basal, lipopolysaccharide (lps) and silica-stimulated

Lipopolysaccharide andsilica-stimulated mononuclear cellprostaglandin production inulcerative colitis

Neville A. Punchard1, CA, John Cason2,Jonathan Mullins1, Chaman Chander1 andRichard P. H. Thompson2

1Department of Biology and Health Science, Facultyof Science, Technology & Design, University ofLuton, Park Square, Luton LU1 3JU, UK; and2Gastrointestinal Laboratory, The Rayne Institute, StThomas’ Hospital, London SE1 7EH, UK

CA Corresponding AuthorTel: 01582 489212Fax: 01582 734111Email: [email protected]

BASAL, lipopolysaccharide (LPS) and s ilica-stim ulatedprostaglandin (PG) production were com paredbe tw een peripheral blood m ononuclear ce lls(PBMNC) from UC patien ts and healthy subjects (HS).Basal and LPS-stim ulated PBMNC PGI2 , but not PGE2 ,production w as greater in UC. LPS stim ulated bothPGE2 and PGI2 by PBMNC from HS and UC patien ts .Silica s tim ulated production of both PGs by cells fromHS but on ly PGE2 by ce lls from UC patien ts . Th edifferences in response s to s ilica and LPS m ay re sultfrom difference s in activation of NF k B or, alte rna-tively, pr ior sensitisation to one of th ese agents . Th atPBMNC PGE2 production is not in creased in UC, as itis in Crohn ’s disease, sugges ts that the re are differ-ences in PBMNC behaviour between the se twodisease s .

Key w ords: Colitis, Leukocytes, Mononuclear, Prosta-glandins, Lipopolysaccharide, Silica, NFkB

Introduction

The accumulation of leukocytes that produce prosta-glandins (PG), specifically mononu-clear cells (MNC),within the inflamed mucosa is closely associatedwith, and responsible for, the elevated levels of PGsreported in luminal contents and mucosal incubationsin inflammatory bowel disease (IBD). However, therehave been relatively few studies on MNC PG produc-tion in IBD, and only one of peripheral blood MNC(PBMNC) PG production in UC.1 In addition, differentstimuli have different effects on PG production andprevious studies have either measured unstimulatedproduction or used only lipopolysaccharide (LPS).There have been no previous studies into thedifferences in MNC cell responses to various stimuliin IBD.

Thus the aim of this study was to investigatewhether the production of both stimulated andunstimulated PG production by peripheral blood MNcells (PBMNC) is increased in UC in response to twodifferent stimuli, namely silica and LPS. PG produc-tion was measured as PGE2 and PGI2 , as differentialproduction of cyclo-oxygenase products by differentstimuli is well recognised.

Materials and methods

PBMNC were separated from 21 UC patients (14 male,median age 36 years, range 22–84) and 14 healthysubjects (controls; 10 male, median age 30 years,range 21–41).2 Healthy subjects (HS) had not takenany medication, including non-steroidal agents, for atleast five days prior. Of the 21 patients: 13 hadinactive disease;3 two [inactive] were receiving notreatment; 10 were receiving sulphasalazine, and 12corticosteroids. The study was approved by the StThomas’ Ethical Committee and all patients gaveinformed written consent.

Heparinised, peripheral venous blood samples(40 ml) were centrifuged at 200 g for 10 min. Theplasma was then discarded and the cells resuspendedto the original volume in Dulbecco’s calcium, magne-sium-free phosphate buffered saline and RPMI–1640tissue culture medium, 1:1 (v/v) mixture with addi-tives.2 Ten ml was then layered onto 8 ml FicollHypaque and centrifuged at 400 g for 30 min at roomtemperature.

PBMNC were aspirated, washed three times andresuspended in 15 ml of the same solution. Aliquots ofcells were taken for total cell counts, viability (trypan

ISSN 0962-9351 print/ISSN 1466-1861 online/00/030189-03 © 2000 Taylor & Francis Ltd 189DOI: 10.1080/09629350020002903

Short Communication

Mediators of Inflammation, 9, 189–191 (2000)

blue exclusion) and percentage monocyte composi-tion (non-specific esterase staining). Basal (unstimu-lated), LPS (10 mg/ml) – stimulated or silica (Gasel 23,Crossfields Chemicals, Warrington, Cheshire) – stimu-lated incubations of 1 ´ 106 viable PBMNC ml–1, wereperformed in 50 mmol l–1 2-mercaptoethanol supple-mented RPMI–1640 medium, at 37°C, in a humidifiedatmosphere of 5% CO2 in air, for 24 h. Incubationswere centrifuged at 350 ´ g for 7 min at 4°C and cell-free supe rnatants store d at –20°C for le ss than tw omonths .

Sample s we re assayed, in duplicate , by radio-immunoassay.2 Measures of quality control w erew ithin pre viously de termined value s and comple teinhibition of PG production by indomethacin con-firmed assay specificity.

The Wilcoxon 2-sample te st for paired data w asused to te st w hether stimulation has an e ffe ct on PGproduction, and the Mann-Whitney te st for unpaireddata used for comparisons be tw een groups.

Results

Basal PGI2 production by PBMNC from UC patientswas greater than that by cells from HS. LPS stimula-tion increased both PGE2 and PGI2 production byPBMNC from patients with UC and HS (Table 1).Stimulation with silica increased PGE2 production bycells from patients with both UC and HS but onlystimulated PGI2 production by cells from HS. Therewas no difference in basal PGE2 production byPBMNC from patients with UC and HS.

Stimulated PG production was independent ofbasal production, with no correlation between basaland either LPS-, or silica-stimulated production in UCpatients or HS. Thus PG production in response to

stimulation alone, derived by subtracting basal fromstimulated PG levels, was compared, thereby possiblyeliminating any effects of stimulation arising from ordue to preparation techniques. After correcting forbasal production, there was no difference in eitherLPS- or silica-stimulated PGE2 production between HSand UC patients, or in LPS-stimulated PGI2 produc-tion. However, PBMNC from UC patients producedsignificantly less silica-stimulated PGI2 than did cellsfrom HS.

No effects of disease activity, or either steroid orsulphasalazine therapy, on PG production were dis-cernible. There were no differences in viability, purityor percentage monocyte composition of the PBMNCpreparations from UC patients and HS; thus, inagreement with previous studies, any differences donot arise from different numbers of monocytes in theincubations.

Discussion

PBMNC from patients with UC had an increasedability to synthesise PGI2 but not PGE2 . Unlike cellsfrom HS, PBMNC from UC patients failed to increasePGI2 production in response to silica. This responseappeared to be specific to PGI2 as cells from both UCpatients and HS increased PGE2 production inresponse to silica. The previous lack of detection ofPGI2 production by PBMNC in UC,1 whereas thesecells have been widely reported to produce thiseicosanoid, may result from differences in incubationor preparation techniques, or different assay sensitiv-ities, all of which have been reported to influence thedetection of PGs.

In agreement with previous studies of peripheralblood and intestinal MN cells, PBMNC production ofPGE2 by was not increased in UC.1,4 Although,

N. A. Puncha rd et al.

190 Mediators of Inflammation · Vol 9 · 2000

Table 1. PG production in response to stimulation with lipopolysaccharide (LPS) and silica byPBMNC from patients with ulcerative colitis (UC), compared with that by cells from healthysubjects (HS), with and without correction for basal production. Data expressed as medians andlower to upper quartiles

Prostaglandin pg/106 PBMNC; median (lower–upper quartiles)

Basal or stimulated HS UC

PGE2 Basal 380 (268–458) 439 (324–557)LPS e652 (497–888) e745 (563–884)LPS – basal 260 (182–452) 326 (141–390)Silica e685 (512–793) c702 (524–849)Silica – basal 318 (129–398) 166 (58–278)

PGI2 Basal 110 (63–132) **156 (122–183)LPS c137 (106–182) e183 (148–223)LPS – basal 27 (12–38) 44 (16–82)Silica c150 (108–192) 161 (150–183)Silica – basal 43 (17–92) *13 (–22–37)

Lipopolysaccharide (LPS) or silica stimulation vs none (basal): c = p < 0.005, d = p < 0.002, e = p < 0.001.Ulcerative colitis (UC) vs healthy subjects (HS): * = p < 0.05, ** = p < 0.005. PGI2 measured as 6KF1a.

previous studies have identified effects of sulphasala-zine on PBMNC PG production in vitro , these werenot confirmed in patients, possibly owing to con-founding factors of disease activity and othertreatments.2

The different effects of the two stimulants upon thecyclo-oxygenase pathway may arise from the differenteffects of LPS and silica on PBMNC NFkB activation,6,7

involved in regulation of cyclooxygenase activity.Increased levels of NFkB activation occur in MN cellsin the bowel.5 Alternatively, silica has been implicatedin the pathogenesis of IBD,8 while there could beincreased passage of endotoxins across the inflamedbowel. Moreover, previous studies in animals haveshown that prior exposure in v ivo to such stimulantscan effect responses on cells in v itro .9,10 Theresponse of PBMNC to silica is selectively altered inUC, which may indicate changes in cellular activationof MNC, while the differences in PG productionbetween CD and UC may indicate some intrinsicdifferences in MNC activity in the two IBDs.

ACKNOWLEDGEMENTS. The authors would like to thank Nick Taub,Department of Public Health Medicine, St Thomas’ Hospital for his statisticaladvice and analysis. They are also grateful to Dr B. Creamer for allowing themto study patients under his care. They would like to thank the Special Trusteesof St Thomas’ Hospital, the National Association for Crohn’s and Colitis, andthe South East Thames Regional Health Authority for financial assistance.

References1. Rachmilewitz D, Ligumsky M, Haimovitz A, Treves AJ. Prostanoid synthe-

sis by cultured peripheral blood mononuclear cells in inflammatorydiseases of the bowel. Ga s tro e nte ro lo g y 1982; 82:673–9

2. Punchard NA, Boswell DJ, Greenfield SM, Thompson RPH. The effects ofsulphasalazine and its metabolites on prostaglandin production byhuman mononuclear cells. Bio chem Pharm a co l 1992; 45:2369–76

3. Truelove SC, Witts LJ. Cortisone in ulcerative colitis. Final report on atherapeutic trial. BMJ 1955; 2:1041–8

4. Zifroni A, Treves AJ, Sachar DB, Rachmilewitz D. Prostanoid synthesis bycultured intestinal epithelial and mononuclear cells in inflammatorybowel disease. Gut 1983; 24:659–64

5. Ellis R., Goodlad JR, Limb GA, Thompson RPH, Punchard NA. Activationof NFkB in Crohn’s Disease. Infla m m Re s 1998; 47:440–5

6. Chen F, Sun S-C, Kuh DC, Gaydos LJ, Demers LM. Essential role of NFkBactivation in silica-induced inflammatory mediator production in macro-phages. Bio chem Bio phy s Re s Co m m un 1995; 214:985–92

7. Chen F, Kuh DC, Sun S-C, Gaydos LJ, Demers LM. Dependence andreversal of nitric oxide production on NFkB in silica and lipopolysacchar-ide-induced macrophages. Bio chem Biophy s Re s Co m m un 1995;214:839–46

8. Powell JJ, Harvey RS, Thompson RPH. Microparticles in Crohn’s disease:has the dust settled?. Gut 1996; 39:231–3

9. Rogers TS, Halushka PV, Wise WC, Cook JA. Differential alteration oflipoxygenase and cyclooxygenase metabolism by rat peritoneal macro-phages induced by endotoxin tolerance. Pro s tag landin s 1986;31:639–50

10. Cantin A, Dubois F, Begin R. Lung exposure to mineral dust enhances thecapacity of lung inflammatory cells to release superoxide. J Le uko cy teBiol 1988; 43:299–303

Received 14 March 2000;accepted 23 May 2000

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