[62]

I N T E R C E L L U L A R T R A N S F E R OF L T A 4

567

metabolism of L T A 4 to L T C 4 c a n be demonstrated even after relatively weak stimulation. [35S]LTC4 Production during PMNL/Vascular Cell Coincubation. To demonstrate that the conversion of PMNL-derived LTA4 to L T C 4 o cc u r r e d within the vascular cell cultures, [35S]cysteine is used to label the cellular GSH pools. Prelabeled PMNL incubated alone or with unlabeled vascular cells releases low levels of [35S]LTC4 after A23187 stimulation. However, [35S]cysteine-prelabeled EC 1 or SMC z incubated with A23187stimulated unlabeled PMNL produces significantly m o r e [358]LTC4 than in the reverse-labeling experiment (Fig. 2). Conclusions Using the methods described in this chapter, it has been possible to demonstrate that activated PMNL release LTA4 which can be converted to LTC4 by adjacent cultured vascular cells. The level of LTC4 produced depends on the activating stimulus. Weak PMNL activators, such as fMLP, may permit the observation of a feedback regulation loop in which PMNL leukotriene synthesis is inhibited by a vascular cell product which is probably a prostaglandin. Acknowledgments Some of the experiments described in this chapter were supported by AI-26702, HL-38312, and HL-21006. Portions of this work were done during the tenure of an Established lnvestigatorship award from the American Heart Association and Boehringer lngelheim, Inc. The author would like to thank Dr. J. Brett for the morphological examination of the cultured cells and Dr. R. R. Sciacca for statistical analyses and critical reading of this manuscript.

[62] R e l e a s e a n d M e t a b o l i s m o f L e u k o t r i e n e A4 in Neutrophil-Mast Cell Interactions

By CLEMENS A. DAmNDEN and URs WmTHMUELLER Introduction The cellular sources, biosynthesis, structure, and different biological activities of the 5-1ipoxygenase (5-LOX) metabolites leukotriene (LT) B4, L T C 4 , LTD4, and L T E 4 a r e well established. However, there is an interesting particularity of the 5-LOX pathway in that at each metabolic step the precursor molecules are formed in amounts largely exceeding the METHODS IN ENZYMOLOGY, VOL. 187

Copyright (~3 1990by Academic Press, Inc. All rights of reproduction in any form reserved.

568

CELL MODELS OF LIPID MEDIATORPRODUCTION

[62]

capacity of the next enzyme. When released outside the cell, the different precursor molecules can be taken up and further metabolized by different cell types, resulting in the generation of lipid mediators that are not produced by an individual cell type alone. Indeed, there is increasing evidence from work of different laboratories, that such cellular cooperations and intercellular transfer of precursor lipids exist among a variety of leukocytes and tissue cells. In contrast to the cyclooxygenase and other lipoxygenases, the 5-LOX is a calcium-dependent enzyme, and requires agonist-induced increases in intracellular calcium for activity.l'2 Since the activity of the phospholipase(s) liberating arachidonic acid, and of the 5-LOX are regulated by different second messengers,~ cell cooperation by exchange and metabolism of free arachidonic acid may already be present at this first step of the cascade. 5-Hydroxy-l-eicosatetraenoic acid (5-HETE) formed by reduction of excess 5-HPETE, can be further metabolized by the 12-LOX of platelets or the 15-LOX, i.e., of eosinophils. Alternatively, 5-HETE may be reincorporated into phospholipids, possibly altering membrane properties and cellular functions. Finally, in neutrophils (PMN) LTA4 is formed in excess of what is enzymatically hydrolyzed to LTB4. This is clearly indicated by the fact that whenever LTB4 is formed in vitro, the all-trans stereoisomers of LTB4 are also present. Here, we review our findings that LTA4 formed by Ca 2+ ionophore-stimulated PMN does not appreciably decay within the cell, but is released into the extracellular medium as the intact epoxide in relatively large amounts. Low concentrations of albuminbound LTA4 are efficiently and rapidly taken up by mast cells and metabolized into LTC4. It is remarkable that more recent studies showed that neutrophil-derived LTA4 can be metabolized into biologically active leukotrienes even by cells (like erythrocytes, platelets, and endothelial cells), which are lacking the 5-LOX and thus are unable to form LTA4. Therefore, interactions between various cell types that release or utilize LTA4 may provide an important metabolic pathway for the production of leukotrienes (Fig. 1). Release of LTA4 by Neutrophils

Materials. LTA4 methyl ester, LTB4, LTC4, LTD4, and LTE4 are a gift of Dr. Rockach (Merck-Frosst, Pointe Claire, PQ, Canada). Organic solvents are HPLC-grade and chemicals of the highest purity available are used as supplied. Synthetic all-trans-LTB4 derivatives are prepared by C. A. Dahinden, J. Zingg, F. E. Maly, and A. L. De Weck, J. Exp. Med. 167, 1281 (1988). 2 T. Puustinen, M. M. Scheffer, and B. Samuelsson, Biochim. Biophys. Acta 960, 261 (1988).

[62]

INTERCELLULARTRANSFEROF LTA4

I

Cell Membrane

Phospholipids

_ _ _

~

l I

569

®

( Phospho/ipase( ) s)

ArachidonicAcid

|

Uptake and metabolism by other cells

•~ t ' ~ [

'

//

I (~rA's,,,h,,, 3

//fPr°s"a/"a~"Synthases'~ //( rh~oxa,8 Syn~ase)

1

I l

¥

HPETE'sand HETE's] Incorprnotion into Modul~ion of ~ fz,~'timl Modulation of Lipox~em~e activity Lipoxyganasesubsti'ate in [ LipoxygenaseNetwork ]

,v^

LTA4 Stai~ilized by AIl~mn Uptake and r~etabeliun by other cells

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PGEz PGF2a

P°°.

LTC4 LTB4

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I P,.cur.o, .,-.,.]

Fro. 1. Hypothetical scheme representing the proposed consequences of precursor release in the 5-LOX pathway. Asterisks indicate that the enzyme is not activated in resting cells.

reacting synthetic LTA4 methyl ester with water, methanol, or ethanol acidified with HCI, respectively, and purification by reversed-phase highperformance liquid chromatography (RP-HPLC) before and after base hydrolysis of the ester.

Preparation of Neutrophils Portions of blood, 20 ml, anticoagulated with 20 U/ml preservative-free heparin (Novo Industries, Copenhagen, Denmark) are carefully layered over 15 ml Methocel metrizoate [10 g Methocel MC 25cP (Fluka AG, Buchs, Switzerland) is dissolved in 500 ml distilled water, degased, and mixed with Isopaque (Nyegaard & Co., Oslo, Norway) to obtain a density of 1.080 at 20°]. The erythrocytes which agglutinate at the interface are allowed to sediment at room temperature. The leukocytes are then aspirated leaving 0.5 cm of plasma at the interface to avoid contaminating the leukocyte suspension with Methocel, and further purified by means of Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) density separation (30 min, 400 g, room temperature). After hypotonic lysis of residual eryth-

570

CELL MODELS OF LIPID MEDIATOR PRODUCTION

[62l

rocytes (65 mOsmol, 20 sec, at 0 °) PMN are washed twice in Gey's solution at 4 ° and suspended in Dulbecco's PBS (both from GIBCO, Glasgow, Scottland) supplemented with 10 mg/ml fatty acid-free BSA (Pentex) and 2 mM glucose (DPBS/A) at a cell density of 107 to 108 ml. This PMN preparation is essentially free of platelets as judged by the abundant production of 20-COOH-LTB4, 20-OH-LTB4, LTB4, all-transLTB4, and 5-HETE in the almost complete absence of 20-OH-5(S), 12(S )DiHETE, 5(S),12(S)-DiHETE, HHT, and 12-HETE (determined by HPLC as described below), after stimulation with 10 ~ M Ca 2+ ionophore for 5 min. We believe this to be a better estimation of platelet contamination from morphological methods, due to their high capacity of 12-HETE and H H T formation and the difficulty inherent in microscopically counting the platelets, particularly when they are adherent to PMN. Platelet contamination may be considerable in isolation procedures that involve mixing of the blood with erythrocyte-agglutinating agents. Also cooling below room temperature has to be avoided before PMN are separated from the platelets. Platelets may affect LTA4 recoveries since they are able to transform LTA4 into LTC43,4 Another unsolved problem is the influence of eosinophils in the PMN preparation. Eosinophils are the major (or possibly even the sole) source of LTC4 in stimulated polymorphonuclear cells, but it is not yet known if they are able to metabolize low concentrations of albumin-bound LTA4.

HPLC Analysis of Lipoxygenase Products The HPLC system is composed of a U6K injector, two model 510 pumps, gradient controller, a column oven, and a variable-wavelength detector (Waters, Milford, MA). In more recent studies, we also used a diode array detector (System 990-plus, from Waters), which permits online spectral analysis of individual peaks of the chromatograms and even allows the distinction of the discrete spectral differences of LTB4 isomers and derivatives at the picomole level, i The neutrophil products are separated on a Nucleosil C18 column (250 x 4.6 mm; Alltech, Deerfield, IL) with a mobile phase of methanol/water/acetic acid (75 : 25 : 0.01, v/v/v) at a flow rate of 1 ml/min at 35° (Fig. 2). 5 Retention times (in minutes) of synthetic trienes are: to-oxidized leukotrienes, 4.55; PGB2 (internal standard), 7.81; 6-trans-LTB4, 9.5; 12-epi-6-trans-LTB4, 10.0; LTB4 and 5(S),12(S)-DiHETE, 10.8; LTB4 lactone, 15.0; 5,6-DiHETEs, 16.6 and 3 j. A. Maclouf and R. C. Murphy, J. Biol. C h e m . 263, 174 (1988). 4 C. Edenius, K. Heidvall, and J. A. Lindgren, Eur. J. Biochem. 178, 81 (1988). 5 C. A. Dahinden, R. M. Clancy, M. Gross, J. M. Chiller, and T. E. Hugli, Proc. Natl. A c a d . Sci. U . S . A . 82, 6632 (1985).

[62]

INTERCELLULARTRANSFEROF LTA4

571

ID

JI ! !

! ! .o

II

II II II

I

I

II

II

II

I

I

I

lO

20

30

I 40

RT Minutes FIG. 2. H P L C c h r o m a t o g r a m s of extracts from s u p e r n a t a n t s trapped with either methanol (solid line) or ethanol (interrupted line) obtained after stimulation o f 108 P M N with ionophore for 5 min.

18.0; 12-O-methyl-6-trans-LTB4isomers, 19.2 and 19.7; and 12-O-ethyl-6trans-LTB4 isomers, 27.2. More recently, we have established the following HPLC procedure for the analysis of LOX metabolites of PMN and platelets~: Stationary phase: Nucleosil C~8-100-5 /zm in a 25 × 0.4 cm column and a 5 × 0.4 cm precolumn, thermostatted at 35°. Mobile phase: Buffer A, water/acetonitrile/tetrahydrofuran/acetic acid (75 : 25 : 0.15 : 0.01, v/v/v/v); Buffer B, acetonitrile/methanol/acetic acid (57 : 43 : 0.01, v/v/v). Flow rate 1 ml/min. Gradient program: Initial, 100% A, 0% B; 0.01 min, 80% A, 20% B; isocratic until 7.5 min; 7.5-8.0 min, linear to 45% A, 55%

572

CELL MODELS OF LIPID MEDIATOR PRODUCTION

[62]

B; isocratic until 17.5 min; 17.5-18 min, linear to 25% A, 75% B; isocratic until 27.7 min; 27.7-28.2 min, back to initial. Retention times with baseline separation for each compound are as follows (in min): 20-COOH-LTB4, 12.39; 20-OH-LTB4, 13.17; 20-OH-5,12-DiHETE, 14.06; PGB2 (internal standard), 18.03; 6-trans-LTB4,20.36; 12-epi-6-trans-LTB4,20.66; LTB4, 21.05; 5(S ), 12(S)-DiHETE, 21.55; HHT (cyclooxygenaseproduct), 23.71; 5,6-DiHETEs, 25.42 and 25.76; 12-O-methyl-6-trans-LTB4, 26.54; 15HETE, 27.09; 12-HETE, 27.71; 5-HETE, 28.43.

Measurements of LTA4 Release by Alcohol Trapping Trapping is performed according to the procedure of Borgeat and Samuelsson. 5'6 Five minutes after exposure of PMN to 10/zm ionophore A23187 (Calbiochem, La Jolla, CA) the cells are sedimented in microfuge tubes (15,000 g, 30 sec, room temperature) and the supernatant immediately mixed with 9 volumes of methanol or ethanol previously acidified with sufficient HC1 to lower the pH of the aqueous buffer to 3. After 3 min, the samples are neutralized with NaOH and the alcohol evaporated until approx. 2 volumes alcohol is left, PGB2 is added as an internal standard, and precipitated proteins are removed by centrifugation. (It should be noted that prolonged exposure of the leukotrienes to acidified alcohol can result in the formation of lactones). The.sample is mixed with 2.5 volumes of diethyl ether and after a second acidification to pH 3 with HCI, a biphasic mixture is formed by the addition of 4 volumes water. The organic phase is dried under nitrogen and further purified by silicic acid chromatography [Silicar CC-4, Mallinckrodt; the sample is applied and washed in diethyl ether/hexane, 20 : 80 (v/v) and eluted with ethyl acetate]. Recoveries of LTB4 and the LTA4 hydrolysis products vary between 75-90% and are identical to that of the PGB2 internal standard. Clean up of the samples by silicic acid chromatography is not always necessary, and other extraction methods may also be useful, since any method that allows equally good recoveries of LTB4 and 5-HETE will also be suitable for alcoholtrapped LTA4, these products being intermediate in hydrophobicity. The LTA4 derivatives can be identified by (1) coelution with synthetic standards in HPLC using different solvents; (2) the typical UV absorption spectra with hmaxat 268.0 nm and shoulders at 258.5 and 279.5 nm indicative of an all-trans-triene configuration (Fig. 3); (3) gas chromatographymass spectrometry (GC-MS) of the purified compounds as described. 5"6 However, we feel that GC-MS is not always necessary for identification of trapped LTA4, if the products are only formed after alcohol trapping and 6 p. Borgeat and B. Samuelsson, Proc. Natl. Acad. Sci. U.S.A. 76, 3213 (1979).

[62]

INTERCELLULAR TRANSFER OF LTA4

280

573

A

268280

B

!,p

1'2 j¢

12-O-Met~J/ ~I

LTC4

p

I

220

I

I

240

I

I

260

I

I

280

I

I

300

i

I

I

320

220

I

I

240

I

I

260

I

I

280

I

I

I

300

I

320

FIG. 3. UV spectra of trienes in methanol. (A) UV spectra of synthetic LTA4 and of the products (12-O-methyl derivatives) which are immediately formed after addition of HCI. (B) UV spectra of 12-O-methyl-6-trans-LTB4 purified from methanol-trapped supernatants of stimulated PMN (interrupted line), and of LTC4 purified from MC after exposure to albuminbound LTA4 (solid line).

are absent under otherwise identical conditions. Also, 12-O-methyl-6trans-LTB4 epimers always elute after the 5,6-DiHETEs (hmax at 272 nm) in all solvent systems examined so far, and we never detect a leukocytederived triene with a hmax at 268 nm other than trapped LTA4 in this region of the chromatogram. Figure 2 demonstrates that the epoxide LTA4 is released by ionophorestimulated PMN without considerable intracellular nonenzymatic hydrolysis. In seven experiments performed in duplicates we found that a mean of 136 pmol LTA4/10 7 PMN (range 40-300) could be trapped by alcohols in the supernatants of PMN 5 min after ionophore stimulation. 5 These data demonstrate that LTA4 is a major LOX product released by stimulated PMN. However, these values are only minimal estimates, since (1) some LTA4 may be released but remain cell-associated; (2) some LTA4 may decay until trapping is performed even in the presence of albuminT; (3) a 7 A. Fitzpatrick, D. R. Morton, and M. A. Wynalda, J. Biol. Chem. 257, 4680 (1982).

574

CELL MODELS OF LIPID MEDIATOR PRODUCTION

[62]

fraction of LTA4 will react with residual water after acidification. Our findings have been confirmed and extended by several groups which showed that PMN-derived LTA4 can be metabolized into LTB4 and LTC4 by cells which are by themselves unable to produce leukotrienes. 3"4'8-~° The enhancement of LTB4 or LTC4 formation through intercellular transfer of LTA4 is (values expressed per 107 PMN): 250-300 pmol LTB4 (PMN-erythrocytes)8; 350 pmol LTC4 by coincubation 8 min after PMN activation, or 105 pmol from PMN supernatants (PMN-platelets)3; 72 pmol LTC4 from coincubations (PMN-platelets)4; 90 pmol LTC4/D4/E4 and 75 pmol LTB4 by coincubation of PMN with endothelial cells. 9 Although other types of cellular interactions may exist in coincubation experiments, 4"9 these values are in good agreement with our trapping experiments. It is interesting that a recent report demonstrated the formation of lipoxins in coincubation experiments of PMN and platelets stimulated with ionophore, probably through the intermediate of LTA4 released by PMN. ~~ Up to now, lipoxins were only formed under very artificial conditions. Therefore, intercellular transfer of LTA4 may also be the major pathway of lipoxin formation. There are no other established methods for the measurement of cellderived LTA4. Gut et al.12 recently described a procedure for the measurement of low amounts of LTA4 which involves reversed-phase HPLC in a volatile buffer followed by direct chemical ionization mass spectrometry, but methods for efficient LTA4 extraction from complex biological fluids have not been worked out. It is quite possible, however, that intracellular decay of LTA4 does not occur at all. Indeed, no all-transLTB4 isomers were detected and large amounts of LTC4 were formed when ionophore-stimulated PMN were infused in lung preparations, ~3 indicating that all the PMN-derived LTA4 was enzymatically metabolized by the tissue cells. Thus, an estimate of LTA4 release may be simply made by measuring the sum of the nonenzymatic products. However, estimates of LTA4 release or consumption based upon the measurement of all-transLTB4 isomers should be interpreted with caution if alcohols are added to a sample that has not been previously acidified (see, i.e., Refs. 4 and 9; in 8 j. E. McGee and F. A. Fitzpatrick, Proc. Natl. Acad. Sci. U.S.A. 83, 1349 (1986). 9 H. E. Claesson and J. Haeggstrom, Eur. J. Biochem. 173, 93 (1988). lo S. J. Feinmark and P. J. Cannon, J2 Biol. Chem. 261, 16466 (1986). 11 C. Edenius, J. Haeggstrom, and J. A. Lindgren, Biochem. Biophys. Res. Commun. 157, 801 (1988). 12 j. Gut, J. R. Trudell, and G. C. Jamieson, Biomed. Enoiron. Mass. Spectrom. 15, 509 (1988). ,3 F. Grimminger, M. Menger, G. Becker, and W. Seeger, Blood 72, 1687 (1988).

[62]

INTERCELLULARTRANSFER OF LTA4

575

these papers the possible formation of 12-O-ethyI-LTA4 derivatives has not been considered). M e t a b o l i s m of A l b u m i n - B o u n d L T A 4 into L T C 4 b y M a s t Cel l s

Analysis of Sulfidoleukotrienes Cellular reactions are stopped by adding 0.6 volumes of 2-propanol and allowing the mixture to stand at room temperature (T) for at least 5 min (leukotrienes are stable in this mixture for at least several hours at 4°). Immediately after acidifying with 0.03 volumes of 5 M formic acid, 1.5 volumes of ether is added, resulting in the development of two phases. The organic phase is harvested and 0.015 volumes of 10 N NH4OH is added. After evaporation to dryness under nitrogen the residue is dissolved in 0.5 ml of HCCl3/methanol (1 : 1) and 0.22 ml of 10 mM NH4OH is added. The upper water/methanol phase containing the sulfidoleukotrienes. 14 is collected, dryed, and dissolved in HPLC solvent. The sulfidoleukotrienes are separated on a Nucleosil C~8-100 5-~m column (250 x 4.6 cm) with a mobile phase of methanol/3.5 mM ammonium acetate in water, pH 5.7 (apparent pH) (65 : 35, v/v), at a flow rate of 1 ml/min. 5"15 LTC4

Formation by Mast Cells

Mast cells (MC) are obtained from cultures of bone marrow cells of BDFI mice in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated horse serum and 10% WEHI 3 supernatant (containing interleukin 3). 15 After 8 weeks of culture and selection of the nonadherent cells every 3-4 days, the cells are >95% MC as judged by staining with toluidine blue, the presence of F c d receptors, and the production of L T C 4 upon activation with ionophore or IgE receptor cross-linking. 5 MC are washed and suspended in DPBS/A at 10 7 cells/ml. After evaporation to dryness under nitrogen, LTA4 methyl ester (I raM) is hydrolyzed in ethanol/50% NaOH in water (9: 1), at 4° for 3 hr. Alternatively, the lithium salt is obtained as described. 8 The concentration, integrity, and purity of LTA4 can be estimated by UV spectroscopy (Fig. 2), HPLC, TM and trapping with alcohols followed by HPLC. LTA4, which is prepared fresh for each experiment, is rapidly mixed with ice-cold DPBS/A at the desired concentration, warmed to 37° just before use, and added to MC in DPBS/A in a l : ! volume ratio (the final concen14 R. M. Clancy and T. E. Hugli, Anal. Biochem. 133, 30 (1983). t5 E. Razin, L. C. Romeo, S. Krilis, F.-T. Liu, R. A. Lewis, E. J. Corey, and K. F. Austen, J. lmmunol. 133, 983 (1984).

576

CELL MODELS OF LIPID MEDIATOR PRODUCTION

[62]

AI

=e

1

o ¢o N

== o

I 10

! 20

30

RT (Minutes)

MC + LTA4

LTC, pm01es

+FPL

sythetcSS 1

2.5

F

10

LTC4

25 pm01es

LTC,

LTC,

~

~'

~

pmoles

1

2.5

10

I

C5a

100 ng

I 1 Minute

FIG. 4. LTC4 produced by mast cells from albumin-bound LTA4. (A) HPLC chromatogram of the extract from 5 x 106 MC after incubation with 100 pmol LTA4 for 10 min. (B) Ileum contractions induced by synthetic LTA4 and MC-derived LTC4 purified by HPLC. The response to LTC4 but not C5a is abolished by 5/zM FPL 55712.

[62]

INTERCELLULARTRANSFEROF LTA4

577

tration of ethanol does not exceed 0.2%). After the indicated time, the reaction is stopped by adding 0.6 volumes of 2-propanol. The metabolism of albumin-bound LTA4 by MC is very efficient and rapid. 5 More than 50% of 2 nmol LTA4 is metabolized into LTC4 by 10 7 MC within I0 to 15 min and no conversion to LTD4 and LTE4 is observed during up to 30 min incubation at 370.5 The production of L T C 4 is linearly correlated with the concentration of LTA4 added from 0. I to 2/zM LTA4. Most important, when 5 x 106 ME are exposed to 100 pmol albuminbound LTA4 for 10 min, 80-87 pmol L T C 4 (mean 83, N = 6) could be measured in the cell cultures (Fig. 4). Thus, MC are able to transform TLA4 even if bound to albumin and in amounts shown to be released by stimulated PMN. The pronounced stabilizing effect of albumin 7 together with the exceedingly short half-life of LTA4 in neutral buffers indicates that LTA4 is bound to albumin with high affinity. The efficient and rapid metabolism of low amounts of LTA4 by MC is, therefore, surprising and may suggest the presence of an LTA4 binding site on MC membranes. Labeling experiments demonstrate that L T C 4 produced by MC derived exclusively from exogenous LTA4. 5 When tritiated LTA4 at variable concentrations is added to MC, the L T C 4 produced has the same specific activity. Furthermore, MC are labeled with 0.5/zCi tritiated arachidonic acid per milliliter (86 Ci/mmol, Amersham) overnight, washed, and cultured for an additional 3 hr in label-free medium. After two washes, the labeled MC are then exposed to either 5/xM ionophore or 1 nmol LTA4 for l0 min. After purification by HPLC, radioactivity is found only in the LTC4 fraction from ionophore-stimulated MC (95,000 cpm/nmol). LTC4 produced by MC after exposure to albumin-bound LTA4 is identified after purification by HPLC by the following criteria: coelution with s y n t h e t i c LTC4 on reversed-phase HPLC (Fig. 4A); UV spectroscopy (Fig. 3B), bioactivity on guinea pig ileum (Fig. 4B); and hydrolysis of 4 nmol o f L T C 4 in 6 M HC1 at 110° for 24 hr and amino acid analysis with a Beckman model 121M analyzer, which demonstrates the presence of glutamic acid and glycine in equimolar concentrationsJ Acknowledgments This work was supported in part by the Swiss National Science Foundation grant #3.058-0.87. We are grateful for the contributions ofR. M. Clancy, M. Gross, J. M. Chiller, and T. E. Hugli.