Tumour necrosis factor-alpha production stimulated by heat shock protein 70 and its inhibition in circulating dendritic cells and cells eluted from mucosal tissues in Crohn's disease

Clinical and Experimental Immunology doi:10.1111/j.1365-2249.2006.03010.x

550

© 2006 British Society for Immunology,

Clinical and Experimental Immunology

,

143:

550–559

et al.

Accepted for publication 7 December 2005

Correspondence: Thomas Lehner, Mucosal

Immunology Unit, Kings College London at

Guy’s Hospital, Guy’s Tower Floor 28, Guy’s

Hospital, London SE1 9RT, UK.

E-mail: [email protected]

*These authors contributed equally to the study.

OR IG INAL ART I C L E

Tumour necrosis factor-

a

production stimulated by heat shock protein

70

and its inhibition in circulating dendritic cells and cells eluted from mucosal tissues in Crohn’s disease

T. Whittall,*

†

Y. Wang,*

†

C. G. Kelly,

‡

R. Thompson,

§

J. Sanderson,

§

M. Lomer,

§

S. Y. Soon,

§

L. A. Bergmeier, M. Singh,

¶

and T. Lehner

†

Departments of

†

Mucosal Immunology Unit and

‡

Oral Immunology, Guy’s, King’s and St Thomas’

Medical and Dental Schools, Guy’s Hospital,

London,

§

Department of Gastroenterology, Guy’s

and St Thomas’ NHS Trust, London, UK, and

¶

Lionex Diagnostics and Therapeutics GmbH,

Braunschweig, Germany

Summary

The objectives were to study the effect of microbial 70 kDa heat shock protein(HSP70) on the production of tumour necrosis factor (TNF)-αααα

and interleu-kin (IL)-12 by dendritic cells (DC) from patients with Crohn’s disease. TNF-αααα

concentration was increased significantly when DC from Crohn’s diseasewere stimulated with HSP70 or CD40L and this was associated with signallingby the extracellular signal regulated kinase (ERK) 1/2 and p38 mitogen acti-vated protein (MAP) kinase pathway. IL-12 production was also increasedwhen DC were stimulated with HSP70. Cells eluted from inflamed intestinalmucosa from Crohn’s disease, stimulated with HSP70, CD40L or lipopolysac-charide produced significantly greater TNF-αααα

and IL-12 concentrations thancells from uninflamed mucosa. Significant inhibition of TNF-αααα

productionwas demonstrated when DC from peripheral blood mononuclear cells or cellseluted from intestinal mucosa of Crohn’s disease were treated with either theHSP70 inhibitory peptide (aa 457–496) or peptides derived from CD40 andCD40L. These inhibitory peptides target the CD40–CD40L and the emergingCD40–HSP70 co-stimulatory pathway. Our findings offer a novel strategy toprevent excessive production of TNF-αααα

in Crohn’s disease.

Keywords:

Crohn’s disease, dendritic cells, HSP70, TNF-

α

Introduction

The immunopathogenesis of Crohn’s disease (CD) has notbeen elucidated so far, but a dysregulated immune responsedirected against gut bacteria has been postulated (reviewedin [1]). Genetic predisposition to CD is clearly important [2–4]. Increased frequency of

Mycobacterium paratuberculosis

and its DNA have been demonstrated in CD [5,6]. However,many Gram-negative and -positive microorganisms andviruses have been associated with CD [7,8]. A breakdown intolerance to the bacterial flora of the gut may be presentbecause of an increased T cell proliferative response to theintestinal flora in the inflamed bowel [9].

Heat shock protein (HSP) is found in most Gram-positiveand Gram-negative intestinal microorganisms. Increasedexpression of the host 70 kDa HSP (HSP70) has been dem-onstrated in the intestinal mucosal and submucosal mono-nuclear cells, as well as epithelial cells in CD [10]. Circulatingantibodies and T cell responses to HSP have been reported ininflammatory bowel disease (IBD) [11,12]. A protective roleof HSP70 in intestinal epithelial cells against oxidant and

other stressful agents has been suggested [13,14]. In non-human primates rectal mucosal administration of HSP70 orHSP65 elicits T and B cell responses and the production ofCC chemokines by circulating mononuclear cells [15].

There is compelling evidence that in CD TH1 responsesare generated with interleukin (IL)-12 [16–18], interferon(IFN)-

γ

[19,20] and tumour necrosis factor (TNF)-

α

[21–24] by intestinal mononuclear cells. One striking clinicaldevelopment has been the beneficial effect of administrationof anti-TNF-

α

antibodies to patients with CD but not ulcer-ative colitis (UC) [25,26]. In the cytokine network TNF-

α

iscritical in the control of CD, rheumatoid arthritis and otherautoimmune diseases [27]. However, it might be more desir-able to prevent rather than neutralize the production ofTNF-

α

, in order to control the immunopathogenesis of CD.This concept was explored, based on the finding that the C-terminal portion of HSP70 (aa359–609) stimulates humanmonocytes or DC to produce TNF-

α

and IL-12 [28,29], andthat a peptide epitope (aa 457–496) inhibits TNF-

α

and IL-12 production by DC stimulated with HSP70 or CD40L[30].

Dendritic cell maturation and TNF-

α

stimulated by HSP70 in Crohn’s disease



,

143:

550–559

551

The aims of this investigation were to study the effect ofHSP70 on cytokine production by DC from patients withCD, compared with those from UC and controls. A furtheraim was to see whether the interaction between CD40 andCD40L can be inhibited by peptides derived from thesequences of HSP70, CD40 and CD40L. HSP70 or CD40Lsignificantly enhances TNF-

α

production by DC frompatients with CD, and this was inhibited significantly byHSP70-derived peptide 457–496 or those from CD40–CD40L.

Materials and methods

Reagents

Human recombinant granulocyte–macrophage colony-stimulating factor (GM-CSF) was obtained from Leucomax(Sandoz Pharmaceuticals, Surrey, UK) and human recombi-nant IL-4 from R&D Systems (Abingdon, Oxon, UK). Solu-ble CD40 ligand trimer (CD40LT) was kindly donated by DrF. Villinger (Atlanta, GA, USA). Lipopolysaccharide (LPS)from

Escherichia coli

strain 0111B4 was obtained from Sigma(Poole, Dorset, UK).

Preparation of microbial HSP70

The recombinant

Mycobacterium tuberculosis

HSP70 wasprepared from the

E. coli

pop strain using the pJLA603 vector[31]. HSP70 was purified by ion exchange chromatographyusing Q-Sepharose resin, followed by ATP-affinity chroma-tography. The preparation was treated further with poly-mixin B-coated beads (Sigma-Aldrich, Dorset, UK) toremove LPS. The LPS content of the HSP preparation wasdetermined by the

Limulus

amoebocyte lysate assay (Sigma-Aldrich) and showed

<

0·0006 units/

µ

g of HSP70 or 5 pg/

µ

gof the HSP preparation.

Investigation of contamination of the HSP70 preparations with LPS

Any contamination of HSP70 with LPS was examined asdescribed previously [28]. Briefly, using the intracellular cal-cium chelator BAPTA/AM, TNF-

α

production was inhibitedin a dose-dependent manner when monocytes were stimu-lated with HSP70 or the two peptide-binding fragments butnot with LPS. Proteinase K differentiated the inhibitoryeffect of TNF-

α

production by HSP70 and its two C-termi-nal fragments but not that of LPS. We then determined theminimal concentration of LPS, which affects the stimulatingactivity of DC by HSP70. We have shown previously that10 ng/ml of LPS was required to double the concentration ofIL-12 by the two HSP70 preparations or CD40LT [30]. TheLPS contamination of the HSP70 preparations was 0·0006units (or 5 pg) per

µ

g of HSP70, which is 2000 times lower

than would be required for LPS to stimulate production ofIL-12.

Preparation of synthetic peptides

Peptide 457–496 was synthesized by Bachem Ltd (Buben-dorf, Switzerland) to a purity of 93·7%, determined by highpressure liquid chromatography, the sequence of which is:IVHTAKDKGTGKENTIRIQEGSGLSKEDIDRMIKDAEAH.We have also studied peptides derived from the CD40 N-terminal tandem repeat (aa 27–36 and 39–58) with theCD40L extracellular domain 18

mer

(aa 102–119) to find out ifthese peptides inhibit the CD40–CD40L co-stimulatorypathway. These were synthesized as above and the sequencesof these peptides are as follows: CD40 aa 27–36 REKQY-LINSQ; aa 39–58 SLSQPGQKLVSDSTEFTETE; residuesunderlined indicate substitution of Cys with Ser, to preventaggregation through disulphide bonds. CD40L aa 102–119KEETKKENSFEMQKGDQN.

Selection of patients and biopsy procedure

Patients were selected for this study from the gastrointestinalout-patient clinics at Guy’s and St Thomas’ Hospitals. Thestudy was approved by the Local Research Ethical Commit-tee and written informed consent was obtained from thepatients. A total of 29 patients with Crohn’s disease (CD), 22with ulcerative colitis (UC) and 13 healthy control subjectswere included in this investigation. The diagnosis of CD orUC was determined by standard clinical, radiological, endo-scopic and histopathological criteria. About 50 ml of bloodwas taken from each subject for the immunological studies.An additional six colonic, rectal or ileal biopsy specimenswere taken at colonoscopy during routine investigation ofpatients for diagnosis, assessment of disease activity and can-cer surveillance. Biopsy specimens were obtained in 13patients with CD and 12 patients with UC; when biopsieswere available from more than one site, these were pooledseparately for inflamed and non-inflamed mucosal tissues.Five patients with iron deficiency or altered bowel habitsshowed normal mucosa on endoscopy and biopsies weretaken which also failed to show any histopathologicalchanges. Cells eluted from these five patients were used ascontrols.

Preparation of human monocytes and monocyte-derived dendritic cells (DC)

Human primary monocytes were isolated from peripheralblood mononuclear cells (PBMC) by centrifugation on aFicoll-Hypaque density gradient (Amersham Biosciences,Little Chalfont, Bucks, UK). The CD14

+

monocytes wereenriched by depletion of CD14

–

cells using a monocyte iso-lation kit (MACS, Miltenyi Biotec, Surrey, UK). The purityof isolated monocytes was consistently greater than 90%

T. Whittall

et al.

552



,

143:

550–559

when analysed by flow cytometry with antibody to CD14.Human DC were generated by culturing monocytes withGM-CSF (400 U/ml) and IL-4 (100 U/ml) for 5 days [32].These monocyte-derived DC were generally considered to beimmature DC, defined by surface expression of DC markersCD83, CD80, CD86, CD40, and were CD14 negative.

Assays of TNF-αααα

and IL-12 production

Immature DC were stimulated with HSP70 (10

µ

g/ml),CD40LT (10

µ

g/ml) or LPS (1

µ

g/ml) and cultured for2 days. IL-12 and TNF-

α

in the culture supernatants wereassayed by enzyme-linked immunosorbent assay (ELISA)using specific antibodies (BD Pharmingen, Cowley, UK),with sensitivity limits of 8 pg/ml for TNF-

α

and 15 pg/ml forIL-12. The supernatants were diluted

×

2 for IL-12 or TNF-

α

assay. The results were expressed in pg/ml.

The effect of co-stimulation of monocytes or DC with the synthetic peptides and HSP, CD40L or LPS, inducing TNF-αααα

and IL-12 production

To study the effect of the synthetic peptides, human mono-cytes (1

×

10

6

/ml) or monocyte-derived DC (2

×

10

5

/ml)were incubated with 12·5 and 25·0

µ

g/ml of the peptides andthe cells were stimulated with 10

µ

g/ml HSP70, 10

µ

g/mlCD40LT or 500 ng/ml LPS. After culture for 3 days TNF-

α

and IL-12 were assayed in the supernatants.

Assay of p38 and extracellular signal regulated kinase (ERK) 1/2 phosphorylation MAP kinases

For the phospho-p38 and ERK 1/2 assays, DC were treatedwith HSP70, CD40L or LPS in the presence or absence ofp38 inhibitors (SB 203580; Sigma) or ERK 1/2 inhibitor(PD 098059) for 30 min, then lysed in isotonic buffer with 6

M

urea and 0·1% Triton X-100. The phospho-p38 and ERK1/2 were assayed by ELISA using IC duo-set reagents (R&DSystems, Oxford, UK), according to the manufacturer’sinstructions. Samples were also subjected to sodium dode-cyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and Western blotting to demonstrate p38 specificphosphorylation.

Elution of mononuclear cells from mucosal tissues

Biopsy material was collected in sterile saline. The cells werepassed through a 70

µ

m sterile nylon cell strainer (BectonDickinson Biosciences, Oxford, UK Ltd) and treated by col-lagenase digestion. Briefly, the tissue was suspended in warmRPMI-1640 supplemented with 10% fetal calf serum (FCS),2 mM

l

-glutamine and penicillin/streptomycin (100 U/ml),and digested with collagenase at 2 mg/ml for 2 h at 37

°

C(Sigma-Aldrich UK Ltd). The cells were then filteredthrough the cell strainer, washed in complete medium,

counted using trypan blue exclusion and resuspended in theappropriate medium. The cells were cultured with HSP70,CD40L or LPS as above. In parallel, PBMC were stimulatedwith the same reagents, cultured for 2 days and the culturesupernatant assayed for TNF-

α

and IL-12. The possibilitythat the enzyme treatment might have affected the functionof eluted mucosal cells was examined by treatment of PBMCby the same procedure as the mucosal tissue, and theircapacity to generate TNF-

α

and IL-12 was unaffected whencompared with untreated PBMC.

Statistical analysis

The data between the groups were analysed by the non-para-metric Wilcoxon’s signed rank test or Mann–Whitney confi-dence interval test. The results of inhibition studies wereanalysed by the paired Student’s

t

-test.

Results

Comparative investigation of TNF-αααα

and IL-12 production by stimulating immature DC and monocytes with HSP70, CD40L or LPS

Production of TNF-

α

was increased significantly (Fig. 1a)when immature DC from patients with CD were stimulatedwith HSP70 (431

±

112 pg/ml), compared with controls(133

±

35 pg/ml;

t

=

2·55,

P

=

0·02) or CD40L (1303

±

422)compared with controls (363

±

94;

t

=

2·18,

P

=

0·046). Incontrast, no significant increase in TNF-

α

production wasfound in cells from UC whether stimulated with HSP70 orCD40L (Fig. 1a). Stimulation of DC with LPS failed to showsignificant differences in TNF-

α

concentrations, althoughthese were lower in CD than in controls.

Stimulation of monocytes with HSP70 or CD40L showedincreased production of TNF-

α

with cells from CD (Fig. 1c),but this did not reach statistical significance when comparedwith controls. Cells from UC were not significantly differentfrom controls in the concentration of TNF-

α

when stimu-lated with HSP70 or CD40L. In contrast, LPS elicited lessTNF-

α

, both in CD and UC. A comparison between DC andmonocytes stimulated with HSP70 or CD40L showed no sig-nificant difference in TNF-

α

production in any of the threegroups of subjects.

Production of IL-12 by monocyte-derived DC was alsoincreased in CD and UC when stimulated with HSP70(Fig. 1b). However, stimulation of DC with CD40L or LPSelicited a much greater increase in IL-12 concentration thanthose stimulated with HSP70. Surprisingly, IL-12 produc-tion by CD40L stimulated by DC in CD (4594

±

1068 pg/ml)was significantly less than that from controls (8812

±

907 pg/ml;

P

=

0·025, Fig. 1b). Although LPS stimulated a similardecrease in concentration of IL-12 by DC, this was not sig-nificant (

P

=

0·065). DC from patients with UC also showed

Dendritic cell maturation and TNF-

α

stimulated by HSP70 in Crohn’s disease


Clinical and Experimental Immunology, 143: 550–559 553

decreased IL-12 production, but again this was not signi-ficant (P = 0·294) (Fig. 1b).

A parallel study of monocytes stimulated with HSP70showed an increased production of IL-12 in cells from CD,but not in those from UC (Fig. 1d). However, as with DC,CD40L and LPS induced less IL-12 in monocytes from CD.None of the differences in IL-12 concentrations betweenhealthy controls and the two groups of patients was statisti-cally significant. Overall, the results suggest that significantdifferences in TNF-α and IL-12 production by monocyte-derived DC stimulated with HSP70 or CD40L were foundonly in DC from CD, compared with those from healthycontrols.

Inhibition of HSP70-stimulated TNF-αααα and IL-12 production by DC with HSP70-derived peptide 457–496

Peptide 457–496 derived from the sequence of HSP70 hasbeen shown to reduce TNF-α and IL-12 production by nor-mal DC or monocytes stimulated with HSP70 or CD40L[35]. With this set of experiments we wanted to assessHSP70-peptide-mediated reduction of TNF-α and IL-12production by DC from CD and UC patients, compared withcontrols. The results are presented for optimum inhibition ofTNF-α or IL-12 production in healthy controls and the twogroups of patients (Table 1a,b). The concentration of TNF-αor IL-12 from DC of healthy controls stimulated with HSP70was reduced significantly by p457–496 (P = 0·001 and 0·004,respectively; Table 1a,b). In CD both TNF-α and IL-12 weresignificantly inhibited by p457–496 (P = 0·020 and 0·050,respectively). DC from patients with ulcerative colitis,however, failed to show significant decrease in TNF-α

production with p457–496. It is important to note that theconcentrations of TNF-α and IL-12 produced by cells fromCD were decreased by p457–496 to 18% and 31%, respec-tively, below the levels of these cytokines produced by cellsfrom healthy controls (Table 1a,b). Thus, TNF-α and IL-12production can be reduced significantly by treatment withp457–496 in DC from CD but not UC.

Inhibition of HSP70-stimulated TNF-αααα and IL-12 production by DC with pCD40/CD40L

We have also used peptides derived from the CD40–N ter-minal tandem repeat 30mer (p27–36/39–58) and the CD40Lextracellular domain 18mer (p102–119) to investigate whetherthese peptides can inhibit TNF-α and IL-12 production inthe CD40–CD40L co-stimulatory pathway. As with p457–496, significant inhibition of TNF-α production by DC waselicited with the CD40/CD40L peptides (Table 1c) in thecontrols (P = 0·001) and CD (P = 0·009) but not UC. Inhi-bition of IL-12 by the CD40/CD40L peptides was again sim-ilar to that for TNF-α, with significant inhibition of DC fromcontrol subjects (P = 0·009) or CD (P = 0·004), but not UC(Table 1d).

P38 and ERK 1/2 phosphorylation MAP kinases

Monocyte-derived DC from CD patients stimulated withCD40L showed a significant increase in TNF-α production(Fig. 1a) but a significant decrease in IL-12 concentration(Fig. 1b). A similar response, although not statistically signif-icant, was also seen with UC (Fig. 1a,b). This dissociatedresponse between TNF-α and IL-12 production was

Fig. 1. Stimulation of monocyte-derived den-

dritic cells (DC) with heat shock protein

(HSP)70, CD40L or lipopolysaccharide (LPS)

producing (a) tumour necrosis factor (TNF)-α

and (b) interleukin (IL)-12 in Crohn’s disease

(CD) � (n = 16 and 12), ulcerative colitis (UC)

(n = 9 and 8) and healthy controls � (n = 13

and 14), respectively; statistical analyses were

performed between CD and controls and

UC and controls for each stimulant (*P < 0·05,

**P < 0·02). Stimulation of monocytes in these

groups producing (c) TNF-α (n = 10–16) and

(d) IL-12 (n = 7–9), respectively.

2000

4000

6000

8000

10000

500

2500

1000

3000

1500

2000

(b)(a)

TN

F-α

(m

ean

+ s

.e.m

.) p

g/m

l

IL-1

2 (m

ean

+ s

.e.m

.) p

g/m

l

TN

F-α

(m

ean

+ s

.e.m

.) p

g/m

l

IL-1

2 (m

ean

+ s

.e.m

.) p

g/m

l

**

* *

200

400

600

800

1000

HSP70 HSP70CD40L CD40LLPS LPS

1000

1500

2000

(d)(c)

T. Whittall et al.

554 © 2006 British Society for Immunology, Clinical and Experimental Immunology, 143: 550–559

investigated further by the ERK 1/2 and p38 phosphorylationMAP kinase pathways of DC from patients with CD. DCstimulated by HSP70, CD40L or LPS elicited a dose-depen-dent inhibition of TNF-α production with the ERK 1/2 inhib-itor (PD098059) (Fig. 2d,e,f), but not with the p38 inhibitor(SB023580) (Fig. 2a,b,c). However, production of IL-12 wasdissociated when the DC were stimulated with HSP70 orCD40L as a dose-dependent decrease of IL-12 (Fig. 2a,b)resulted with the p38 inhibitor (Fig. 2a,b), whereas ERK 1/2inhibitor enhanced IL-12 production (Fig. 2d,e). The effectof the ERK inhibitor on LPS-induced IL-12 production wasinconclusive. The data presented in Fig. 2 are themean ± s.e.m. of three experiments. These responses did notappear to be specific to DC from CD, as those from normalcontrol subjects showed similar results (data not presented).

Stimulation of TNF-αααα and IL-12 production by HSP70, CD40L or LPS of mononuclear cells eluted from intestinal biopsy specimens of patients with CD, UC and normal subjects

Eluted cells from five normal colonic mucosal specimensfailed to be stimulated by HSP70, CD40L or LPS to produceTNF-α (0–3·5 pg/ml). In CD and UC the mucosal tissueswere divided into inflamed and uninflamed, based on endo-scopic appearance and then histological examination. In CDstimulation of eluted cells from uninflamed mucosal tissuewith the three reagents produced very little TNF-α; themeans were 7·3 ± 3·4, 8·7 ± 4·9 and 12·7 ± 4·7 pg/ml, respec-tively (Fig. 3a). However, cells eluted from the inflamedmucosa of patients with CD, compared with those from

uninflamed mucosa, produced significantly increasedconcentrations of TNF-α when stimulated by HSP70(41·7 ± 13·7 pg/ml, P = 0·001), CD40L (41·0 ± 14·7 pg/ml,P = 0·055), which is just over the significant level, and LPS(51·9 ± 21·0 pg/ml, P = 0·007).

Cells eluted from UC mucosa produced similar concen-trations of TNF-α to CD cells when they were stimulatedwith the three reagents (Fig. 3b). However, a significantlyhigher concentration of TNF-α (P < 0·05) was found withmucosal cells eluted from inflamed compared with thosefrom uninflamed mucosa when stimulated with CD40L butnot with HSP70 or LPS (Fig. 3b). Intraepithelial and subep-ithelial (lamina propria) cells were not studied separately, asthe specimens of mucosal tissues were inadequate to yield asufficient number of cells.

IL-12 production was then evaluated by stimulating thecells eluted from inflamed colonic mucosa of CD or UC. Theconcentration of IL-12 was greater in cells from CD than UCwhen stimulated with HSP70, CD40L or LPS, but none ofthese differences reached significant levels (Table 2a).

Inhibition of HSP70-stimulated TNF-αααα and IL-12 production by cells eluted from intestinal biopsy specimens by peptides derived from HSP70 (p457–496) or CD40/CD40L (p27–36/p39–58 and 102–119)

Both peptides inhibited TNF-α production by more than50% from a mean of 41·9 ± 14·1 to 10·8 ± 4·8 pg/ml(P = 0·016) with p457–496, and from 41·9 ± 14·1 to15·2 ± 5·1 pg/ml with p27–36/39–58/p102–119 (P = 0·018),when the cells eluted from inflamed mucosa of CD were

Table 1. The effect of heat shock protein (HSP)70-stimulated dendritic cells (DC) on (a) tumour necrosis factor (TNF)-α and (b) interleukin

(IL)-12 production when treated with p457–496 derived from HSP70, and (c) TNF-α, and (d) IL-12 production when treated with pCD40 (p27–36/

39–58) and pCD40L (p102–119).

n

Mean ± s.e.m. pg/ml

Before After t P

(a) Effect of p457–496 on TNF-α1 Controls 13 187 (64) 78 (41) 4·15 0·001

2 Crohn’s disease 13 308 (85) 153 (54) 2·67 0·020

3 Ulcerative colitis 11 330 (162) 274 (170) 1·84 0·096

(b) Effect of p457–496 on IL-12

1 Controls 9 436 (159) 246 (119) 4·06 0·004

2 Crohn’s disease 10 523 (178) 301 (119) 2·27 0·050


(c) Effect of pCD40/CD40L on TNF-α1 Controls 13 192 (63) 88 (43) 4·06 0·001

2 Crohn’s disease 13 309 (90) 131 (45) 3·1 0·009


(d) Effect of pCD40/CD40L on IL-12

1 Controls 5 212 (43) 113 (24) 4·62 0·009

2 Crohn’s disease 10 405 (134) 249 (105) 3·81 0·004


Dendritic cell maturation and TNF-α stimulated by HSP70 in Crohn’s disease

© 2006 British Society for Immunology, Clinical and Experimental Immunology, 143: 550–559 555

stimulated with HSP70 (Fig. 3b). Cells from uninflamedmucosal tissue produced little TNF-α, but even this wasinhibited significantly (Fig. 3b). Cells eluted from bothinflamed and uninflamed mucosal tissues of UC also showedinhibition of TNF-α production with the two sets of

peptides, but surprisingly, significant inhibition was foundonly in those from uninflamed mucosa (Fig. 3d).

Similar investigation of HSP70 induced IL-12 production(Table 2a) showed significant inhibition by both p457–496(P = 0·022) and pCD40/CD40L (P = 0·023) for cells eluted

Fig. 2. Monocyte-derived dendritic cells (DC)

from Crohn’s disease stimulated with heat shock

protein (HSP)70, CD40L or lipopolysaccharide

(LPS) to produce tumour necrosis factor (TNF)-

α and interleukin (IL)-12p40, and treated with

increasing concentrations of p38 inhibitor

(SB023580) (a, b, c) or extracellular signal regu-

lated kinase (ERK) inhibitor (PD098059) (d, e,

f); presented as mean ± s.e.m. pg/ml.

200

100

0

TNF-α

IL-12

HSP70(a) (d)

(b) (e)

(c) (f)

2000

3000

1000

0

TNF-αIL-12

CD40L

300

200

100

0 TNF-α

IL-12

HSP70

1000

2000

3000

4000

00 1 5 25 50

TNF-αIL-12

LPS

0100020003000400050006000700080009000

TNF-α

IL-12

CD40L

1000

2000

3000

4000

5000

00 1 5 25 50

TNF-α

IL-12

LPS

P38 inhibitor concentration µg/ml

Con

cent

ratio

n pg

/ml

ERK inhibitor concentration µg/ml

Table 2. Stimulation of interleukin (IL)-12 production by heat shock protein (HSP)70, CD40L or lipopolysaccharide (LPS) of mononuclear cells eluted

from colonic biopsy specimens from patients with Crohn’s disease or ulcerative colitis, and inhibition of HSP70-stimulated cells with p457–496 or

pCD40/CD40L.

Mucosal cells n

Concentration of IL-12, mean ± s.e.m. pg/ml

n

HSP70-stimulated cells inhibited with

P457-496 PCD40/CD40L

HSP70 CD40L LPS Pre- Post- Pre- Post-

(a) Crohn’s disease

Uninflamed 8 24·9 (10·2) 39·9 (11·6) 32·6 (11·9) 8 20·3 (8·0) 2·8 (1·8) 20·3 (8·0) 6·6 (3·8)

Inflamed 10 28·1 (7·3) 30·9 (12·4) 58·0 (30·5) 10 24·3 (8·0) 6·6 (2·9) 24·3 (7·2) 11·5 (2·9)

P-value 0·753 0·396 1·0 Uninflamed

Inflamed

0·061

0·022

0·043

0·023

(b) Ulcerative colitis

Uninflamed 6 6·1 (2·8) 7·2 (5·1) 16·2 (8·9) 6 19·2 (12·1) 4·5 (2·9) 19·2 (12·1) 5·0 (2·9)

Inflamed 5 19·9 (12·7) 3·4 (3·2) 23·2 (14·4) 5 17·3 (9·2)

Uninflamed

Inflamed

5·8 (4·2)

0·18

0·30

17·3 (9·2)

0·19

0·17

12·5 (7·2)

T. Whittall et al.


Fig. 3. Stimulation by heat shock protein

(HSP)70, CD40L or lipopolysaccharide (LPS) of

tumour necrosis factor (TNF)-α production by

cells eluted from mucosal biopsies from patients

with (a) Crohn’s disease (inflamed n = 13 and

uninflamed n = 13) or (b) ulcerative colitis

(inflamed n = 8, uninflamed n = 12) and inhibi-

tion of HSP70-stimulated cells with p457–496 or

pCD40/CD40L (c) in Crohn’s disease (inflamed

n = 11, uninflamed n = 12) and (d) in ulcerative

colitis (inflamed n = 7, uninflamed n = 11).

HSP70

0·264 0·029 0·061 0·032 0·025

0·040·030·0180·0160·0070·0550·001

0·11 0·087

95

80

70

60

50

40

30

20

10

+++ _ _InflamedP value

CD40L LPS

P value

Crohn’s Disease

Ulcerative Colitis

CD40/CD40L

457-496

CD40/CD40L

457-496

+ + +_ _ _Inflamed

+ ++ _ __

+ + + _ __

_

70

60

50

40

30

20

10

(a)

(c)

(b)

(d)

Fig. 4. A novel strategy for inhibition of CD40–CD40L or CD40–heat

shock protein (HSP)70 interaction by peptides derived from HSP70 or

CD40 and CD40L, stimulating production of tumour necrosis factor

(TNF)-α, interleukin (I)-12 and CCL-5.

CD40L

CD40

HSP70

T Cell

Inhibition by peptides:HSP70 (aa 457-496)CD40 (27-36/39-58)CD40L (102-119)

Dendritic Cell

TNF-α

IL-12

CCL-5

from inflamed mucosa of CD; cells from uninflamed mucosaalso showed significant inhibition with pCD40/CD40L.However, significant changes in IL-12 production were notfound with cells eluted from UC (Table 2b). It should benoted that the enzyme treatment of mucosal specimens usedto elute cells did not affect the functional capacity of thetreated cells to generate TNF-α or IL-12, as equally treatedPBMC, stimulated with HSP70, CD40L or LPS showed sim-ilar production of these cytokines as untreated PBMC (datanot presented).

Discussion

DC have been accorded a central role in the interphasebetween immunity and tolerance [33,34]. We have pursuedthe present study of DC in Crohn’s disease with special



of interest that mycobacterial and other microbial HSP70show extensive homology with human HSP70 [46] andmycobacteria have been implicated directly in the pathogen-esis of CD [5,6]. Furthermore, microbial HSP70 elicitschemokines and cytokines via the CD40 co-stimulatory mol-ecules [28,29], and these may affect host immunity. Indeed,the alternative HSP70–CD40 co-stimulatory pathway hasbeen invoked in priming T cells, in the control of tubercu-losis [44] and in converting T cell tolerance to autoimmunity[45].

It was of interest to find out if there is any homologybetween the mycobacterial p457–496 and that found inhuman HSP70. Aligning the mycobacterial (457–496) withhuman HSP70 (p485–524) sequences revealed that 29/40(73%) residues are conserved. However, the critical residueswithin the 40mer peptide have not, as yet, been defined. Thismight be significant, as the mycobacterial N-terminal por-tion of the inhibitory peptide (aa 457–471) shows that 12/15(80%) residues are identical or conserved when comparedwith those of the human HSP70 (aa 485–499). The C-termi-nal portion (aa 480–494) shows 15/15 (100%) homology.

An alternative strategy to inhibiting TNF-α and IL-12stimulated by the CD40–CD40L or CD40–HSP70 pathwaywas to use peptides derived from the CD40, N-terminal tan-dem repeat (aa 27–36 and 39–58) and from the CD40L cyto-plasmic tail (aa 102–119). Significant inhibition ofproduction of both cytokines was found with these two pep-tides in DC from CD and healthy controls, but not UC. ThusTNF-α and IL-12 production by HSP70-stimulated DCfrom CD can be inhibited by a peptide derived from HSP70that targets CD40 [30] and by peptides from the CD40 andCD40L molecules.

It was important to establish whether cells eluted fromintestinal mucosa affected by CD respond to stimulationwith HSP70, CD40L and LPS in a similar manner to thatfound with PBMC. Indeed, cells eluted from the inflamedtissue when stimulated with HSP70, CD40L and LPS showeda significant increase in TNF-α production in CD but notUC. As both CD and UC are mucosal ulcerative lesions, theraised TNF-α concentration is unlikely to be due to a greaterinflux of microbial HSP70 through the damaged mucosa,increasing stimulation of TNF-α production. We favour theconcept that the increased number of CD40+ cells in CDenables microbial HSP70 to potentiate TNF-α production.We also studied the effect of the two sets of inhibitory pep-tides on the HSP70-stimulated cells eluted from CD and UC.Significant inhibition of TNF-α and IL-12 production wasfound, with both p457–496 and p27–36/39–58/102–119using eluted cells from inflamed CD but not UC mucosa.The small number of cells eluted from the intestinal biopsyspecimens obliged us to use the entire cell population, unlikethe circulating cells from which monocyte-derived DC wereisolated. Cells eluted from the mucosal tissues contain epi-thelial cells, in addition to lymphocytes, macrophages, DCand neutrophils, and these may have a differential effect on

reference to responses to HSP70, which is found in Gram-positive and Gram-negative gut organisms. As CD40 is areceptor for microbial HSP70 [28], the CD40L (CD154) tri-mer was used as a positive control. LPS is present in Gram-negative gut organisms and binds to CD14 [35], and wastherefore used as a relevant CD40 negative control.

Stimulation by HSP70 of monocyte-derived DC showedsignificant increases in TNF-α and IL-12 production in CDbut not UC. However, stimulation with CD40L resulted in asignificant increase in TNF-α but a decrease in IL-12 in DCfrom CD. The dissociation between TNF-α and IL-12 pro-duction by stimulating DC from CD with CD40L might beaccounted for by the ERK 1/2 phosphorylation MAP kinasepathways, as the inhibitor enhanced IL-12, while inhibitingTNF-α production. The increase in TNF-α concentrationproduced by DC from CD stimulated with HSP70 or CD40Lis consistent with that found in the literature [21–23].Although a decrease in IL-12 production by stimulation withCD40L, but not with HSP70, in the circulating monocyte-derived DC was demonstrated in the present study, a slightincrease in IL-12 was found with cells eluted from inflamedtissues and this is consistent with the data found in the lit-erature [17,18]. It should be pointed out that HSP70 stimu-lated relatively modest amounts of TNF-α and IL-12,compared with those stimulated by CD40L or LPS.

There is a great deal of evidence that the CD40–CD40L co-stimulatory pathway is involved in the immunopathogenesisof inflammatory bowel disease [36]. An increase in CD40+

cells was reported in mononuclear, endothelial and mesen-chymal cells from patients with intestinal lesions in CD andUC [37–39]. CD40L+ CD4+ cells and some CD8+ T cells werealso increased in CD and UC [38,40]. The proportions of cir-culating CD40+ lymphocytes and monocytes are alsoincreased in patients with CD [41]. Furthermore, colitis wasreported in a transgenic CD40L mouse model [42]. BecauseHSP70 may function as an alternative to CD40L in the inter-action with CD40 [28,29,43–45], this may enhance thealready heightened CD40–CD40L interaction in CD. CD40–CD40L and the alternative CD40–HSP70 pathway are espe-cially relevant in the intestinal mucosa, which is exposed toHSP70 from gut bacteria. These potent co-stimulatory path-ways provide a mechanism for recruitment of T and B cells,monocytes and DC into the mucosa and production ofproinflammatory cytokines, which may maintain chronicinflammation, especially in genetically susceptible subjects.

As TNF-α is critical in the control of CD and autoimmunediseases [27], and HSP70-derived peptide 457–496 inhibitsTNF-α production by DC from healthy control subjects[30], we explored the possibility that p457–496 might alsoinhibit TNF-α in CD. Indeed, addition of p457–496 toHSP70 significantly inhibited TNF-α production by DCfrom CD but not those from UC. It is noteworthy that p457–496 inhibits TNF-α production to below the level found inhealthy controls, but did not prevent TNF-α productioncompletely, thereby allowing normal function of TNF-α. It is

T. Whittall et al.


TNF-α and IL-12 production, especially when comparingepithelial cells from inflamed and uninflamed tissues. How-ever, irrespective of any contributory role of epithelial cells,TNF-α and IL-12 production were inhibited significantly byboth the HSP70-derived peptide (aa 457–496) and CD40/CD40L peptides (aa27–36/39–58/102–119), which may havea therapeutic effect in CD. Further work is required to estab-lish whether mucosal DC are involved in these changes, butthe results are consistent with DC playing a role in theimmunopathogenesis of CD.

An increase in TNF-α production was found to be stim-ulated by HSP70 or CD40L, with both mucosal cells and cir-culating monocyte-derived DC. The inhibition experiments,targeting the CD40-CD40L or CD40-HSP70 co-stimulatorypathway, raise the possibility that the two sets of inhibitorypeptides may offer an alternative strategy for inhibition ofTNF-α production in CD by direct rectal or oral adminis-tration of either peptide, or possibly both sets of peptides(Fig. 4). This peptide inhibitory strategy might preventexcessive TNF-α production, compared with anti-TNF-αantibodies that neutralize any TNF-α already produced.Such an approach may complement existing treatment withmonoclonal antibodies to TNF-α, which have to be given byinjection, and usually with cytotoxic drugs.

Acknowledgements

This work was supported by the Guy’s & St Thomas’ Char-itable Foundation. We wish to thank Drs Wendy Clarke,Abdul Mohsen and Bijay Baburajan in helping with theintestinal biopsy specimen.

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Documents

Tumour necrosis factor-alpha production stimulated by heat shock protein 70 and its inhibition in circulating dendritic cells and cells eluted from mucosal tissues in Crohn's disease