Journal of Leukocyte Biology 49:283-288

6-Trans-Leukotriene Donald

J. Fretland,

B4 Is a Neutrophil Guinea Pig Dermis

Deborah

L. Widomski,

Charles

and Timothy Gastrointestinal

Diseases

Research,

Chemotaxin

P. Anglin,

Robin

E. Walsh,

(1991)

in the Stuart

Levin,

S. Gaginella GD.

Searle

and

Co.,

Skokie,

Illinois

The products of the 5- and 12-lipoxygenase (5-LO, I 2-LO) pathways of arachidonic acid metabolism are implicated as proinflammatory mediators in a number of disease states. 12(R)-hydroxyeicosatetraenoic acid [12(R)-HETE] is present in large quantities in human psoriatic

lesional

skin

and can be further

metabolized

by 5-LO

to 5(S),12(R)-dihydroxy-

(6E,8Z,1OE,14Z)-eicosatetraenoic acid (6-trans-LTB4). Furthermore, leukotriene B4 (LTB4) and the sulfidopeptide leukotrienes (LTC4, LTD4) can be transformed to 6-trans-LTB4. When injected into the guinea pig dermis, 6-trans-LTB4 (1 .0, 10.0, 20.0 ig/lntradermal site) caused a significant (P < 0.02) infiltration of polymorphonuclear leukocytes (PMN) at 4 hr as assessed by histology and the levels of the PMN marker enzyme myeloperoxidase. 6-Trans-LTB4 IS a more potent PMN chemoattractant than 12(R)-HETE in the guinea pig dermis but is far less potent production or receptor binding

than LTB4. Pharmacological interdiction of leukotriene should take into account the proinflammatory activity of

6-trans-LTB4. Key words:

chemotaxis,

inflammation,

myeloperoxidase

INTRODUCTION There are a plethora of agents that stimulate polymorphonuclear leukocyte (PMN) diapedesis [26,36]: bacterial N-formyl peptides [2,40], the CSa cleavage product of the fifth component of the serum complement system and CSa des arg [17] interleukin 1 and tumor necrosis factor [35], casein [47], and platelet-activating factor [43]. Prominent also as chemotaxins are the proinflammatory products of arachidonic acid metabolism, leukotriene B4 (LTB4) and 12-hydroxyeicosatetraenoic acid ( 1 2-HETE) Injected LTB4 induces neutrophil accumulation within the skin ofthe guinea pig [13,22] and rabbit [1 1] and into aqueous humor [3]. LTB4 caused PMN recruitment when instilled into human lung [33] Intradermal injection of LTB4 in the human caused neutrophil infiltration determined by punch biopsy histological analysis [10,45]. l2(R)-HETE is a neutrophil chemotaxin in a variety of species [23]. 12-HETE is chemotactic to human neutrophils and is present in elevated quantities in psoriatic scale [12, 18]. The 12-HETE present in psoriatic lesional skin has been shown to be of the 12-R stereochemistry [49] and 12(R)-HETE is more potent than 12(S)-HETE as an enhancer of neutrophil chemoattraction [48,50]. 5(5), 1 2(R)-dihydroxy-(6E,8Z, lOE, 14Z)-eicosatetraenoic acid (6-trans-LTB4) arises in PMNs through the nonenzymatic hydrolysis of leukotriene A4 (LTA4) [4,7,8], through the myeloperoxidase (MPO)-dependent metabolism of the sulfidopeptide leukotrienes (LTC4, ,

-D4, E4) [31], through the calcium ionophore stimulation of 5-lipoxygenase products [28], and through the action of 5-lipoxygenase on 12(R)-HETE [42]. 6-TransLTB4 is further metabolized to dihydro-6-trans-LTB4 [39] . 6-Trans LTB4 (Fig. 1 ) has been reported to be a chemotaxin in vitro to human PMNs [42] and rat peritoneal macrophages [5 1] in modified Boyden chamber experiments. The objective of this study was to test whether 6-trans-LTB4 possessed PMN chemotactic activity in vivo.

.

.

,

© 1991 Wiley-Liss,

Inc.

MATERIALS Materials

AND METHODS

LTB4 and 6-trans-LTB4 were purchased from Bio Mol (Plymouth Meeting, PA). Analytical high-performance liquid chromatography (HPLC) was done on a 15 cm ji-porasil column with a mobile phase of methanol:water:acetic acid:ammonium hydroxide (67:33:0.08:0.07), pH 6.8. l2(R)-HETE was synthesized in these laboratories as previously reported [ 16] . 0-dianisidine and hexadecyltrimethyl ammonium bromide (HTAB) were purchased from Sigma (St. Louis, MO). Male Hartley guinea pigs [Crl:(HA)BR], 250-400 g body weight, were Received

July

Reprint requests: Research, GD. 60077.

5, 1990;

accepted

July

15, 1990.

Timothy S. Gaginella, Gastrointestinal Searle and Co. , 4901 Searle Parkway,

Diseases Skokie, IL

Fretland et al.

284

iw

cient of 0.979. Intradermal injections of 35 and 100 ng 6-trans-LTB4 did not evoke chemotaxis at 4 hr. When 10 jig 6-trans-LTB4 was injected intradermally, peak MPO activity was observed at 3 hr, followed by gradual decline over the next 96 hr (Fig. 3). MPO levels were significantly (P < .05) elevated over their respective time controls from 30 mm to 24 hr after intradermal injection.

OH

OH Fig. 1 . Structure of 6-trans-LTB4 (5(5], [6E,8Z,1OE,14Z]-eicosatetraenoic acid).

12[RJ-dihydroxy-

Histological 6-Trans-LTB4 purchased from Charles River Laboratories (Wilmington, MA), housed 10 per cage, allowed food and water libitum, and kept on a light/dark cycle of 14/10 hr.

Intradermal

ad

Chemotaxis

Methods for intradermally stimulated neutrophil chemotaxis have been previously reported [20,44]. So lutions of chemotaxins were made up in 0.9% NaCi with never more than 1% ethanol. Various doses in 0. 1 cc were injected intradermally with a 26-G allergists needle into the shaved backs of guinea pigs lightly anesthetized with pentobarbital. There were four to six injections per animal. Appropriate vehicle controls were used throughout. After 4 hr, the animals were killed by carbon dioxide anoxia and excorticated, and the skin was frozen at -70#{176}C.

MPO Assay MPO levels were determined as previously reported 14,30,44] . Skin punches ( I 2 mm diameter) of frozen tissue were minced, homogenized with HTAB detergent buffer, sonicated, and freeze-thawed. After centrifugation, extracted MPO was measured spectrophotometrically using 0-dianisidine-hydrochioride as the substrate. Data were recorded as the absorbance (460 nm) at 15 mm and are expressed as mean ± SEM. [

Histological Dermal mounted, eosin.

Statistical

Assessment tissues sectioned,

were and

fixed stained

in Carson’s solution, with hemotoxylin and

Analysis

Statistical evaluation of dose-response studies was by linear regression analysis and analysis of variance. N is always the number of animals in a particular group.

RESULTS Neutrophil

Diapedesis

Induced

by 6-Trans-LTB4

When doses from 1 to 25 jig 6-trans-LTB4 (96-98% pure by analytical HPLC) were injected intradermally and assessed by MPO activity at 4 hr, there was significant (P < .02) dose-related diapedesis (Fig. 2). Linear regression analysis yielded a correlation coeffi-

Evaluation

of Intradermal

The vehicle control was essentially normal at 4 hr as assessed histologically (Fig. 4A), although under higher magnification ( x 130) a few inflammatory cells can be seen in the dermis and subcutis (Fig. 4B). Twenty micrograms of 6-trans-LTB4 induced mild PMN infiltration, slight eosinophil infiltration (< 1% of the infiltrate) and slight edema in the dermis and subcutis (Fig. 4C,D).

DISCUSSION It is accepted that tissue MPO activity correlates well with the number of infiltrating neutrophils [ I ,20,22]. Granulocyte infiltration of tissues is a prominent feature of inflammatory diseases such as inflammatory bowel disease [32,41 ,46], psoriasis [24,25], and asthma [9,29]. The resultant degranulation, superoxide formation, and vascular leakage may well add to the tissue pathology seen in these diseases. Selective antagonists ofthe sulfidopeptide leukotrienes [34] LTB4 [ 15] and 12-HETE [ 19,2 1 ,27] have been reported with the intent of utilizing them as antiinflammatory agents. We have demonstrated for the first time that 6-trans-LTB4 is indeed a PMN chemotaxin in the guinea pig dermis. Interdermal injections of 35 ng LTB4 or 30 jig 12(R)-HETE have been reported to induced comparable PMN infiltration in the guinea pig [23]. Whether these observations extend into other species remains for further investigation. In comparison with these other chemotactic metabolites of arachidonic acid, our data suggest that 6-trans-LTB4 is more potent in stimulating directed PMN migration than is 1 2(R)-HETE [23], but the 6-trans form is much less active than LTB4 itself. These differences in potency could be explained by issues involving lipophilicity, distribution, or relative affinity for neutrophil receptors and are the subject of further study. Indeed, recent reports indicate that 6trans-LTB4 is much less potent than LTB4 itself in inducing phospholipid methylation in human PMNs and that this was directly correlated to chemotactic ability [5]. Furthermore, 6-trans-LTB4 was a log less potent in displacing tritiated LTB4 from its receptor on human PMN membranes [6]. This is in marked contrast to the 5,6-di-HETEs, which do not compete for LTB4 binding sites [38]. The slight eosinophil infiltrate observed is probably nonspecific; whereas LTB4 is itself a potent ,

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TIME (HOURS) Fig. 3. Time course of PMN dermal infiltration in response to 6-trans-LTB4. Ten micrograms of 6-trans-LTB4 was injected into guinea pig dermis, and infiltration of PMN was evaluated over time by the levels of myeloperoxidase (MPO). Data points represent mean MPO (U/gm) ± SEM, (n 8-12/point). < .05 compared to vehicle time-paired control. =

*

96

286

Fretland et al.

Fig. 4. Histological assessment of guinea pig dermis. A (x 50) and B (x 130) represent vehicle control at 4 hr. C (x50) and D (x 130) represent 20 .tg 6-trans-LTB4 injected intradermally and assessed at 4 hr.

6-Trans-Leukotriene eosinophil chemotaxin, 6-trans-LTB4 is not in blind-well Boyden chamber experiments at concentrations as high as 1 jiM [37]. These data suggest the following caveat. Receptor antagonists, by blocking the interaction of sulfidopeptide leukotrienes and l2(R)-HETE at target cells, may permit the accumulation of precursors subject to further metabolism to 6-trans-LTB4, which is capable of activating PMN diapedesis. If this observation is confirmed in other species, receptor antagonists and/or biosynthesis inhibitors of 6-trans-LTB4 may well be of value in the treatment of inflammatory diseases in which high levels of the sulfidopeptide leukotrienes and 12(R)-HETE have been implicated as mediators, such as inflammatory bowel disease, asthma, and psoriasis.

ACKNOWLEDGMENTS The expert secretarial Fraser and the graphics acknowledged.

assistance of of Todd Minske

Mrs. Jeanette are gratefully

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