism™ 7700 Sequence Detection System was used for RT-PCR ampliﬁ cations. During ampliﬁ cation, ther-mal cycler conditions were as follows: each sample was analysed in

duplicate for 2 min at 50 ° C, 10 min at 95 ° C, 15 s at 95 ° C and

1 min at 60 ° C. The PCR ampliﬁ cation was correlated against a

standard curve.

Quantitative Determination of Cytokines and Chemokines in Serum-Free Culture Medium

The BD human cytometric bead array kits (human chemokine-1, human Th1/Th2 cytokine CBA kit II and human inﬂ ammation CBA kit; BD Biosciences) were used to quantitatively measure cy-tokine/chemokine expression levels in culture media. Fifty micro-litres of conditioned medium were used and the assay was per-formed according to the manufacturer’s instructions and analysed on the FACSCalibur ﬂ ow cytometer (Becton Dickinson).

Statistical Analysis

The nonparametric Wilcoxon signed ranks test was used to as-sess differences between groups. Differences were accepted as sig-niﬁ cant at a level of p ! 0.05.

Results

Cytokine Protein Expression in Media from LPS-Treated Carotid Lesions

Specimens from 6 patients were taken from plaques removed during carotid endarterectomy. All patients were operated on due to symptomatic carotid artery ste-nosis greater than 70%. Carotid lesions were incubated for 6 h at 37 ° C in serum-free medium. After incubation, culture media were collected and analysed using cytomet-ric bead array to quantify protein levels of IL-1

, IL-2, IL-4, IL-6, IL-10, IL-12, TNF-

and IFN-

. Cytokine pro-tein expression was detectable in the medium, i.e. ex-pressions of IL-6, IL-1

, IL-10 and TNF-

. The mean expression of IL-6 was highest, 9,000 pg/ml (range 1,900– 28,600 pg/ml) followed by TNF-

100 pg/ml (range 13– 317 pg/ml), IL-1

50 pg/ml (range 14–204 pg/ml) and IL-10 20 pg/ml (range 2–52 pg/ml). We were not able to detect IL-2, IL-4, IL-12 or IFN-

in the culture media. LPS induced a 50- and 15-fold increase in the expressions

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of TNF-

and IL-10, respectively, compared to untreated lesions ( ﬁ g. 1 ), whereas IL-1

as well as IL-6 showed mere-ly a 5- and 3-fold increase, respectivemere-ly, in expression levels after LPS treatment ( ﬁ g. 1 ). Even after treatment with LPS, we were not able to detect expressions of IL-2, IL-4, IL-12 or IFN-

.

Chemokine Protein Expression in Media from Cultured Carotid Lesions Treated with LPS

Medium that contained atherosclerotic lesions showed expression of all the chemokines analysed in the study (IL-8, IP-10, MCP-1, MIG and RANTES). The average protein expression of MCP-1 was 5,300 pg/ml (range 2,600–13,300 pg/ml), an amount that is twice the quantity expressions of IL-8 (2,600 pg/ml, range 940– 9,630 pg/ml) and MIG (2,400 pg/ml, range 700–7,400

pg/ml) and almost four times more than RANTES (1,300 pg/ml, range 142–5,720 pg/ml) and IP-10 (900 pg/ ml, range 444–1,986 pg/ml).

LPS treatment induced a 5- and 2-fold increase, re-spectively, in the expressions of IL-8 and MCP-1. Despite a high expression level of RANTES, IP-10 and MIG in media from non-LPS-treated carotid lesions, we were not able to observe any signiﬁ cant elevation of them after LPS administration ( ﬁ g. 2 ).

mRNA Expression of Cytokines and Chemokines in LPS-Stimulated Renal Arteries and Carotid Lesions We compared the induction of cytokines and chemo-kines in atherosclerotic lesions after LPS stimulation with the response in normal artery, where we used renal artery obtained preoperatively from patients scheduled

Fig. 1. Cytokine expression in media from cultured atherosclerotic lesions exposed to LPS. Samples from human atherosclerotic ca-rotid plaques (n = 6) were incubated for 6 h in LPS-treated (100 ng) and untreated culture media. Media were collected and analysed

by cytometric array for cytokine expression. IL-1 , IL-6, IL-10 and

TNF- expressions were signiﬁ cantly upregulated (* p ! 0.05) in

the LPS-treated compared to untreated culture media. Data are given as fold-induced cytokine expression in LPS-treated versus untreated culture media. Individual samples are shown as boxes and medians are expressed as solid bars.

Fig. 2. Chemokine expression in media from cultured atheroscle-rotic lesions exposed to LPS. Samples from human atheroscleatheroscle-rotic carotid plaques (n = 6) were incubated for 6 h in LPS-treated (100 ng) and untreated culture media. Media were collected and analysed by cytometric array for chemokine expression. IL-8 and MCP-1 expressions were signiﬁ cantly upregulated (* p ! 0.01) in the LPS-treated media compared to untreated culture media. There was no signiﬁ cant upregulation of IP-10, MIG or RANTES. Data are given as fold-induced chemokine expression in LPS-treated ver-sus untreated culture media. Individual samples are shown as box-es and medians are exprbox-essed as solid bars.

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for nephrectomy. We also used a new set of carotid le-sions. As in carotid lesions, LPS signiﬁ cantly induced TNF-

(p ! 0.05), IL-1

(p ! 0.05), 8 (p ! 0.001), IL-10 (p ! 0.01) and MCP-1 (p ! 0.05) mRNA expressions in normal renal artery. However, in contrast to carotid

lesions, LPS did not induce IL-6 expression in normal renal artery ( ﬁ g. 3 b).

Furthermore, LPS induces signiﬁ cantly higher levels of TNF-

(p = 0.05), IL-1

(p = 0.001) and IL-10 (p ! 0.01) mRNA in carotid artery lesions compared to nor-mal renal artery ( ﬁ g. 3 ).

Discussion

The pathophysiological progress of atherosclerosis includes activation of the immunologic response leading to an increase in production and release of cytokines and chemokines [7] . In the present study, we detected pro-tein expressions of IL-1

, IL-6, IL-10, TNF-

, IL-8, IP-10, MCP-1, MIG and RANTES in culture media that had contained carotid lesion. These ﬁ ndings give clear indications of substantial activity of the immune com-petent cells found in the atherosclerotic lesions [8, 9] . To demonstrate the consistency of our ﬁ ndings, we found that cytokines/chemokines that showed highest protein expression were also most abundant in mRNA expressions. IL-1

, IL-6, IL-10 and TNF-

are cytokines that have been implicated in atherosclerosis [10] . Ani-mal models of atherosclerosis have shown that IL-6 is expressed in the atherosclerotic plaque and that exoge-nous IL-6 administration enhanced fatty lesion develop-ment [11] . Subsequent studies conﬁ rmed these ﬁ ndings from immune histochemical studies where protein ex-pressions of IL-6 are found in human atherosclerotic lesion [12, 13] .

In this study, we have shown an overall high protein expression of the chemokines IL-8, IP-10, MIG, RAN-TES and MCP-1 compared to the levels of cytokines, with the exception of IL-6. This indicates that cells in human carotid plaques produce chemokines that may serve to recruit additional inﬂ ammatory cells into the plaque en-hancing the inﬂ ammatory reaction that in turn may result in accelerated plaque development. There are a multitude of studies that have suggested a pathogenic role of IL-8 and MCP-1 in atherosclerosis by demonstrating their ex-pression in areas of atherosclerotic lesions [14–16] . A more direct evidence for the role of MCP-1 was shown in animal models where combined MCP-1/LDLR knockout mice had 80% decrease in lesion size, 55% decrease in macrophage content in the plaques and less lipid accu-mulation throughout their aortas compared to control MCP-1 +/+ /LDLR –/– mice [17] . However, less is said about the role of IP-10, MIG and RANTES in atherogenesis compared to the role of IL-8 and MCP-1 [18] .

Fig. 3. mRNA expressions of TNF- , IL-1 , IL-10 ( a ), IL-6, IL-8

and MCP-1 ( b ) in LPS-stimulated human renal arteries and

ca-rotid lesions. Signiﬁ cant induction of TNF- (p ! 0.01; p ! 0.05),

IL-1 (p ! 0.01; p ! 0.05), IL-10 (p ! 0.01; p ! 0.01), IL-8 (p ! 0.01;

p ! 0.01) and MCP-1 (p ! 0.01; p ! 0.05) were observed in

LPS-treated carotid lesions and renal arteries, respectively ( a , b ).

Induc-tion of IL-6 (p ! 0.01) was only seen in carotid lesions and not in

renal arteries ( b ). LPS signiﬁ cantly induced higher levels of TNF-

(p ! 0.05), IL-1 (p ! 0.01) and IL-10 (p ! 0.01) mRNA in carotid

lesions compared to renal arteries. a , b Data are expressed as

rela-tive values. All mRNA values are normalised to the expression of

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Studies have linked novel risk factors including infec-tions as contributing factors in the pathogenesis of ath-erosclerosis [19] . The presence of micro-organisms such as Chlamydia pneumoniae and herpes viruses within ath-erosclerotic lesions has been documented and they may act as additional risk factors for the development and progression of atherosclerosis in experimental models [19–21] .

The present study demonstrates that LPS administra-tion promotes substantial activity within the atheroscle-rotic plaque, with release and expression of various cyto-kines such as TNF-

, IL-10 and chemokines such as IL-8 and MCP-1. In the normal renal artery, LPS also induces mRNA expression of all chemokines and cytokines inves-tigated in this study, with the exception of IL-6. This is in line with the ﬁ ndings by Rice et al. [22] which demon-strated increased expressions of IL-8 and MCP-1 in hu-man saphenous veins after exposure to endotoxin. How-ever, the level of cytokines was higher in the carotid le-sions compared to normal renal artery after LPS treat-ment. These results suggest that low levels of circulating endotoxins may affect the levels of pro-inﬂ ammatory cy-tokines much more in atherosclerotic blood vessels than in the normal vessels and may contribute to the develop-ment of atherosclerotic lesions.

The key role of cytokines/chemokines such as IL-1

, IL-8, IL-10, TNF-

and MCP-1 in plaque development has been conﬁ rmed in several studies both in human and in animal models [23] . Since LPS enhances their expres-sion, we therefore suggest a pivotal role for LPS in plaque development. However, the mechanisms by which LPS exerts its action on the immunologic reaction are undoubt-edly complex and multifactorial, differing probably be-tween patients and environmental conditions. Neverthe-less, in 2001, Medzhitov [24] suggested that initial recog-nition of microbes as they enter the body is based on the ability of the cells of the innate immune system such as macrophages to recognize common and conserved struc-tural components of microbial origin by pattern recogni-tion receptors, i.e. toll-like receptors (TLRs). Further-more, studies of Edfeldt et al. [25] showed that TLRs are colocalized with NF-k

in endothelial and macrophage-rich areas in human atherosclerotic lesions, suggesting a possible TLR-NF-k

pathway that may accelerate immu-nologic activity within atherosclerotic plaque through the activation of endothelial cells and macrophages by bacte-rial endotoxin such as LPS; however, further studies on this postulation remain to be performed.

We speculate that the limited induction of IL-6 and all chemokines measured after LPS administration may be

due to their already high expression prior to LPS expo-sure. However, a more complete assessment of the role of administered LPS and incubation times are to be consid-ered. It is noteworthy to point out that according to our knowledge, this in vitro pilot study is the ﬁ rst attempt be-ing made to quantitatively verify comparative quantities of cytokine and chemokine expression in culture media that had contained human carotid plaque either unstim-ulated or stimunstim-ulated with LPS.

In summary, the present study demonstrates that LPS exposure on human carotid plaque and normal renal ar-tery in vitro leads to induction of cytokines and chemo-kines. We suggest that endotoxin of gram-negative bacte-ria may accelerate an already ongoing inﬂ ammatory pro-cess as in atherosclerosis by the induction of proathero-genic cytokines, and it should be of importance to further investigate the contribution of pathogenic components such as LPS in the pathophysiological process of athero-genesis.

Acknowledgments

The study was supported by grants from the Swedish Health Care Sciences Postgraduate School (NFVO) Karolinska Institutet, Swedish Medical Research Council (K2002-71X-02042-36A) and the Swedish Heart Lung Foundation.

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