Changes in eicosanoid necrosis · 2019. 8. 1. · W. M. Pruimboomet al. Cellisolation: Onday 1...

Research Paper

Mediators of Inflammation 3, 335-340 (1994)

CrIAGES and correlations in cytokine and eicosanoid pro-duction by blood monocytes, non-purified and purifiedperitoneal cells during a carrageenin-induced peritonitiswere investigated for a period of ten days. The cells wereisolated and stimulated in vitro. Cytokine and eicosanoidproduction of the non-purified fraction increased stead-ily during peritonitis. During the whole episode of peri-tonitis the production capacity of granulocytes was verylow and hardly any effect on the production capacityof macrophages (M) was observed. Cytokine andeicosanoid production of the non-purified fraction wasmainly due to the presence ofM. The production capac-ity of the peripheral blood monocytes was not similar tothat of the peritoneal M.

Key words: Eicosanoids, Peritoneal macrophages, Peritonitis,Tumour necrosis factor-

Changes in eicosanoid and tumournecrosis factorproduction by ratperitoneal macrophages duringcarrageenin-induced peritonitis

W. M. Pruimboom,1,cA A. Verdoold,C. J. A. M. Tak, A. P. M. van Dijk,M. van Batenburg, J. H. P. Wilson, andF. J. Zijlstra

Dept of Pharmacology, Erasmus University,Rotterdam and Dept of Internal Medicine II,University Hospital Dijkzigt Rotterdam, TheNetherlands

CA Corresponding Author

Introduction

Inflammation is the reaction to an injury such as aninvasion by infectious agents. The blood supply tothe inflamed area increases, venular permeabilityincreases and leucocytes migrate out of venules intothe surrounding tissue. At the site of an acute inflam-mation polymorphonuclear granulocytes (PMNs)produce large amounts of reactive oxygen interme-diates, release a variety of hydrolytic enzymes andphagocytose pathogens. By generating chemotacticfactors, the migration of mononuclear cells(monocytes/M and lymphocytes) to the site of in-flammation is stimulated. ’,3 During the chronic phaseof the inflammatory response, M, mononuclearphagocytes derived from blood monocytes, pre-dominate over PMNs. In addition to acting as anonspecific defence, activated M5,6 will initiate andcontrol the specific defence by presenting processedantigens to lymphocytes and by cytotoxity throughdirect cell-cell contact with tumour cells or infectedcells,8,9 during which they secrete cytokines andeicosanoids. 1,11 These inflammatory mediators alsoinfluence and regulate functions of other cells partici-pating in the immune response,l’aCytokines are proteins with regulatory functions.

They can induce other cytokines with overlappingeffects and form interactive networks with hormonesand eicosanoids. M are a potent source of a widevariety of cytokines,4 whereas granulocytes are re-ported to produce only a limited spectrum ofcytokines. 15-17

( 1994 Rapid Communications of Oxford Lid

Eicosanoids are bioactive lipids derived fromarachidonic acid (AA) by cyclooxygenase andlipoxygenase pathways.8a9 Eicosa.noid production isspecies and tissue dependent, and often stimulusdependent.2,21

The aim of this work was to study cytokine(tumour necrosis factor-m, TNF) and eicosanoid(leukotriene B4, LTB4; prostaglandin E2, PGE2;prostacycline, detected as 6-keto-prostaglandin Fire6kPGF=; and thromboxane, TXB,) production capac-ity of peritoneal PMNs, M and blood monocytesafter the induction of peritonitis with a carrageeninsolution, a sulfated polygalactan which stimulatescell-mediated immunity.22 It was of interest to deter-mine whether, during an episode of peritonitis,changes occurred in (a) the differentiation ofinfluxed cells; (b) the cytokine and eicosanoid pro-duction capacity of blood monocytes and peritonealcells; (c) interactions between different types ofperitoneal cells concerning their production capacity;and (d) if there was a correlation between the pro-duction of eicosanoids and TNF by bloodmonocytes and peritoneal M.

Materials and Methods

Animals and treatment: Young male Wistar rats (13weeks, approximately 200 g, six rats per group) wereinjected intraperitoneally with 2 ml of a carrageeninsolution (Marine Colloids Inc., USA, 1 mg/ml) on day0. Six rats received an injection of saline (controlgroup).

Mediators of Inflammation. Vol 3. 1994 335

W. M. Pruimboom et al.

Cell isolation: On day 1 (that is, 24 h after inducingthe peritonitis with the carrageenin solution), three,seven and ten peritoneal cells and monocytes fromthe blood were isolated. With each group, cells werealso isolated from a control animal, which was con-sidered as day 0. Blood (_+ 7 ml) was collected intubes with 1 ml EDTA (0.1 M) after decapitation. Theblood was centrifuged (400 xg, 4C, 10 min) andfrom the leucocyte layer the monocytes were isolatedinto a ’monocyte’ fraction by density gradientcentrifugation using Percoll (d 1.064 g/ml,Kabi-Pharmacia, Sweden; 400xg, 4C, 25 min).Monocytes were washed three times with phosphatebuffer solution (PBS, 400 xg, 4C, 10 min) and sus-pended in Dulbecco’s modification of Eagle’s me-dium (DMEM + HEPES (GIBCO, UK)+ penicillin/streptomycin (5 x 10 U/1 per 50 mg/1, Flow Lab,UK) + foetal calf serum (10% FCS, GIBCO, UK) + L-

glutamine (600 mg/1 Flow Lab, UK), 1 x 106 cells/ml).The peritoneal cavities, were washed twice with

20 ml PBS (pH 7.4, 4C, Oxoid, UK). The washingsper rat were pooled, centrifuged (400 xg, 4C, 10min) and suspended in 5 ml DMEM. 5 x 106 cells ofthis non-purified fraction were kept separate, the restof this ’crude’ fraction was separated on Percoll intoa ’macrophage’ and a ’granulocyte’ fraction. Thesetwo purified fractions were washed three times withPBS (400 xg, 4C, 10 min), and suspended in DMEM(i x 106 cells/ml).A small sample of cells from each fraction was

stained by Hemacolor (Merck, Germany) and thedifferent cell types were counted using a microscope(Zeiss, standard 25, Germany). The viability of thecells was determined by trypan blue exclusion.

Cell incubation: One million leucocytes per mlDMEM were plated on plastic culture dishes (Costar,UK). The cells were triggered for 15 min by calciumionophore A23187 (Calbiochem, USA), 1 t.tM finalconcentration in dimethylsulfoxide (0.1% DMSO,Sigma, USA), 37C, 7.5% CO,.). Controls were incu-bated with DMSO (0.1%).The cells were also incubated for 24 h in the

absence or presence of lipopolysaccharide (LPS,10 l.tg/ml final concentration in PBS, LPS from E. coli0111:B4 in PBS (Sigma, USA), 37C, 7.5% COg). As ablank, PBS was added. At the end of the incubationthe supernatant was centrifuged and kept at -80Cuntil required for analysis.

Cytokine production: TNFc production in the sam-ples was determined directly in the supernatant bybioassay. For this bioassay the TNF0t sensitive cellline WEHI-164 was used. The WEHI-164 cells wereplated out in 96-well plates (2 x 10 cells/50 btl/well,Costar, UK) and the samples (50 l.d/well) or thehuman recombinant TNFz (hr-TNFc) standards(0.1-1000u/ml hr-TNFc, 50 l.d/well) were added.After 24h incubation (37C, 7.5% CO2), MTT

336 Mediators of Inflammation Vol 3. 1994

(tetrazolium salt, Sigma, USA) was added (0.125 mg/well) and after an incubation of 3 h the cells werelysed with buffer (20% sodium dodecyl sulfate (SDS)in 50% N,N-dimethylformamide (DMF), pH 4.7,100 l.d/well) for 18 h. The absorbance was measuredat 595 nm with an ELISA reader (BIO-RAD, model3550, UK). The TNFz production by the Me wasexpressed as the cytotoxicity against WEHI-164 cellscompared to the blank (DMEM complete).23

Eicosanoid production: Eicosanoid production(LTB4, PGE2, TXB2 and 6kPGFI= from endogenousarachidonate of the samples was determined directlyin the supernatant by radioimmuno assays (antibod-ies were obtained from Advanced Magnetics, USA;standards from Sigma, USA; and tritiated antigensfrom Amersham, UK). Cross-reactivities for individualantigens on antibodies were negligible.

Statistical analysis: Data are expressed as the mean_+ standard error of the mean (S.E.M.). Data wereanalysed statistically with ANOVA followed by theDunnett test or Student’s t-test. The correlations weredetermined by the Pearson Correlation test. Datawere considered significant when p < 0.05.

Results

Concentration and differentiation of cells obtainedfrom theperitoneal cavity. Induction of peritonitis inrats caused an increase of cells in the peritonealcavity on day 1. The cell count dropped on thefollowing days (Fig. 1A). A pronounced influx ofgranulocytes was seen on the first day which wasfollowed by an influx of Me, resulting in a Me/granulocyte ratio of 4:1 from day 3 to 10 (Fig. 1B).

Production capacity of cells to generate inflamma-tory mediators: The inflammatory mediator produc-tion per million cells stimulated in vitro (LTB4, 15 minA23187; and PGE2, TXB2, 6kPGFI=, TNFo:, 24 h LPS)by the peritoneal ’crude’, ’macrophage’ and’granulocyte’ fractions and the peripheral blood’monocyte’ fraction is shown in Fig. 2A, 2B, 2C and2D.Cytokine and eicosanoid production by the crude

fraction increased steadily during peritonitis. TNFcand TXB2 reached peak levels after 3 days, whereasPGE2 and 6kPGFI= reached a plateau 7 days afterinduction of the peritonitis. In comparison with lev-els of other eicosanoids the LTB4 production was lowbefore and on the first day of the peritonitis anddecreased significantly thereafter. By day 10 the pro-duction capacity returned to the initial level.The production pattern of the cells from the

macrophage fraction (Fig. 2B) was similar to that ofcells from the crude fraction (Fig. 2A). Inflammatorymediators were also produced by the granulocytefraction (Fig. 2C), although the production capacity

Changes in eicosanoid and TNF(z production

25

20

15

10

12

0 ) " 1) 0 ( i’ : " 1Time (days) Time (days)

FIG. 1. Time course (0-10 days) of cell number (A) and differentiation of the cells (B) during a peritonitis in rats. M (e); granulocytes (e). Mean + S.E.M.;n 6 per group; *p < 0.05 macrophages vs. granulocytes.

Crude fraction

160 80

0 ,’ 03 7 10

120

80

40

60

40

20

Macrophage fraction

160B

80

so 40

40 20

0 00 a 7

Granulocyte fraction Monocyte fraction

0

0

160

120

80-

40

0 3 7 10

8O 2O 8O

-60

40

20

0 0

E 15

O

o

10

5

0

#

0 3 7 10

6O

40

20

Time (days) Time (days)

FIG. 2. Production capacity of stimulated ’crude’ (A), ’macrophage’ (B), ’granulocyte’ (C) and ’monocyte’ (D) fraction to produce the eicosanoids LTB (+);PGE (,); 6kPGFI (e); TXB2 (B); and the cytokine TNFx (e). LTB4 production of 10cells/ml determined in supernatant after 15 min, IM A23187stimulation. PGEv TXBv 6kPGFI, TNF(x production of 106 cells/ml determined in supernatant after 24 h 10 lg/ml LPS stimulation; mean + S.E.M.;",#,= p < 0.05vs. day 0; n 4-6.



of these cells was very low in comparison with cellsfrom the crude and macrophage fraction. The patternof inflammatory mediator production of thegranulocyte fraction did not significantly change intime. On the tenth day after induction of the perito-nitis, the concentration of the cells in the granulocytefraction was too low to permit in vitro incubations.The capacity of cells from the peripheral blood

monocyte fraction to produce inflammatory media-tors (Fig. 2D) was at least four times lower in com-parison with both the crude and macrophage frac-tions. The production capacity of the cells from themonocyte fraction was about the same with or with-out stimulus. The patterns of the TNFz, PGE and6kPGFI= production of cells from the monocyte frac-tion were different from those of the macrophageand crude fraction. After induction of peritonitisthe TNF0t production of cells from the monocytefraction increased significantly only on the tenth day,whereas PGE2 and TXB,. reached their highestlevel on the third day. The 6kPGFlu level did notchange during peritonitis. The LTB4 production wasnegligible.

Correlations between inflammatory mediators pro-duced in the samefraction: In the crude fraction (Fig.2A) there was a significant positive correlation be-tween the production of PGE,. and 6kPGFI= and PGE2and TXB,. (r= 0.6365 and r= 0.7145). Similar signifi-cant correlations were found in the macrophagefraction (Fig. 2B, r= 0.5580 and r= 0.6106). In themonocyte fraction (Fig. 2D) the only significant posi-tive correlation between the inflammatory mediatorswas between PGE and WXB (r= 0.8881).

Correlation between inflammatory mediators in

different fractions: In Fig. 2A and 2B the patternsof inflammatory mediators produced by the stimu-lated crude and macrophage cell fractions are pre-sented. There was always a significant positivecorrelation between these two fractions. The corre-lation varied from r= 0.5671 for LTB to r 0.8586 forTNFo:. (PGE,., r= 0.6254; 6kPGFI=, r= 0.7536; andTXB2, r= 0.7502). This correlation remained signifi-cant when we assumed that production of the cellinflammation mediators in the crude andmacrophage fraction were only derived from the Me(calculated to 100% macrophages, LTB4=0.4649;PGE2 0.5855; 6kPGFI= 0.5506; TXB2 0.7810; andTNFo: 0.8674).There were, however, some significant differences

between the calculated (to 100% Me) productionlevels of the crude and macrophage fractions. Whenseparate days were considered the production levelsof the calculated crude fraction in comparison withthe calculated macrophage fraction was on day 1lower for PGE2 and 6kPGF=, and on day 3 higher forgWB4, TXB2 and TNFo: (Fig. 3 A-E).

Correlation coefficient between inflammatory media-torsfrom monocyte and macrophagefraction: Onlythe basal TNFz production of the cells from themonocyte and macrophage fraction were clearlycorrelated (r= 0.5421).

Discussion

Characterization of inflammatory mediators pro-duced by granulocytes and monocytes/macrophagesduring induced inflammation in animal models, maybe helpful to the understanding of inflammatorydiseases and their treatment. The present study in-vestigated the changes and correlations in cytokineand eicosanoid production capacity of peripheralblood monocytes, non-purified (’crude’ fraction) andpurified (’macrophage’ and ’granulocyte’ fraction)peritoneal cells during a carrageenin-induced perito-nitis in rats.

In the present study, where an influx of leucocytesinto the peritoneum was achieved after carrageenininjection, the number of PMNs increased in the acutephase (day 1) and thereafter decreased quickly. Onday 0, before inducing the peritonitis, there was areasonable number of Me present in the peritonealcavity. The number of Me increased from day 1 until7 days after induction of the peritonitis, resulting ina mainly Me cell population. Previous histologicalstudies had similar results,’4,25 although a quantitativeand kinetic method showed that both monocytesand PMNs from rat migrate rapidly to the inflamedsite, the migration of PMNs, however, also declinedlong before the migration of monocytes started todecrease. The pattern of this migration can beexplained with the function of the leucocytes ininflammation. In the initial phase both PMNs and Meare involved in nonspecific defence mechanisms.,2

In addition, Me will also initiate and control thespecific defence.7-1 The mechanism of this selectivemigration is not clear. Leucocyte adhesion tothe blood vessel endothelium, which is followedby transendothelial migration, is a multistepprocess in which several adhesion molecules areinvolved, z6,z7

Our experiment showed that the inflammatorymediator production capacity of the non-purifiedfraction, which was harvested from the inflamedperitoneal cavity, changed with time. The productionof inflammatory mediators in the non-purified frac-tion was mainly caused by the presence of M) duringcarrageenin-induced peritonitis. This is, first of all,based on the significant correlation between theeicosanoid and TNFot production capacity of themacrophage fraction and the crude fraction, thesecorrelations hardly changed when it was assumedthat these fractions only contained M. Secondly, theproduction capacity of the granulocytes appeared to

338 Mediators of Inflammation Vol 3 1994

Changes in eicosanoid and TNF production

10

LTB PRODUCTION

A

0 3 7 10

Time (days)

PGE= PRODUCTION

200

150

100

50

B

o "Time (days)

6kPGF PRODUCTION

80

" 60

0: 40

C

Time (days)

TxB PRODUCTION TNF( PRODUCTION80

60

40

20

lOOD

x0 50

0’6; o

E #

Time (days) Time (days)FIG. 3. Production capacity of the stimulated ’crude’ (B) and ’macrophage’ fraction (e) (calculated to 100% M) to produce the eicosanoids LTB (A), PGE(B), 6kPGFI (C), TXB2 (D) and the cytokine TNFo (E). LTB production of 106 cells/ml determined in supernatant after 15 min, IM A23187 stimulation.PGE, TXB, 6kPGFI, TNFo production of 106 cells/ml determined in supernatant after 24 h 10 g/ml LPS stimulation; mean +_ S.E.M.; "p < 0.05 vs. day 0;"p < 0.05 granulocytes vs. macrophages; n 4-6.

be very low. Moreover the production capacity of thegranulocyte fraction did not correlate with the crudefraction.When the production levels of the non-purified

fraction was compared with the macrophage fraction,assuming that the inflammatory mediators were onlyproduced by M, it was observed that the productionof PGE,. and 6kPGFI= had decreased on day I and theproduction of LTB4, TXB and TNF had increasedon day 3. The results of these effects could be anamplification of the inflammation, due to the de-crease of anti-inflammatory substances and the sub-sequent increase of pro-inflammatory substances.The low production capacity of the crude fraction

in the acute phase followed by an increasing produc-tion capacity in the chronic phase could also beexplained by presence of the M. Blood monocytesand resident M transform from primed into active

M at the side of the inflammation, after whichprofound changes occur in morphology and functionof these cells. The M present at day 1 in theperitoneal cavity, probably were not primed in vivoat this time, which could be the reason that theeicosanoid and cytokine production capacity of

these M were so low after stimulation in vitro withLPS. Once these M were primed in vivo, also thenew monocytes that migrate into the peritoneal cav-ity, the production capacity of these monocytes/Msteadily increased, showing in vitro a differentiatedpattern for the eicosanoids and TNF. This patternprobably depends on the function of the metabolite.As peripheral blood monocytes transform into

peritoneal m, the correlation between these cellswas investigated. The eicosanoid and TNF produc-tion capacity of blood monocytes was not similar tothat of M during peritonitis. It was clearly shownthat through influx and transformation of monocytesinto M the synthesis of mediators completelychanged.

In conclusion, M are the main source of theinflammatory mediators whose production is de-pendent on the episode of the carrageenin-inducedperitonitis.Characterization of purified M in vitro inanimal and human models will be helpful to theunderstanding of inflammatory diseases and theirtreatment. Peripheral blood monocytes do not reflectmediator production ofM present at the inflamma-tory site.



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Received 28 March 1994;accepted in revised form 2 May 1994

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