* Department of Virology and
Department of Medical Microbiology & Immunology, Göteborg University, Sweden
Correspondence: 
sa Betten, The Phagocyte Research Laboratory, Department of Medical Microbiology & Immunology, Göteborg University, Göteborg, Sweden. E-mail:
Aasa.Betten{at}microbio.gu.se
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Key Words: monocytes • hydrogen peroxide • scavenger • respiratory burst • myeloperoxidase
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(IFN-) are effective
activators of NK-cell-mediated cytotoxicity against malignant cells in
vitro and in vivo, and these effects are believed to mediate, at least
in part, the therapeutic benefit of these cytokines in neoplastic
diseases [4
5
6
7
]. NK cell activation is also assumed to
contribute to the antitumor properties of IL-12, IL-15, and IL-18 in
vivo in experimental tumor models and in humans [8
9
10
]. In recent years, several investigators have reported that the tissue within or adjacent to malignant tumors is frequently subjected to oxidative stress, presumably mediated by tumor-infiltrating cells of the monocyte/macrophage lineage. The oxidative stress, which is defined as toxicity inflicted by reactive oxygen species (ROS), is assumed to contribute to the state of immunosuppression in the tumor area by inhibiting the function of NK cells and other lymphocytes of relevance to protection against neoplastic cells [11 12 13 ]. Typically, lymphocytes within or adjacent to tumors show a high degree of apoptotic cell death [14 ] and frequently display deficient cell surface expression of signal-transducing molecules [12 , 15 16 17 ]. A critical point is that IL-2 and other NK-cell-activating cytokines are ineffective in an environment of oxidative stress [12 , 18 , 19 ]. Hence, studies of the interactions between ROS-generating monocytes and NK cells may lead to the identification of compounds that can rescue NK cells from oxidative inhibition, which may be exploited therapeutically.
Serotonin is a biogenic amine, which is stored in peripheral tissues in platelets and in neurochromaffin cells of the gut mucosa [20 , 21 ]. In humans, serotonin is released from activated platelets at inflammatory sites and in ischemic tissues, and it may reach local concentrations of 100 µM at the immediate site of release [22 ]. Earlier in vitro studies have revealed that serotonin is an activator of human NK cells by regulating an interaction between NK cells and monocytes [23 24 25 ], but the mechanistic details of these activating properties are not known. Here we show that serotonin protects NK cells from monocyte-derived inhibitory and apoptosis-inducing signals conveyed by ROS; in the presence of serotonin, NK cells remain viable and functionally active and can be activated by IL-2 despite the presence of suppressive monocytes. Our data suggest that a target for serotonin is ROS derived from myeloperoxidase (MPO), a monocyte enzyme that utilizes hydrogen peroxide (H2O2) to generate toxic oxygen radicals [26 , 27 ].
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The mononuclear cells were further separated into lymphocytes and
monocytes using the counter-current centrifugal elutriation technique,
as described in detail elsewhere [18
, 28
].
Briefly, the mononuclear cells were resuspended in elutriation buffer
containing 0.5% bovine serum albumin and 0.1% EDTA in buffered NaCl
and fed into a Beckman J2-21 ultracentrifuge with a JE-6B rotor
(Beckman Coulter Inc., Fullerton, CA) at 2,100 rpm. A fraction with
>90% monocytes was obtained at a flow rate of 18 mL/min. A lymphocyte
fraction enriched for NK cells (CD3
-/56+
phenotype) and T cells (CD3+/56- phenotype)
was recovered at flow rates of 14–15 mL/min. The latter fraction
consisted of CD3-/56+ NK cells (45–50%),
CD3+/56- T cells (35–40%),
CD3-/56- cells (5–10%), and
CD3+/56+ cells (1–5%) with <3%
contaminating monocytes, as judged by flow cytometry. The mixture of NK
and T cells was exposed to autologous, elutriated monocytes in
microplates. Lymphocytes (100,000 cells/well in 200 µL) were
incubated with or without monocytes (5,000–100,000 cells/well) for
16 h at 37°C after which they were assayed for cytotoxic
function, phenotype, and viability.
Target cells and microcytotoxicity assay
Cells from an NK-cell-sensitive cell line (K562) originating
from patients with chronic myelogenous leukemia in blast crisis
[29
] were used as target cells. These cells (5 to
10 x106 cells/mL) were loaded with 51Cr at
a concentration of 150 µCi/mL of cell suspension for 2 h at
37°C. Excess 51Cr was removed by centrifugation and
resuspension of the target cells in culture medium. Finally, 10,000
51Cr-loaded target cells in 50-µL portions were added to
the mononuclear cells in 96-well microplates (Nunc, Roskilde, Denmark).
The NK-cell-enriched lymphocytes and target cells were incubated in
sextuplicates in microplates in a total volume of 200 µL in the
presence or absence of monocytes. The compounds were added at the onset
of incubation with the exception of
formylmethionyl-leucyl-phenylalanine (fMLF), which was added at time
t = 15 min. After incubation at 37°C for 16 h,
supernatant fluids were collected by a tissue-collecting system
(Amersham Pharmacia Biotech AB, Uppsala, Sweden) and assayed for
radioactivity in a 
-counter. Maximum 51Cr release was
determined in target cell cultures treated with Triton X-100. NK-cell
cytotoxicity was calculated using the formula 100 x
[(experimental 51Cr release - spontaneous
release)/(maximum release - spontaneous release)] = cell lysis
%.
In accordance with earlier studies [18 , 19 ], >90% of the lymphocyte cytotoxicity against K562 cells was depleted by the removal of CD56+ NK cells (by use of anti-CD56-coated beads [18 , 19 ]); in contrast, the removal of CD3+ T cells (by use of anti-CD3-coated beads) from the effector lymphocyte preparations did not significantly reduce cytotoxicity.
Determination of NADPH-oxidase activity
Monocyte superoxide production was determined using a
luminol/isoluminol-amplified chemiluminescence (CL) technique
[30
]. Samples containing 500,000 elutriated monocytes in
Krebs ringer glucose buffer were incubated in a six-channel Berthold
Biolumat LB 9505 (Berthold Technologies Co., Wildbad, Germany) at
37°C. Release of superoxide was measured in the presence of 10
µg/mL of isoluminol and 4 U/mL of horseradish peroxidase (HRP), and
the intracellular production of ROS was measured in the presence of 10
µg/mL of luminol, 20 U/mL of superoxide dismutase (SOD), and 2000
U/mL of catalase. The activity was determined without any additive, or
cells were activated by the addition of 0.1 µM fMLF for induction of
extracellular radical production or by 0.1 µM ionomycin for
intracellular production of ROS.
H2O2 consumption
The consumption of H2O2 by serotonin or
5-hydroxytryptophan (5-HTP) in a cell-free environment was measured
using a p-hydroxyphenylacetate (PHPA)-HRP fluorescence
system [31
]. PHPA was excited at a wavelength of 317 nm,
and a Perkin-Elmer luminescence spectrometer (LS 50 B; Perkin-Elmer
Inc., Norwalk, Connecticut) registered light emitted by oxidized PHPA
at 400 nm.
Apoptosis assays
Apoptosis was detected by flow cytometry [28
].
The fluorescein-activated cell sorter gate was set to comprise
lymphocytes with a reduced forward scatter and an increased right-angle
scatter characteristic of apoptosis [32
]. Two additional
methods were used for the determination of apoptosis in NK cells:
analysis of DNA strand breaks by terminal deoxynucleotidyl
transferase-mediated bromolated deoxyuridine triphosphate nick-end
labeling (TUNEL assay) of DNA fragments (APO-BRDUTM kit, PharMingen,
San Diego, CA) [19
] and detection of extracellular
expression of phosphatidyl serine [Annexin V-fluorescein
isothiocyanate (FITC) apoptosis detection kit I, Pharmingen]
[19
].
Detection of surface antigens
One million cells were incubated with appropriate FITC- and
phycoerythrin (PE)-conjugated monoclonal antibodies (1
µL/106 cells) on ice for 30 min. The cells were washed
twice in PBS, resuspended in 500 µL of sterile filtered PBS, and
analyzed by flow cytometry on a FACSort with a Lysys II software
program (Becton Dickinson, Stockholm, Sweden). Lymphocytes were gated
on the basis of forward and right-angle scatter. The flow rate was
adjusted to <200 cells s-1, and at least 10,000 cells were
analyzed for each sample.
Compounds
The following compounds were used: serotonin hydrochloride,
5-HTP, luminol, isoluminol, fMLF, and PHPA (Sigma Chemical Co., St.
Louis, MO); human recombinant IL-2 (Genzyme, Stockholm, Sweden); a Fas
ligand inhibitor [comprising the extracellular domain of human Fas,
amino acids 1 through 154, fused to the Fc portion of human
immunoglobulin (Ig) G1; Kamiya Biomedical Co., Seattle, WA]
[19
]; ionomycin (Calbiochem, La Jolla, CA); MPO (kindly
provided by I. Olsson, Lund, Sweden); HRP, SOD, and catalase
(Bohringer-Mannheim, Mannheim, Germany); Dextran (Kabi Pharmacia,
Stockholm, Sweden); acid citrate dextrose (Baxter, Deerfield, IL);
51Cr (Amersham); bovine serum albumin (ICN Biomedicals,
Inc., Aurora, OH); and EDTA and H2O2 (KEBOLab,
Göteborg, Sweden). All compounds were readily dissolved in
Iscove’s culture medium. FITC- and PE-conjugated monoclonal antibodies
(mAbs) against CD3, CD56, and CD69 were purchased from Becton
Dickinson. Reagents and media were regularly checked for the presence
of endotoxin using the Limulus amoebocyte assay.
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 View larger version (40K): [in a new window] |
Figure 1. Inhibition of NK-cell cytotoxicity by monocytes: reversal by serotonin
and catalase. Eighty thousand NK-cell-enriched lymphocytes were admixed
with 80,000 monocytes and assayed for cytotoxicity against K562 cells
(104 cells/well) in a 16-h assay. The cell cultures were
treated with culture medium (control, open bars), serotonin (100 µM;
dark-gray bars), catalase (100 U/mL; light-gray bars) or serotonin plus
catalase (black bars), added at the onset of incubation. Inset A:
titration of monocytes [10–50% of all mononuclear cells (abscissa)]
with a fixed amount of NK-cell-enriched lymphocytes (80,000/well); open
circles, culture medium; closed circles, serotonin (100 µM). Inset B:
dose-response experiment using serotonin concentrations of 1–100 µM
(abscissa) at a monocyte-to-lymphocyte ratio of 1:1. All data are cell
lysis (%) ± SE of sextuplicate determinations, and
similar results were obtained in eight experiments, using blood from
different blood donors. In these experiments, the NK-cell-inhibitory
effect of monocytes was statistically significant at 25–50% monocytes
(P<0.01), and the effect of serotonin at a 1:1 ratio
between monocytes and lymphocytes was statistically significant at
final serotonin concentrations of 1-100 µM (Mann-Whitney U-test,
P<0.05-0.001).
|
 View larger version (16K): [in a new window] |
Figure 2. Protection of NK cells from monocyte-induced inhibition by catalase and
serotonin. Eighty thousand NK cell-enriched lymphocytes were admixed
with 80,000 monocytes and assayed for cytotoxicity against K562 cells
(104 cells/well) in a 16 hr assay. The cell cultures were
treated with catalase at indicated concentrations (open circles), alone
or together with serotonin (100 µM; filled circles), added at the
onset of incubation. Data are cell lysis % ± SE of
sextuplicate determinations, and similar results were obtained in four
experiments using blood from four different blood donors.
|
Role of ROS
Taken together, these data suggest that serotonin protects NK
cells against monocyte-induced inhibition by interference with ROS
produced by monocytes. Next we studied the effects of serotonin on
NK-cell function in a model designed to simulate suppression by
monocyte-derived ROS. In these experiments, monocytes were replaced by
exogenously added H2O2. In accordance with
earlier studies [28
], H2O2 was
found to strongly suppress NK-cell cytotoxicity. However, NK cells were
not protected from the H2O2-mediated inhibition
by the addition of serotonin, even at high concentrations (Fig. 3
).
 View larger version (25K): [in a new window] |
Figure 3. Serotonin protects NK cells against inhibition of cytotoxicity
induced by H2O2 and MPO. Eighty thousand NK
cell-enriched lymphocytes were assayed for cytotoxicity against K562
cells (104 cells/well) in a 16 hr assay. The cell cultures
were treated with culture medium (control, open circles), serotonin
(100 µM; filled circles), MPO (open triangles), or serotonin +
MPO (filled triangles). The final concentrations of
H2O2 are indicated on the abscissa. All
compounds were added at the onset of incubation. Similar results were
obtained in seven experiments using blood from seven different blood
donors; in these experiments, the protection achieved by serotonin + MPO was statistically significant as compared with MPO alone
(P < 0.001; Student’s t-test). The inset shows one
representative experiment out of four, where A is the control, and B
the suppression of NK cell cytotoxicity induced by 150 µM
H2O2 and HRP. The cell cultures were treated
with culture medium (control, open bars), serotonin (100 µM; dark
grey bars), HRP (light grey bars) or serotonin + HRP (filled
bars). Data are cell lysis % ± SE of quadruplicate
determinations.
|
To determine whether serotonin protected NK cells from peroxidase-derived radicals in general or whether the protection was specific for MPO-derived products, we introduced HRP into the experimental system. Serotonin reversed the inhibition of NK-cell cytotoxicity induced by HRP plus H2O2 in a fashion similar to that observed for MPO + H2O2 (Fig. 3 , inset).
Monocyte-induced apoptosis
Earlier studies revealed that monocytes can induce programmed cell
death or apoptosis in NK cells and that catalase or inhibitors of ROS
formation, such as diphenyleneiodonium or histamine, afford protection
against monocyte-triggered apoptosis [19
,
28
]. Therefore, we investigated whether serotonin
protected NK cells against monocyte-induced apoptosis. In these
experiments, flow cytometry was used to estimate the frequency of
apoptotic NK cells by gating a viable and an apoptotic lymphocyte
population, based on size and granulation of cells (forward and side
scatter) [19
, 28
]. Apoptosis was confirmed
by two methods: the Annexin V-binding assay and DNA fragmentation (by
use of the TUNEL assay). These experiments revealed that serotonin
effectively rescued NK cells from apoptosis induced by autologous
monocytes as well as apoptosis induced by H2O2
plus MPO (Fig. 4A
).
 View larger version (19K): [in a new window] |
Figure 4. Serotonin rescues viability and inducibility of NK cells from
inhibition triggered by H2O2 and MPO. Cells
were prepared and treated with serotonin, MPO and
H2O2 as described in the legend to Figure 3
.
After incubation for 16 hrs, the frequency of apoptotic lymphocytes was
determined fluorimetrically by use of an Annexin V binding assay (A).
Apoptosis was confirmed by TUNEL assay and by the appearance of
apoptotic morphology. The inset shows the morphological apoptosis (%)
induced by H2O2 at 150 µM, and the bars
represent the mean ± SE of data obtained using blood
from three different blood donors; in these experiments, MPO alone
protected lymphocytes from apoptosis (P<0.02), and the
protection against apoptosis achieved by serotonin + MPO was
statistically significant as compared with that achieved by MPO alone
(P<0.02; Student’s t-test). The cells were also
treated with IL-2 (100 U/ml; 16 hr), and gated viable NK cells were
assayed for expression of the CD69 activation antigen (B). The inset
shows the percentage of cells with CD56+/69+
phenotype at a H2O2 concentration of 150 µM
and are the mean ± SE of data obtained using cells
from four different blood donors; in these experiments, the enhanced
CD69 expression achieved by serotonin + MPO was statistically
significant as compared with that induced in cells treated with MPO
alone (P<0.05; Student’s t-test).
|
Maintenance of NK-cell activation
In the next series of experiments, we investigated whether the
fraction of NK cells that remained viable after treatment with
H2O2 plus MPO could be activated or instead
were in a state of anergy. We used IL-2, which is a recognized inducer
of the CD69 activation antigen in NK cells [19
,
35
], and studied the acquisition of this antigen on the
cell surface of viable NK cells incubated with monocytes. IL-2 induced
the appearance of CD69 on >70% of NK cells, and this response was
almost completely abolished by exogenous H2O2
alone or by H2O2 plus MPO. Serotonin did not
maintain IL-2-induced activation in NK cells treated with
H2O2 alone, but it effectively protected NK
cells from inhibition induced by H2O2 plus MPO
(Fig. 4B)
.
Scavenger activity of serotonin
These data suggest that serotonin protects NK cells from
functional inhibition and subsequent apoptosis induced by
monocyte-derived ROS and specifically that serotonin rescues NK cells
from oxidative damage induced by H2O2 and a
peroxidase such as MPO. Earlier studies demonstrated that serotonin and
structurally related compounds can be oxidized by
H2O2 [36
], but conflicting data
suggest that serotonin is instead a pro-oxidant
[37
38
39
]. To establish whether serotonin is a scavenger
of ROS and in particular of MPO-derived ROS, we determined ROS-induced
CL after treatment with fMLF, which induces extracellular ROS
generation in monocytes [40
], or with ionomycin, which
triggers intracellular ROS formation [41
]. We also
studied cell-free systems in which serotonin was coincubated with
H2O2, alone or together with a peroxidase
[31
].
The extracellular fMLF-induced ROS in monocytes was strongly suppressed by serotonin (Fig. 5A and B ). To reveal whether serotonin was acting via a serotonin receptor on monocytes, we first compared the efficiency of serotonin to inhibit extracellular ROS with that of its precursor, 5-HTP, which has low activity at serotonin receptors [42 ]. Serotonin and 5-HTP were equally effective in scavenging extracellular ROS, regardless of whether ROS were generated by fMLF (Fig. 5A) or occurred spontaneously (Fig. 5C) . Neither of these compounds suppressed the intracellular radical production induced by ionomycin (Fig. 5D) .
 View larger version (30K): [in a new window] |
Figure 5. Effect of serotonin and its precursor 5-HTP on monocyte radical
formation, measured by luminol/isoluminol-amplified CL. In experiment
A, the activity was measured using 0.1 µM fMLF-treated monocytes
(500,000/well) as source of extracellular radicals. Experiment B is a
dose-response analysis for serotonin and 5-HTP in this experimental
system. In experiment C, the scavenger properties of serotonin/5-HTP
were monitored without the addition of fMLF (spontaneous radical
release). Serotonin or 5-HTP did not affect intracellular radical
production induced by fMLF, PMA, or the Ca2+ ionophor
ionomycin; D shows an experiment using ionomycin (0.5 µM) as the
inducer. Similar results were obtained in five separate experiments.
|
 View larger version (37K): [in a new window] |
Figure 6. The scavenging activity of serotonin (5 µM; filled bars) or 5-HTP (5
µM; grey bars) was measured in a
PHPA-H2O2-HRP system. To determine whether
H2O2 (10 µM) was consumed by serotonin/5-HTP,
HRP was added either at the onset of incubation with
H2O2 or after a 2 min pre-incubation with
H2O2. The ratios between the fluorescence
values obtained with preincubation periods of 0 and 2 minutes,
respectively are shown in the inserted table. The figure shows mean
values ± SD of three different experiments.
|
We therefore compared the effects of serotonin and 5-HTP on the cytotoxicity of NK cells against K562 cells in the presence of monocytes. Despite the similarities in scavenging oxygen radicals, there was a clear difference between the abilities of serotonin and 5-HTP to protect NK cells from the down-regulatory effects induced by monocytes. Serotonin was at least 10-fold more efficient than 5-HTP in preventing inhibition of cytotoxicity (Fig. 7A ) as well as in protecting NK cells against H2O2 plus MPO (Fig. 7B) .
 View larger version (11K): [in a new window] |
Figure 7. Effects of serotonin and 5-HTP on monocyte-induced or
H2O2/MPO-induced inhibition of NK cells. In A,
cells were prepared and analyzed as described in the legend to Figure 1
. In B, NK cell-enriched lymphocytes were treated with
H2O2 (50 µM) and MPO, as decribed
in the legend to Figure 3
. Data are cell lysis % ± SE of
sextuplicate determinations, and similar results were obtained in five
experiments using blood from five different blood donors. In these five
experiments, the cytotoxicity of serotonin-treated cells was
significantly superior to that of medium-treated control cells, both in
the presence of monocytes and when cells were treated with
H2O2/MPO, at final serotonin concentrations of
1, 5, 10, 50 and 100 µM (P<0.05-0.001, Mann-Whitney
U-test). Serotonin was significantly superior to 5-HTP, both when
measuring monocyte-induced and H2O2-induced
inhibition of NK cells, at final compound concentrations of 10, 50, and
100 mM (P<0.02-0.01). 5-HTP significantly protected NK
cells against monocyte-induced inhibition or inhibition induced by
H2O2/MPO at final 5-HTP concentrations of 500
and 1000 µM (P<0.02, data not shown).
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A role for serotonin in regulating NK-cell function was suggested by the findings that serotonin augments the cytotoxicity, cytokine-producing capacity, and proliferation of NK cells in the presence of autologous monocytes [23 , 24 ]. Subsequent work has revealed that serotonin reverses an NK-cell suppressive, monocyte-derived signal [6 ]. This study, aimed at elucidating the mechanism of action by which serotonin protects NK cells, used three models—one studying effects of serotonin on monocyte/NK-cell interactions, another studying effects of serotonin on NK-cell inhibition induced by exogenous ROS, and a third studying the putative scavenger properties of serotonin.
Our studies of serotonergic regulation of monocyte/NK-cell interactions confirm and extend previous findings by demonstrating that serotonin protects NK cells not only against inhibition of cytotoxicity but also against monocyte-induced apoptosis and anergy to IL-2; thus, serotonin maintained the anti-tumor cytotoxicity of NK cells, preserved the IL-2-induced acquisition of the CD69 activation antigen, and rescued NK cells from cell death by apoptosis despite the presence of ROS-producing monocytes. Serotonin was as effective as catalase, a known scavenger of H2O2, in this regard, and the combination of catalase and serotonin did not further protect NK cells.
Serotonin was also found to protect NK cells from oxidative damage induced by exogenous ROS. Serotonin did not rescue NK-cell function and viability after treatment with H2O2, but it afforded efficient protection against H2O2 in combination with a peroxidase such as MPO or HRP. In line with these findings, our data demonstrate that serotonin is a potent scavenger of ROS derived from H2O2 and a peroxidase but not of H2O2 alone.
MPO is present in monocytes and is released into the extracellular space on activation [40 ]. Earlier studies suggest that a significant part of the monocyte-induced inhibition of NK cells is dependent on the activity of the H2O2-MPO radical-generating system [28 ]. We therefore put forward the hypothesis that the consumption of ROS induced by serotonin significantly contributes to its NK-cell-protective properties; by neutralizing extracellular, peroxidase-derived toxic-oxygen products released by monocytes, serotonin preserves NK-cell function and prevents apoptosis.
The scavenger activity of serotonin can be explained either by an interaction with the peroxidase or, more likely, indirectly via neutralization by serotonin of an unidentified ROS, generated by the MPO-H2O2 system. The ability to consume MPO-H2O2-derived radicals is not unique to serotonin but is shared also by its precursor, 5-HTP. An unexpected finding was that, whereas serotonin and 5-HTP were equally effective scavengers of H2O2-peroxidase-derived radicals, serotonin was >10-fold more effective in protecting NK cells against inhibition induced by monocytes as well as by H2O2 plus MPO. The mechanism explaining this difference between serotonin and 5-HTP should be the focus of further study. Two mechanisms are proposed to account for these findings: (1) the attachment of serotonin to cellular receptors expressed on NK cells may more effectively neutralize MPO-derived ROS in direct connection with the cell membrane, or (2) serotonin triggers an unidentified intracellular signal in NK cells, which protects these cells against oxidatively induced inhibition and apoptosis.
In conclusion, our data reveal a novel immunoregulatory property of serotonin, namely to protect NK cells from oxidant stress. This mechanism may shed further light on the role of serotonin in immune and inflammatory reactions, in which serotonin may serve to neutralize ROS generated by adjacent phagocytes and thereby to preserve the viability and function of NK cells. The protection may be relevant in the host defense against neoplastic cells, since a large number of recent studies report that lymphocytes localized within or adjacent to human and murine cancer tumors frequently display signs of oxidative inhibition. Thus, intratumoral NK cells and other lymphocytes endowed with antitumor activity show reduced viability [14 ] and frequently display reduced cell surface expression of critical signal-transducing molecules [12 , 15 16 17 ]; this form of tumor-induced immunosuppression has been attributed to ROS formation by adjacent monocytes/macrophages [12 , 15 16 17 ]. Therefore, it can be speculated that serotonin or other substances with similar protective properties could be exploited therapeutically as, e.g., adjuncts to immunotherapy with IL-2 or other compounds aimed at activating lymphocyte-mediated tumor cell destruction.
We are obliged to Marie-Louise Landelius for excellent technical assistance.
Received July 5, 2000; revised February 21, 2001; accepted February 22, 2001.
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