Human glomerular mesangial cells inactivate leukotriene B4 by reduction into dihydro-leukotriene B4 metabolites

Life Sciences, vol. 46,

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Pergamon

pp. 1465-1470

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HUMAN GLOMERULAR MESANGIAL CELLS INACTIVATE LEUKOTRIENE B4 BY REDUCTION INTO DIHYDRO-LEUKOTRIENE B4 METABOLITES V. Kaevere, J. BruunsB, J. Wundera, B. Dameraud. G. Zimmerd, J. Faulerb, K. Wessele, J. Floegec, N. Topleya, H. Radekea, and K. Resch* Institutes of aMolecular Pharmacology and bClinical Pharmacology, Department of Pharmacology and Toxicology, CDepartment of Nephrology, Medical School, Hannover, D-3000 Hannover 61, F.R.G., dDivision of Biochemical Pharmacology, Max-Planck Institute for Experimental Medicine, D-3400 Gsttingen, F.R.G. (Received in final form March 9, 1990)

Summary Due to its potent chemotactic properties leukotriene B4 is an important mediator of inflammatory reactions. Cultured human kidney converted exogenously added leukotriene BS mesangial cells efficiently into three different more llpophilic metabolites, two of This them probably representing dlhydro-leukotriene B4 isomers. metabolic contrast to represents an alternative pathway, in found in human polymorphonuclear leukotriene B4 omega-oxidation dihydro-leukotriene B4 isomers had nearly leukocytes. Both leukocyte chemotaxis as completely lost their abillt,y to induce compared to leukotriene B4. Elevated levels of leukotriene B4 (LTB4) observed in various pathophysiological situations as for example in different forms of glomerulonephritis (1,2) may result from an enhanced synthesis as well as from an impaired Whereas biological effects of LTB4 on metabolism of this endogenous mediator. leukocytes, such as chemotactic and chemokinetic movement, aggregation, to endothellal cells, and lysosomal enzyme release, have been adherence extensively studied (31, the precise mechanisms whereby LTB4 is degraded and inactivated are much less understood in human cells. In the monkey LTB4 is almost, totally degraded into volatile products several hours after infusion (4). Therefore, the liver might represent the site of terminal LTB4 metabolism (51, but the primary inactivation may also take place in other cell types more close to the site of LTB4 production. In isolated human polymorphonuclear leukocytes (P%NL) LTB4 is readily oxidized to 20-hydroxy (20-OH)-LTB4 and 20-carboxy (20-COOH)-LTB4 (61, among which only 20-COOH-LTB4 is totally inactive in biological assay systems (7). We (8) and others (9,lO) have described previously the existence of alternative metabolic pathways for LTB4 depending on the reduction of one double bond in different murine cell types and porcine leukocytes, respectively. The primary dihydro-LTB4 metabolite found in murine cells was shown to be biologically much less active than LTB4 itself (11). Very recently it has been demonstrated that dihydro-LTB4 was also generated from LTB4 in highly purified human peripheral monocytes (12) and alveolar macrophages (13). Dihydro-LTB4 could be separated into two dihydro-LTB4 isomers by using an improved HPLC gradient system (12). In this study we report that in cultured human mesangial cells LTB4 is initially metabolized into two dihydroLTB4 isomers identical to those found in human monocytes and an additional not yet identified more lipophilic metabolite. Compared to LTB4 both dihydro-LTB4 isomers exhibited a marked decrease in chemotactic activity. 0024-3205/90 $3.00 + .oo Copyright (c) 1990 Pergamon Press

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LTB4 Metabolism

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Materials

Cells

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46, No. 20, 1990

and Methods

Mesangial cells were isolated from normal human kidney tissue obtained at the time of elective nephrectomy for encapsulated renal carcinomas and cultured as described elsewhere in detail (14). Both dlhydro-LTB4 isomers were prepared through conversion of LTB4 by these cells. To about 5 x lo6 mesanglal cells in monolayer culture 10 ug LTBI (Bayer Diagnostic and Electronic, Miinchen. F.R.G.) together with 0.1 uCi 13H]LTB4 (NEN, Dreleich, F.R.G.) were added for 24 hours under serum-free conditions. LTB4 and its metabolltes were purified by solid phase extraction (8) and reverse phase-HPLC (RP-HPLC) using different chromatographic systems. In RP-HPLC system I (Nucleosil Cla, 4.6 x 250 mm, 5 pm particle size, mobile phase consisting of acetonltrlle / methanol / 0.2 M acetic acid, sodium acetate, 30:30:40, v/v, pH 5.5, flow rate 1 ml/mln) LTBI (retention time 18-20 mln), dlhydro-LTB4 isomers (coeluatlng between 20-24 mln), and an unknown metabollte (retention time 30-32 min) were separated. In a successive second RP-HPLC system (Shandon &a, 4.6 x 250 mm, 5 urn particle size, mobile phase consisting of a linear gradient between acetonitrlle / methanol / water / acetic acid, 25:25:50:0.02, v/v, and acetonitrile / methanol / water / acetic acid, 50:20:30:0.02, V/V, within 100 min, flow rate 1 ml/mln) a clear separation of two dlhydro-LTB4 isomers was achieved (retention times: dlhydro-LTB4 isomer I 57.4 mln, dihydro-LTB4 isomer II 60.5 mln). Each metabolite was concentrated and purified by solid phase extraction and stored in phosphate-buffered saline pH 7.4 as 10-g M stock solution (calculation based upon their radioactivity) at -20 “C without significant decomposition during several months. UV absorption and GC-MS spectra were recorded as described elsewhere (8,121. LTB4 and 6-trans LTBI were also purified by RP-HPLC (RP-HPLC system I) and handled in an identical manner as the dihydro-LTB4 isomers. For chemotaxis assays human non-fractionated leukocytes were isolat,ed by dextran sedimentation (15) and suspended in complete Gey’s solution containing 0.5 % human serum albumin to a final concentration of 1 x 106 cells/ml. Chemotactic migration towards concentration gradients of LTB4, 6-trans-LTB4, dihydro-LTBs isomers I and II, and Csa-desarg as standard, was assayed in Boyden chambers as described (15). Results

and Discussion

Addition of LTB4 to human kidney mesangial cells for 24 hours in a high concentration (10 pg, corresponding to a concentration of about 3 pM in the cell supernatant) resulted in an almost total conversion of this eicosanold mainly into different more llpophlllc metabolltes (figs. 1A and 1B). Only 5.8 96 of the total to dlhydro-LTB4 isomers radioactivity was found belonging to LTB4, in contrast, (55.2 %) and a very lipophilic unknown metabolite (19.3 %). Only 5.1 % of the radioactivity was eluted between 4.5 - 6 min representing polar degradation products but their amount increased at longer incubation times (data not shown). When lower amounts of LTBI were used (50 - 500 ng) an increased conversion rate into dihydro-LTB4 isomers and polar degradation products became obvious. Interestingly, under those experimental conditions the very lipophllic metabolite (eluted between 30-32 min) was not observed (data not shown). From these dat,a it can be suggested that under physiological conditions human kidney mesangial cells and various murine cells (8) preponderantly reduce LTB4 to dihydro-LTB4 isomers, which are subsequently degraded via oxidative steps into polar not yet isomers was of two dihydro-LTB4 structurally defined substances. Separation achieved in a second RP-HPLC system (fig. 1C) previously described by Fauler and coworkers (12). By recording UV absorption maxima (232 nm) and GC-MS spectra these two LTB4 metabolites proved to be identical to the described dihydro-LTB4 (12). Calculat,ed on the basis of their isomers formed by human monocyt,es radioactivity dihydro-LTB4 isomer I had been formed by mesangial cells in higher amounts than isomer II (70.2 % versus 29.8 % of recovered radioactivity from RPHPLC system 1).

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LTBI, Metabolism

1990

in Mesangial

Cells

RETENTION TIME (mid

HPLC system II

0

s

10

IS

20

25

30

3s

40

4s

50

55

00

85

RETENTION TIME (mid

FIG.1

Separation of leukotriene RI and its metabolites generated by human kidney mesangial cells. Metabolites formed after addition of LTB4 (10 ug) together with 13HJLTB4 (0.1 pCi) for 24 hours were analyzed using RP-HPLC system I (A and B) as described in Materials and Met,hods. In a successive RP-HPLC system dihydro-LTB4 isomers were separated (C). Radioactivity of 25 1.11 aliquots of the effluent collected in 0.5 ml fractions was determined by liquid sdntillation counting. I ,polsr metabolites; 2,LTBa; 3,dihydro-LTB4 isomers, 4,unknown lipophiiic metabolite; S,dihydro-LTB4 isomer I; G,dihydro-LTB4 isomer II.

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The structure of the third very llpophilic metabolite has not. yet been fully The elucidated. No UV absorbance maximum at 271 nm or at 232 nm was found with peaks observed in the UV recording trace at 232 nm (fig. 1A) coeluatlng . radioactivity (fig. 1B) were due to contaminants from the culture medium not separated by solid phase extraction. Jt, therefore, seems reasonable that this which has lost two or even three product might represent an LTBI metabollte, dlhydro metabolites. double bonds, rather than a lactone form of LTB4 or its However, it should be kept in mind that this product was only formed at high non-physiological concentrations of LTB4 added. In order to evaluate the biological activities of compared to LTB4 and its inactive isomer B-trans-LTB4 chemotactic movement of human PMNL was investigated. TABLE 1 of Human Leukocyte

Induction addition

migration

---

37.5

+

[pm]

M M M

48.1 64.3 94.3

f 17.0 + 15.0 + 15.1

dihydro-LTB4 isomer I

10-g 1O-8

M M M

40.0 40.5

c 11.3 + 5.6

51.6 f 10.0 33.7 t

significance ---

8.7

10-g lo-* lo-’

dihydro-IaTB4 10-g M 1O-8 M isomer II

dihydro-LTB4 isomers their ability to induce As shown in table 1,

Chemotaxis

JJTBI

10-7

the

p p

n.s. < 0.01 < 0.001 n-s. ri . s .

p < 0.05

8.5

n.s.

10-7

M

42.0 38.5

t 12.5 + 8.9

n.s. n.s.

6-trans-LTB4

1O-g 1O-e lo-’

M M M

37.7 40.4 54.2

+ 17.6 f 9.6 t 5.6

n.s. n.s. p < 0.01

CS B -desarg

1O-9 lo-” 10-T

M M M

55.5 93.6 110.2

f 9.8 t 13.1 ?r 2.6

p p p

< 0.05 < 0.001 < 0.001

Results are the mean + SD of 5 experiments with different cells, the maximal distance of 5 determinations of each comprising migration. St,udent’s t-test for unpaired samp\Fes was 11sed t.o assess the level of significance. n.d., not significant.

Csa-desarg and LT84 significantly even at, nanomolar concentrations. and dihydro-LTH4 this bioassay, chemotactic activity only at, the

enhanced the migration of human leukocytes Dihydro-LTH4 isomer II &as totally inactive in isomer I and 6-trans-I,TB4 showed marginal highest concentration t.ested (IO-’ M).

These data provide further evidence that. in human performed potent endogenous mediator LTB4 is not solely PMNL, but. also takes place via reduction to different tissue-bound cells such as kidney rnesangial cells. This an unspecific characteristic of most human cell types.

cells inactivation of thp via omega-oxidation by dihydro-l.TB4 isomers by metabolic pathway is not In conl.rast to rnesangixl

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LTBI, Metabolism

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cells and highly purified peripheral blood monocytes (12) or alveolar macrophages (13) we were unable to detect any LTB4 metabolism in other cultured human cell lines (table 2). Powell and Gravelle have shown recently that porcine leukocytes also convert LTB4 into different less polar dihydro-LTB4 metabolites (10). These were identified as 10,ll -dihydro-LTB4 and 10.11 -dihydro-12-oxo-LTB4 existing in equilibrium with one another, but their biological activities have not been described yet (10).

Occurance

of Dihydro-LTB4

TABLE 2 Isomers In Different

Human Cells

detectable peripheral blood monocytes alveolar macrophages (ref. kidney mesangial cells not

(ref. 13)

12)

detectable

peripheral PMNL (ref. 8) epithelial cells (chorion) endothelial cells (umbilicial fibroblast-like cells (Fldl) various tumor cells (liver, Experiments in Materials

cord) bronchus,

kidney

tubule)

with different human cells were performed as described and Methods for human kidney mesangial cells .

It. is still an open question whether mesangial cells themselves can secrete measurable amounts of LTB4 after addition of appropriate stimuli (16) either in normal or in pathophysiologicai situations. LTBI can be synthesized by whole glomeruli (17) and an enhanced synthesis has been reported in glomeruli isolated from rats with glomerular immune injury (1,2). Changes in the metabolic capacity for LTB4 inactivation may seriously influence inflammatory processes within the kidney and thus be important in the pathogenesis of different forms of glomerulonephritis. As LTB4 is not a circulating hormone it seems reasonable that the primary inactivation of LTB4 by reduction into dihydro-LTB4 isomers directly at the inflammatory site by cells which do not have to infiltrate provides the prefered route compared to the well-known omega-oxidation by PMNL. Acknowledgements This publication is dedicated to Dr. B. Damerau, who unfortunately died in an accident before the study was finished. This work was supported by grants Ka 730/l-2 and SFB 244/Bl from the Deutsche Forschungsgemeinschaft. References 1. 2.

M.A. RAHMAN, M. NAKAZAWA, Invest. 81 1945 - 1952 (1988). J. FAULER, A. WIEMEYER, K.-H. Kidney Int. S 46 - 50 (1989).

S.N.

EMANCIPATOR and

M.J.

DUNN, J.

Clin.

MARX, K. KUHN, K.M. KOCH and J.C. FRCLICH,

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3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

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1990

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