The Journal of Immunology, 1999, 162: 829-836.
Copyright © 1999 by The American Association of Immunologists
Critical Requirement for Aspartic Acid at Position 82 of Myelin Basic Protein 73–86 for Recruitment of Vß8.2+ T Cells and Encephalitogenicity in the Lewis Rat
Ronald B. Smeltz*,
Marca H. M. Wauben
,
Norbert A. Wolf* and
Robert H. Swanborg*
*
Departments of Immunology and Microbiology, Wayne State University School of Medicine, Detroit, MI 48201; and
Institute of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
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Abstract
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We synthesized single amino acid-substituted peptide analogues of
guinea pig myelin basic protein (MBP) 73–86 to study the importance of
aspartic acid at residue 82 (QKSQRSQDENPV), which
previous reports have suggested is a critical TCR contact
residue. Whereas the wild-type 73–86 peptide elicited severe
experimental autoimmune encephalomyelitis (EAE) in the Lewis rat, none
of the peptide analogues with substitutions at position 82 were capable
of inducing EAE. The inability to cause EAE was not due to a failure to
bind MHC or to elicit T cell proliferation and cytokine secretion. T
cells specific for MBP73–86 did not cross-react with any of the
analogues tested, further indicating the importance of this residue in
T cell responses to 73–86. Analysis by flow cytometry showed that only
the wild-type 73–86 peptide was capable of recruiting
Vß8.2+ T cells, which have been shown previously to be
important for disease induction. Reduced expression of the Vß8.2 TCR
was also seen in Lewis rats protected from EAE by coimmunization of
MBP73–86 with 73–86(82D
A), despite an increase in cytokine
production when both peptides were present during in vitro culture. The
data indicate that aspartic acid 82 is a critical TCR contact residue
and is required for the recruitment of Vß8.2+ T cells and
the encephalitogenic activity of MBP73–86.
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Introduction
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Previous
studies have revealed that analogues of the encephalitogenic peptide,
guinea pig (gp)3 myelin basic
protein (MBP) 73–86, with single alanine substitutions at
either position 82 (QKSQRSQAENPV, 82DA) or 83
(QKSQRSQDANPV, 83EA), were not encephalitogenic in
the Lewis (LEW) rat (1, 2). Furthermore, LEW rats could be protected
from experimental autoimmune encephalomyelitis (EAE) induced with whole
MBP or gpMBP73–86 by the coimmunization of MBP73–86 with
82DA (3). However, protection could not be conferred by
coimmunization with 83EA, which lacks an important MHC class
II-binding anchor residue (4, 5). These findings suggest that aspartic
acid at position 82 might be a critical TCR contact residue for the
Vß8.2+ encephalitogenic T cells that predominate in the
response of LEW rats to gpMBP73–86 (6, 7).
In this report, peptide analogues were synthesized with conservative or
nonconservative amino acid substitutions at position 82. Each analogue
was tested for its ability to induce EAE in vivo, to bind MHC class II,
and to induce T cell proliferation and cytokine secretion in vitro. In
addition, expression of the Vß8.2 TCR was determined by flow
cytometry. The ability of the pathogenic MBP73–86-specific T cells to
proliferate and secrete cytokines in response to each analogue was also
tested to determine immunologic cross-reactivity. Finally, because the
82DA analogue has been shown previously to protect LEW rats from EAE
when coimmunized with 73–86, the expression of the Vß8.2 TCR was
determined in coimmunized, protected rats. The results suggest that
aspartic acid at residue 82 is required for the recruitment of
pathogenic Vß8.2+ T cells and for the encephalitogenicity
of gpMBP73–86.
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Materials and Methods
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Animals and immunization
Female LEW rats were purchased from Charles River (Raleigh, NC),
maintained in our American Association for the Accreditation of
Laboratory Animal Care-accredited facility, and used at 8–10 wk
of age. Rats were immunized s.c. with 25 µg of peptide emulsified in
IFA supplemented with 200 µg of Mycobacterium
butyricum (Difco, Detroit, MI) as described previously (8),
and EAE was scored based on the following criteria: 0, no paralysis; 1,
flaccid tail; 2, partial hind limb paralysis; and 3, complete hind limb
paralysis with incontinence. Hematoxylin and eosin-stained spinal cord
sections from representative rats were examined microscopically for the
characteristic mononuclear cell infiltration without knowledge of
experimental treatment and scored on a scale of 0–4 based on the
intensity of inflammation.
Peptides
Peptides were synthesized using fluorenylmethoxycarbonyl
chemistry with an automated Applied Biosystems Synergy model 432A
peptide synthesizer (Perkin-Elmer, Foster City, CA). The m.w. and
purity of each peptide was confirmed by mass spectrometry. All peptide
analogues were synthesized based upon the dominant LEW rat
encephalitogenic MBP epitope, residues 73–86 (QKSQRSQDENPV) of gpMBP.
Cell lines
Draining lymph nodes were removed from rats that had been
immunized 9–10 days earlier, and single-cell suspensions were prepared
as described previously (7). The primary culture consisted of lymph
node cells (LNCs) cultured at 2 x 106/ml in RPMI 1640
supplemented with 2-ME, L-glutamine, antibiotics, 1%
normal rat serum (Life Technologies, Grand Island, NY), and 20 µg/ml
of the immunizing peptide as described previously (8). After 3 days of
incubation at 37°C, lymphoblasts were isolated by Ficoll-Isopaque
(Pharmacia, Uppsala, Sweden) centrifugation. Viable blast cells were
cultured at a density of 2 x 105/ml in RPMI 1640
supplemented as described above but with 10% FCS and with Con A
supernatant containing 20 U/ml of IL-2 in place of Ag. After 4 days of
expansion, T cells were collected and prepared for flow cytometry.
Flow cytometry
Cell lines were prepared for flow cytometry by first incubating
1 x 106 cells with PBS containing 0.02% sodium
azide, 1% BSA (PBS-A-BSA), and 1% normal rat serum for 30 min as a
blocking step (9). Cells were subsequently washed in PBS-A-BSA and
incubated with the appropriate chromogen-labeled Ab for 20 min. The Abs
used were: FITC-labeled anti-rat TCR (R73), FITC-labeled
anti-rat Vß8.2 (R78), phycoerythrin-labeled anti-rat
CD25 (OX39), and FITC anti-rat DNP as an isotype control (all
purchased from PharMingen, San Diego, CA); FITC anti-
4 HP2/1,
which reacts with the 4-chain of very late
Ag-4 (purchased from AMAC, Westbrooke, ME) was also
used. Cells were analyzed in a FACScan flow cytometer using
Lysis software (Becton Dickinson, Mountain View, CA).
Proliferation assay
T cell proliferation assays were performed as described
previously (9, 10). Briefly, plastic nonadherent LNCs were applied to a
T cell column (Biotex, Edmonton, Canada) and collected by washes with
PBS supplemented with 2% FCS. Each well received 5 x
105 T cells and 5 x 105 syngeneic,
irradiated thymocytes (2000 rad) as APCs. Cells were cultured in RPMI
1640 containing 5% FCS. Wells receiving Ag contained a final
concentration of either 5 or 20 µM of the appropriate peptide, which
was determined to be optimal in previous studies (9). Cultures
were incubated for 96 h and subsequently pulsed with 0.5 µCi of
[3H]thymidine for the last 18 h of the assay. Plates
were harvested using a Tomtec Harvester 96 (Orange, CT) and
counted with the 1450 Microbeta Plus liquid scintillation counter
(Wallac, Gaithersburg, MD). Results are presented as cpm.
MHC binding studies
To determine whether peptide analogues bind to
RT1.B1 (the rat equivalent of MHC class II I-A),
each analogue was tested for its ability to compete with biotinylated
73–86 for binding to detergent-solubilized RT1.B1
molecules, as described previously (11). Briefly, biotinylated 73–86
peptide (100 nM) was incubated with MHC class II molecules
(RT1.B1, 3 µM) that had been affinity-purified from the
MBP-specific encephalitogenic Z1A T cell line. Various concentrations
of competitor peptide (ranging from 0 to 256 µM) were subsequently
added. The MHC-peptide mixtures were analyzed by SDS-PAGE followed by
blotting onto nitrocellulose. The ability of each analogue to compete
with biotinylated 73–86 was determined by measuring the reduction in
signal from the biotinylated peptide by enhanced chemiluminescence. As
a control, competition for binding to RT1.D1 (the rat
equivalent of I-E) was determined in a similar fashion; biotinylated
MBP 87–99, which is the minor encephalitogenic
RT1.D1-restricted MBP epitope for LEW rats, was used as a
marker peptide (12).
Cytokine analysis
The 72 h culture supernatants from peptide-stimulated T
cells were evaluated for IFN-
, TNF-, and IL-10 using rat-specific
commercial ELISA kits (Biosource International, Camarillo, CA; Life
Technologies, Gaithersburg, MD) according to the manufacturers’
instructions.
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Results
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Substitution of aspartic acid at position 82 of MBP73–86 abrogates
encephalitogenic activity
Immunizing LEW rats with native gpMBP epitope 73–86
(QKSQRSQDENPV) induces severe clinical EAE (Table I
). Extensive mononuclear infiltration
was present in the spinal cords (Fig. 1A). Demyelination is not a
prominent finding in LEW rats with acute EAE (8). Although tested in
this experiment at 25 µg, the same peptide also induced paralytic EAE
at 2.5 µg (9). Aspartic acid at position 82 has been proposed to be a
TCR contact residue (1). To test the hypothesis that this aspartic acid
residue is important for encephalitogenicity, peptide analogues were
synthesized with substitutions at position 82. The analogues we
prepared included substitution of the native aspartic acid residue with
a neutral aliphatic amino acid (alanine), a bulky hydrophobic ring
(tyrosine), a basic amino acid (lysine), an asparagine (a basic amino
acid in which the native carboxyl group has been replaced by an amino
group), or a glutamic acid, which differs from the native aspartic acid
residue by one additional methyl group. LEW rats were immunized with 25
µg of an individual analogue in CFA and observed for clinical signs
of EAE. None of the analogues tested induced clinical or histologic EAE
(Table I, Fig. 1, B, C, D, and
E). Interestingly, not even the conservative exchange of
aspartic acid for glutamic acid at position 82 could maintain
encephalitogenicity.
Altered peptide ligands of MBP73–86 bind to MHC
One possible explanation for the failure of the various peptide
analogues to cause EAE is that MHC binding had been affected. This
possibility was tested by determining whether any of the analogues
could inhibit the binding of biotinylated 73–86 peptide to
detergent-solubilized MHC class II RT1.B1 molecules (the
homologue of murine I-A). The data confirm that each of the analogues
binds to RT1.B1 at least as well as peptide 73–86 (Fig. 2). As expected, there was no binding to
RT1.D1, the homologue of I-E (Fig. 3).
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FIGURE 2. Competitive binding of MBP analogues to purified RT1.B1.
Inhibition of the binding of 100 nM of biotinylated MBP73–86 to 3 µM
of affinity-purified RT1.B1 is shown. Different
dose ranges (0–256 µM) of competitor peptide were tested.
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FIGURE 3. Competitive binding of MBP analogues to purified RT1.D1.
Inhibition of the binding of 100 nM of biotinylated MBP87–99 to 3 µM
of affinity-purified RT1.D1 is shown. Different
dose ranges (0–256 µM) of competitor peptide were tested.
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Immunogenicity and cross-reactivity of analogue-primed T cells
To determine whether the analogues were capable of inducing an
immune response, lymph node T cells from analogue-primed rats were
tested in vitro for proliferation to the immunizing peptide. As shown
in Fig. 4, each analogue was immunogenic
as reflected by T cell proliferation in vitro. T cells from rats
immunized with 73–86 responded to the priming peptide, but did not
proliferate when stimulated in vitro with any of the analogues (Fig. 4A). Similarly, T cells from rats immunized with 82DA,
82DK, or 82DY responded to the priming peptide but failed to
cross-react with either the native peptide or the other analogues (Fig. 4, B, E, and F). T cells from LEW rats
immunized with 82DE cross-reacted weakly with 82DA (Fig. 4D), whereas T cells primed to 82DN cross-reacted weakly
with 82DK and exhibited a heteroclitic response to 82DA (Fig. 4C). Proliferative responses to a higher concentration of
peptide were similar to those seen at the 5-µM concentration (Fig. 5).
Because the 82DA analogue has been shown to have protective effects
in vivo when coimmunized with 73–86, it was important to more closely
examine cross-reactivity between 82DA T cells and MBP73–86. 82DA
T cells, when stimulated in vitro with 5 µM of either gp73–86 or rat
73–86, did not show any significant proliferation when compared with
the proliferation induced by the 82DA peptide (Table II). Proliferation using 20 µM of
peptide gave similar results (data not shown).
To independently confirm the finding that all analogues induced an
immune response, culture supernatants were also tested for the
production of IFN- and IL-10 by ELISA. As shown in Fig. 6, LNCs from immunized rats secrete
significant levels of IFN- and measurable, albeit lower quantities
of IL-10, when stimulated with the immunizing peptide. Consistent with
the observed lack of cross-reactivity at the level of proliferation,
MBP73–86 T cells did not secrete IL-10 when stimulated with the
different analogues (Fig. 7). Although
IFN- was secreted at levels comparable with altered peptide
ligand-immunized rats as shown in Fig. 6, a direct comparison cannot be
made, because column-enriched 73–86-specific T cells were used in Fig. 7.
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FIGURE 6. Cytokine secretion by LNCs from 73–86- or analogue-primed rats. LNCs
pooled from immunized rats (three rats per group) were stimulated in
vitro with the priming peptide (vertical axis) for 72 h; IFN-
and IL-10 production was measured by rat-specific ELISA.
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FIGURE 7. Cytokine secretion by T cells from 73–86-primed rats. Column-enriched
73–86-specific T cells isolated from pooled LNCs of 73–86-immunized
rats (n = 3) were stimulated in vitro for 72 h
with either wt 73–86 or one of the peptide analogues (vertical axis);
IFN- and IL-10 production was measured by rat-specific ELISA.
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Only MBP73–86 elicits Vß8.2+ T cells
Previous reports have shown that LEW rat T cells that bear the
Vß8.2 TCR predominate in both short-term T cell lines and in T cell
clones specific for gpMBP73–86 (6, 7). TCR peptides consisting of
complementarity-determining region-2 (13, 14) or
complementarity-determining region-3 (15) of the Vß8.2 TCR have been
used successfully to treat EAE in LEW rats. To determine what effect,
if any, the various substitutions at position 82 would have on the
recruitment of T cells bearing the Vß8.2 TCR, short-term T cell lines
were generated from LEW rats immunized with each analogue and evaluated
by flow cytometry. To confirm the dominance of the Vß8.2 TCR in T
cell responses to 73–86, T cells specific for the native 73–86
peptide were also stained with FITC anti-rat Vß8.2. Fig. 8 shows that >50% of the
73–86-specific T cells express Vß8.2. A majority of these T cells
are also -4+ and CD25+,
demonstrating the activation status of the Vß8.2+ T cells
(Fig. 9). In contrast, when T cells
specific for 82DA, 82DN, or 82DE were analyzed, only 2–4%
expressed the Vß8.2 TCR (Fig. 8, B–D). Surprisingly, not
even 82DE, which differs from the wild-type (wt) peptide only by the
addition of one methyl group, expanded the Vß8.2+ T cell
population (Fig. 8C). Because of the dramatic effect
observed with such a conservative substitution, Vß8.2 expression was
not determined for the DK and DY analogues. T cells specific for
each analogue expressed ß-TCR, -4, and CD25 at levels
equivalent to the 73–86 cells. The results of 82DA are shown as a
representative experiment with the analogue peptides (Fig. 9), and
indicate that non-Vß8.2+ T cells are preferentially
expanded by the 73–86 analogues. In support of this,
73–86-specific T cells that are cross-stimulated in vitro with the
82DA analogue and then expanded in T cell growth factor
(TCGF)-containing medium show a high degree of activation by
virtue of CD25 expression but exhibit low Vß8.2 expression (Figs. 11 and
12).
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FIGURE 11. LNCs from LEW rats immunized with 73–86 were stimulated in vitro with
73–86 for 72 h, followed by expansion in TCGF-containing medium
for 4 days. Cells were subsequently stained for FACS analysis as
described previously, using anti-Vß8.2 and anti-CD25
Abs.
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Vß8.2 expression in coimmunized, protected rats
Previously it has been shown that when 73–86(82DA) is
coimmunized with encephalitogenic 73–86, LEW rats are protected from
EAE (1, 2). To confirm this finding, we coimmunized 10 LEW rats with
73–86(82DA) and 73–86 as described previously (1). Consistent with
previous findings, 70% (7 of 10) of the rats were completely protected
from EAE (data not shown). To determine Vß8.2 expression in the
coimmunized group, LNCs were harvested from protected rats and
stimulated in vitro with either 73–86 or both 73–86 and
73–86(82DA) peptides. When analyzed by FACS, T cells stimulated
with 73–86 alone showed a dominance of the Vß8.2 TCR (Fig. 10H, Table III). However, when both peptides were
included during the stimulation, there was a significant decrease in
the percentage of T cells expressing the Vß8.2 TCR (Fig. 10F, Table III). Both cell populations were predominantly
ß-TCR+ (Fig. 10, B and D) and
were activated (CD25+; Fig. 10, E and
G). However, the decrease in Vß8.2 expression was not
accompanied by a decrease in cytokine production, as supernatants from
cultures in which both peptides were present during the stimulation
period showed an additive effect, with a significant increase in IL-10
production in the coimmunized group (Table III).
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Discussion
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Wauben et al. (1) first studied single alanine-substituted
analogues of MBP73–86 and determined that inhibition of EAE in LEW
rats coimmunized with MBP73–86 plus 82DA was due to a mechanism(s)
other than MHC blockade alone. Their results suggested that TCR
antagonism could play a role, although the precise mechanism was not
delineated. The present study reveals that the aspartic acid residue at
MBP residue 82 is essential for encephalitogenic activity in LEW rats,
because no structural modification is tolerated at this position. Thus,
even the conservative change from aspartic to glutamic acid (the
addition of one methyl group), aspartic acid to alanine (the loss of
the carboxyl group), or aspartic acid to asparagine (the change of a
carboxyl to an amino group) rendered the analogues nonencephalitogenic.
Each of the analogues tested was an effective competitor of the wt
MBP73–86 peptide in binding to detergent-solubilized
RT1.B1 molecules, indicating that the aspartic acid residue
at position 82 does not play a crucial role in MHC class II binding.
The failure of these analogues to induce EAE was not due to lack of
immunogenicity, because proliferative responses were elicited in vitro
by lymph node T cells stimulated with the priming peptide. The
analogue-primed T cells did not cross-react in vitro with 73–86, the
encephalitogenic epitope, as determined by proliferation. When 82DA
T cells were further tested for cross-reactivity by measuring
proliferation to rat 73–86, there was also no significant
cross-reactivity. This is an important point to address, because the
82DA analogue has protective effects in vivo. MBP73–86-primed cells
also did not proliferate or secrete IL-10 in response to the analogues.
Despite their immunogenicity, the analogues failed to stimulate the
clonal expansion of T cells expressing the EAE-associated Vß8.2 TCR,
suggesting that aspartic acid at MBP position 82 is a critical TCR
contact residue for Vß8.2+ T cells. Consistent with this
possibility is the observation that a small population of
73–86-specific T cells cross-reacts with the 82DA analogue, and
that these T cells exhibit high CD25 expression but low Vß8.2
expression. This finding helps to explain why 73–86-specific,
encephalitogenic T cells cannot be primed with 82DA in vitro to
transfer disease (2). However, this cross-reactivity is not required
for protection from EAE, because the 82DA analogue will protect LEW
rats from EAE induced with MBP87–99. Weissert et al. (16) recently
compared Vß8.2 T cell activation by gp and rat MBP63–88, which
contains the encephalitogenic 73–86 sequence. However, the rat
sequence differs at position 78 (our numbering system), where threonine
is substituted for serine in rat MBP. Although both peptides were
encephalitogenic for LEW rats, only the gp peptide preferentially
recruited Vß8.2 T cells (16). Thus, two residues influence Vß8.2 T
cell activation, serine at position 78 (16) and aspartic acid at
position 82 (this report), although only the latter residue is
essential for encephalitogenic activity.
Because the 82DA analogue has been shown to protect LEW rats from
EAE when coimmunized with 73–86, it was of interest to determine
Vß8.2 expression in these protected rats. There was a significant
decrease in Vß8.2 expression when both the 73–86 and 82DA
peptides were included in culture, despite an additive increase in
cytokine production, especially IL-10. Although the levels of IL-10 are
relatively low, we cannot exclude a possible contribution of IL-10 in
the protection. Additional experiments will determine whether the
decrease in Vß8.2 expression is responsible for protection from EAE.
Preliminary studies have shown that LEW rats are protected against EAE
induced with MBP73–86 by adoptive transfer of LNCs from donors
immunized with the nonencephalitogenic 82DA (data not shown). This
explanation would be consistent with the findings of Nicholson et al.
(17), who reported that an analogue of proteolipid protein peptide
139–151, in which glutamine was substituted for the native tryptophan
TCR contact residue at position 144, was no longer encephalitogenic but
protected SJL mice against EAE. The WQ modification elicited T cells
that could transfer protection upon adoptive transfer into naive
recipients (17). The results presented here suggest that the mechanism
underlying the inhibition of EAE by 82DA cannot solely be explained
by MHC blockade (1). In support of this possibility, we have observed
that analogue 82DA protected LEW rats from EAE induced with the MBP
87–99 epitope (our manuscript in preparation), which binds to
RT1.D1 rather than RT1.B1 and elicits
non-Vß8.2 encephalitogenic T cells (18). The possibility that 82DA
may inhibit EAE via a bystander mechanism is under investigation.
The analogue-specific T cells described in our study appear to be Th1
cells, because the predominant cytokine produced is IFN-. Although
IL-10 was also detected, the concentrations produced by the
analogue-primed T cells were significantly lower than the quantities of
IFN- present in the same culture supernatants. Furthermore,
encephalitogenic Th1 rat T cell clones reportedly express both IFN-
and IL-10 mRNA (19), suggesting that rat and mouse T lymphocyte subsets
may not be comparable on the basis of cytokine gene expression.
In conclusion, conservative substitutions at the aspartic acid residue
at position 82 of gpMBP abrogate the ability of the epitope to induce
EAE in LEW rats. The analogues are immunogenic, but fail to activate T
cells that express the encephalitogenic Vß8.2 TCR. The effect of the
82DA analogue on the recruitment of Vß8.2+ T cells was
also observed in coimmunized protected rats. IFN--secreting,
non-Vß8.2 T cells are activated. T cells activated in response to
analogue 82DA inhibit EAE, as shown in coimmunization (1) and
preliminary adoptive transfer experiments. Studies are in progress to
elucidate the mechanism by which these T cells mediate suppression.
Nonencephalitogenic analogue peptides have been studied in an effort to
devise therapeutic strategies of potential relevance to human disease.
Although the analogues employed in the present study are devoid of
encephalitogenic activity, caution must be exercised when considering
possible application to human autoimmune disease. Pathogenic T cells
that recognize different TCR contact residues on multiple autoantigenic
molecules (20, 21) might eventually arise to counter the therapeutic
effects exerted by the peptide analogue originally employed for
treatment. Consideration must also be given to the possibility that
analogues that fail to induce autoimmune disease in unprimed hosts may
nevertheless activate memory T cells in animals that have previously
encountered the wt autoreactive epitope (22).
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FIGURE 12. LNCs from LEW rats immunized with 73–86 were stimulated in vitro
with 73–86(82DA) for 72 h, followed by expansion in
TCGF-containing medium for 4 days. Cells were subsequently stained for
FACS analysis as described previously, using anti-Vß8.2 and
anti-CD25 Abs.
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Acknowledgments
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We thank Dr. Myron Leon for critically reading this manuscript and
M. Grosfeld for technical assistance. We also thank Dr. Linda
Hazlett for technical assistance.
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Footnotes
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1 This study was supported by Research Grant NS06985-31 from the National Institutes of Health and Grant RG1073G10 from the National Multiple Sclerosis Society. R.H.S. is the recipient of a Javits Neuroscience Investigator Award from the National Institute of Neurological Diseases and Stroke. The research of M.H.M.W. has been made possible by a fellowship of the Royal Netherlands Academy of Arts and Sciences. 
2 Address correspondence and reprint requests to Dr. Ronald B. Smeltz, Department of Immunology, Wayne State University School of Medicine, 540 East Canfield, Room 7263, Detroit, MI 48201. E-mail address:
3 Abbreviations used in this paper: gp, guinea pig; MBP, myelin basic protein; LEW, Lewis; EAE, experimental autoimmune encephalomyelitis; wt, wild type; LNC, lymph node cell; TCGF, T cell growth factor.
Received for publication July 16, 1998.
Accepted for publication September 29, 1998.
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Abstract
Introduction
