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Vol. 288, Issue 3, 1174-1184, March 1999
the
Disulfide Dimer of N-acetylcysteineIs a Potent
Modulator of Contact Sensitivity/Delayed Type Hypersensitivity
Reactions in Rodents1
Department of Pharmacology, Astra Draco AB, Lund, Sweden
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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Oral N-acetyl-L-cysteine (NAC) is used clinically for treatment of chronic obstructive pulmonary disease. NAC is easily oxidized to its disulfide. We show here that N,N'-diacetyl-L-cystine (DiNAC) is a potent modulator of contact sensitivity (CS)/delayed type hypersensitivity (DTH) reactions in rodents. Oral treatment of BALB/c mice with 0.003 to 30 µmol/kg DiNAC leads to enhancement of a CS reaction to oxazolone; DiNAC is 100 to 1000 times more potent than NAC in this respect, indicating that it does not act as a prodrug of NAC. Structure-activity studies suggest that a stereochemically-defined disulfide element is needed for activity. The DiNAC-induced enhancement of the CS reaction is counteracted by simultaneous NAC-treatment; in contrast, the CS reaction is even more enhanced in animals treated with DiNAC together with the glutathione-depleting agent buthionine sulfoximine. These data suggest that DiNAC acts via redox processes. Immunohistochemically, ear specimens from oxazolone-sensitized and -challenged BALB/c mice treated with DiNAC display increased numbers of CD8+ cells. DiNAC treatment augments the CS reaction also when fluorescein isothiocyanate is used as a sensitizer in BALB/c mice; this is a purported TH2 type of response. However, when dinitrofluorobenzene is used as a sensitizer, inducing a purported TH1 type of response, DiNAC treatment reduces the reaction. Treatment with DiNAC also reduces a DTH footpad-swelling reaction to methylated BSA. Collectively, these data indicate that DiNAC in vivo acts as a potent and effective immunomodulator that can either enhance or reduce the CS or DTH response depending on the experimental conditions.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Oral
N-acetyl-L-cysteine (NAC) has been
used clinically as a remedy for chronic obstructive pulmonary disease
for decades, and it is generally believed that its beneficial effect is
due to a mucolytic activity. However, it has not been possible to demonstrate presence of the drug in epithelial lining fluid after oral
administration (Cotgreave et al., 1987
); this is not fully compatible
with its proposed in vivo mucolytic activity. Moreover, NAC is reported
to primarily reduce exacerbation rates in the disease (Boman et al.,
1983). Considering this, it is possible that NAC acts in chronic
obstructive pulmonary disease patients, at least in part, by enhancing
their host defense.
In support of such an idea, numerous reports show that NAC treatment in
vitro can modulate activities of lymphoid cells. Human T cell IL-4
synthesis and B cell IgE and IgG4 production are decreased by the
compound, whereas T cell IL-2 production is enhanced (Jeannin et al.,
1995). NAC inhibits apoptosis of T cell hybridomas (Jones et al., 1995)
and reverses down-regulation of IL-2 mRNA and IL-2 activity in human
lymphocytes (Flescher et al., 1994). In biochemical terms, NAC has been
generally considered to act as a precursor of glutathione (Dröge
et al., 1994; Jeannin et al., 1995; Jones et al., 1995; Yan et al.,
1995), as an antioxidant (Aruoma et al., 1989), and/or as a reductant
that modulates redox-sensitive transcription factors like AP-1 or
NF-
B or otherwise influences transcriptional events (Schreck et al.,
1991; Dröge et al., 1994; Yan et al., 1995; Xia et al., 1996).
The effects of NAC, seen at close to mM concentrations, conform to the
in vitro stimulatory effects of other low molecular weight thiols, like
2-mercaptoethanol and glutathione, on lymphoid cell function (Hiestand
and Strasser 1985a; Dröge et al., 1994; Jeannin et al., 1995).
Modulation of in vivo immune responses by NAC have also been reported
(Hiestand and Strasser, 1985b; Kinscherf et al., 1994; Jeannin et al.,
1995).
Reports have suggested that the efficacy of in vivo modulation by some
thiols relies on the presence of an intact disulfide bridge (Hiestand
and Strasser, 1985b; St Georgiev, 1988). Therefore, we examined the
effects of the disulfide dimer of NAC,
N,N'-diacetyl-L-cystine (DiNAC) in in vivo systems reflecting various immune responses in
rodents. One such system is the delayed type hypersensitivity (DTH)
reaction, an in vivo reflection of a cell-mediated immune response
(Askenase, 1992). The expression DTH is often used to denote reactions
induced by protein antigens as well as by contact sensitizers. However,
recent reports indicate that different subsets of T cells govern
classical DTH reactions to protein antigens and contact sensitivity
(CS) reactions (Grabbe and Schwarz, 1998). The present report deals
with effects of DiNAC in CS skin reactions induced by
4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (oxazolone), fluorescein isothiocyanate (FITC), and 2,4-dinitrofluorobenzene (DNFB),
as well as in a DTH reaction to methylated BSA (mBSA) assessed
by foot pad swelling.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Synthesis and Sources of Compounds.
Compounds
N,N'-diacetyl-L-cystine
(DiNAC; referred to as D7042) and its
di-L-lysinium salt (referred to as D7193 - preferred for use over D7042 due to pharmaceutical reasons),
N-acetyl-L-cysteine, ethylene-2,2'-bis(dithio)bis(ethanol) (ADA 202-718),
L-homocystine, N,N'-diacetyl-L-homocystine,
N,N'-diacetyl-cystamine,
L-cystathionine, N,N'-diacetyl-L-cystathionine,
racemic lanthionine, and racemic N,N'-diacetyl
lanthionine, were synthesized by Astra Draco AB (Lund, Sweden) or Astra
APP (Södertälje, Sweden). Aminoguanidine bicarbonate salt,
L-buthionine-[S, R]-sulfoximine (BSO),
diethyldithiocarbamate (DDTC), DNFB, FITC-isomer 1, and mBSA were all
obtained from Sigma Chemical Co. (St. Louis, MO).
Bis-(2-hydroxyethyl)-disulfide (HEDS) was from Fluka AG (Buchs,
Switzerland), whereas oxazolone was obtained from either BDH Chemicals
Ltd. (Poole, Dorset, England) or from Sigma. Cyclosporin A (CSA;
Sandimmun) was obtained from Sandoz Pharma AG (Basel, Switzerland) and
rapamycin and
N
-nitro-L-arginine
methyl ester (L-NAME) were obtained from Alexis Corp. (San Diego, CA). Sodium pentobarbiturate (Mebumal) was purchased from Apoteksbolaget (Umeå, Sweden) and enflurane (Efrane) was obtained
from Abbott S.p.A. (Campoverde Lieti, Italy). Sources of
antibodies used in immunohistochemical experiments are given below.
Animals. Male and female mice of the following strains were obtained from Bomholtsgaard (Ry, Denmark) or Charles River Breeding Laboratories (Kent, UK): BALB/c, CD-1, C57BL/6, CBA/J, NOD, MRL/Mp and MRL-lpr/lpr. The mice were used at the weight of 18 to 20 g. Female Sprague-Dawley rats were from Möllegaard (Ejby, Denmark); they were used at 150 g. The animals were caged for at least 8 days after arrival before experiments were initiated. Animals had free access to food (R3, Ewos, Södertälje, Sweden) and water. The light period in the room was 12 h (6:00 AM-6:00 PM). Rabbits of New Zealand strain of both sexes were obtained from HB Rabbit Farm, Lidköping, Sweden. All animal study designs were approved by local ethics committees.
Induction and Assessment of CS to Oxazolone, DNFB, and FITC.
Animals were sensitized (day 0) by a single epicutaneous application of
150 µL (mice), 400 µL (rats), or 1000 µL (rabbits) 3% oxazolone
solution in absolute ethanol-acetone (3:1) on the shaved thorax and
abdomen. Treatment with D7042, D7193, or other compounds was normally
initiated by oral feeding (gavage) of 10 ml/kg body weight of an
appropriate concentration of the compound immediately after
sensitization. Treatment continued once daily up to and including day
6. Control animals were given the corresponding amount of vehicle
(saline or water as indicated in the figure legends). Other treatment
regimens with D7042 or D7193 were examined as specified in the text. In
separate experiments, D7193 was administered in the drinking water
(prepared fresh each day) in free access, injected either i.v. in the
tail vein (20 µL) or i.p. (200 µL), or instilled intratracheally
under light Efrane anesthesia. Eight days after sensitization (i.e., on
day 7), mice were challenged on both sides of both ears by topical
application of a total of 20 µL of 1% oxazolone dissolved in peanut
oil; rats were challenged with 40 µL and rabbits with 400 µL of the
same solution. Ear thickness was measured before and 24 h after
challenge (in some experiments also at 48 h) using an Oditest
spring caliper handled manually (van Loveren et al., 1984) or coupled
to a computer-directed motor device. Challenges and measurements of ear
thickness were performed under light pentobarbital anesthesia. Most
experiments were performed under coded conditions.
). The magnitude of the CS reactions
induced by DNFB and FITC were assessed as described above for
oxazolone-induced reactions.
Immunohistochemical Examinations.
Histological and
immunohistological examinations were performed in blind manner on coded
ears from BALB/c mice untreated, sensitized, and challenged with
oxazolone with or without treatment with D7193. The ears were cut off
at the bases and immediately snap-frozen in isopentane in liquid
nitrogen and stored at 
70°C. The frozen ears were processed for
routine histological examinations and for immunohistochemical
examinations using the ABC-technique according to principles described
previously (Scheynius et al., 1996). The frozen specimens were cut
through the center of the ear extending from the top to the base in a
cryostat. The cryostat sections, 6 µm thick, were acetone-fixed, and
incubated in 0.3% H2O2 in
phosphate-buffered saline for 15 min at room temperature to block
endogenous peroxidase. Sections were then treated with normal rabbit
serum to reduce nonspecific binding and with Avidin D solution and
biotin-solution blocking kit (Vector Laboratories, Inc., Burlingame,
CA). This was followed by incubation with rat anti-mouse monoclonal
antibodies (mAbs) (see below), biotinylated anti-rat IgG (Vector
Laboratories) and avidin-biotin-peroxidase complex (Dako A/S, Glostrup,
Denmark). The peroxidase reaction was developed with
3-amino-9-ethylcarbazole (Aldrich Chemical Co., Steinheim, Germany).
The sections were counterstained with Mayer's hematoxylin. Optimal
dilutions of the mAbs were determined in control experiments with
sections from normal mouse spleen and skin. Each ear specimen was also
processed for hematoxylin and eosin staining. The following rat
anti-mouse mAbs were used: anti-CD4 (L3T4, clone H129.19), anti-MHC
class II I-Ab, d (clone B21.2), anti-CD11a (the
-chain of LFA-1, clone FD441.8), anti-CD11b (the -chain of Mac-1,
C3biR, clone M1/70.15.11.5), anti-CD54 (ICAM-1, clone YNI/1.7.4), and
anti-CD44 (clone IRAWB14.4) all obtained from their hybridoma cells
expanded in vitro, anti-CD8 (Lyt2) obtained from Serotec Ltd.,
(Kidlington, Oxford, UK), and anti-CD106 (VCAM-1) from PharMingen (San
Diego, CA).
Multi-Probe RNase Protection Assay (RPA) Analysis of Cytokine
Profiles in CS Reaction Sites.
Four different groups of
mice were investigated for chemokine/cytokine expression; nonsensitized
mice treated either with vehicle or with 3 µmol/kg D7193 and mice
sensitized to and challenged with oxazolone that were treated either
with vehicle or with 3 µmol/kg D7193. Three individuals in each group
sacrificed 24 h after challenge were analyzed. RNA was prepared as
described (Chomczynski and Sacchi, 1987) from ears that had been cut
from sacrificed mice and snap-frozen in liquid nitrogen. The ears were
ground in liquid nitrogen and the resulting powder was suspended in
homogenization solution (4 M guanidium thiocyanate, 0.1 M Tris-Cl pH
7.5, 1% 
-mercaptoethanol). The slurry was sheared with a disposable
syringe using a 0.9-mm needle. Expression of mRNAs for eotaxin,
glyceraldehyde-phosphate dehydrogenase, IL-2, IL-4, IL-5, IL-6, IL-9,
IL-10, IL-13, IL-15, IFN-
, IP-10, L32, lymphotactin, MCP-1,
MIP-1, MIP-1, MIP-2, RANTES, and TCA-3 were examined with the
Riboquant multi-probe RPA system from PharMingen according to the
manufacturer's protocol.
-32P]UTP, 1 µL GACU pool, 2 µL DTT, 4 µL 5X transcription buffer, 1 µL RPA template set, 1 µL RNasin, 1 µL T7 RNA polymerase) and incubated at 37°C for 1 h. The
reaction was terminated by adding 2 µL of DNAse I and incubating at
37°C for an additional 30 min. Twenty six µL of 20 mM EDTA and 2 µL of yeast tRNA (2 mg/mL) were added, and the reaction was extracted
once with phenol/24:1 chloroform:isoamylalcohol (1:1) and once with
chloroform/isoamylalcohol alone. The aqueous phase was precipitated by
adding 50 µL of 4 M ammonium acetate and 250 µL of ice-cold
ethanol. After centrifugation, the pellet was dissolved in 50 µL of
the hybridization buffer provided in the kit. Incorporation of
radiolabel was quantified using a Bioscan Quick-count and the probe was
diluted to the recommended 3.0 × 105
cpm/µL. Ten µg of total RNA was used for each incubation together with approximately 5 × 105 cpm of probe in
total volume of 20 µL of hybridization buffer. The annealing reaction
mixture was incubated at 56°C overnight. RNase digestion was
performed by adding 100 µL of RNase mix (provided in the kit)
containing 192 pg/µL RNase A, 0.6 U/µL RNase T1, and RNase buffer
to the reaction. The mixture was incubated at 37°C for 45 min. To
stop the digestion, proteinase K was added together with yeast RNA as
carrier. The reaction mixture was extracted once with 130 µL
phenol/24:1 chloroform:isoamylalcohol (1:1). The aqueous phase was
removed to a new tube and precipitated with 120 µL of 4 M ammonium
acetate and 650 µL of ice-cold ethanol. Reactions were precipitated
at 20°C for 1 h; after centrifugation the pellet was dissolved
in 5 µL of formamide-loading buffer. Dissolved precipitates were
heated at 90°C for 3 min before resolution on 5%, 1X TBE, denaturing
polyacrylamide gels. Gels were transferred to Whatman paper and dried
in a Savant vacuum dryer. Dried gels were placed in a phospho-Imager
cassette (Molecular Dynamics) and exposed overnight. The phospho-screen
was scanned in a Molecular Dynamics Storm 860 scanner and analyzed with
Image-Quant software (Molecular Dynamics).
Footpad DTH Responses in Mice Sensitized to mBSA.
A DTH
response to mBSA was induced in BALB/c mice as described (Tarayre et
al., 1990). Briefly, mBSA emulsified in Freund's complete adjuvant was
injected intradermally on day 0 (a total of 0.25 mg of mBSA in a total
volume of 0.1 ml of emulsion was given at three injection sites).
Challenge was performed by injection on day 7 of 0.025 ml mBSA solution
(25 mg/mL) in the right hind paw; the left hind paw, injected with the
corresponding volume of the vehicle, served as a control. Twenty-four
hours later, the animals were deeply anesthetized and both hind paws
were cut. The DTH reaction was estimated from the difference in weight
between the two hind paws.
DTH Granuloma Reaction Induced by mBSA.
A s.c. DTH reaction
induced by mBSA resulting in a quantifiable chronic granulomatous
lesion (Dunn et al., 1989) was used to assess effects of treatment with
D7193 on a chronic cell-mediated inflammatory reaction. Briefly, mice
were sensitized to mBSA (a total of 25 mg of mBSA and 500 mg of Dextran
in a total volume of 0.1 ml of emulsion was given at a total of three
injection sites). The animals were challenged 3 weeks later by s.c.
implantation of Millipore filters (10 mm diameter) soaked in 25 µL of
mBSA solution (3 mg/mL). After 7 days, filter implants were dissected away from the lesions and weighed (wet/dry) for quantification of
inflammation. The results were expressed as the mean ± S.E.M from
groups of 8 to 10 animals. Degree of significance for differences between means of groups was obtained by Student's two-tailed
t test.
Pharmacological Modulation of the CS Skin Reactions to Oxazolone in Mice Mice sensitized to oxazolone as described above were treated orally with D7193 or vehicle and simultaneously with either freshly prepared NAC (3 µmol/kg or 30 µmol/kg orally), CSA (30 µmol/kg orally), rapamycin (3 µmol/kg orally), aminoguanidine (AMG; 813 µmol/kg i.p. once daily from day 2 before sensitization up to and including the day of challenge, day 7), L-NAME (115 µmol/kg orally by gavage from day 5 before sensitization to day 6), or BSO (2 mmol/kg twice a day i.p. and 20 mM in drinking water from day 5 before sensitization up to and including the day of challenge, day 7). The results were expressed as the mean ± S.E.M. from groups of 8 to 10 animals. Degree of significance for differences between means of groups was obtained by Student's two-tailed t test.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Influence of DiNAC (D7042/D7193) on CS Skin Reactions to Oxazolone in Mice. The capacity of D7042 to modulate the CS skin reaction to oxazolone is illustrated in Fig. 1, which shows the results of measurements of ear thickness 24 h after challenge in animals treated with D7042 compared with those from animals treated with NAC. Treatment with both compounds causes a dose-dependent increase of ear thickness; however, the log dose-response relations indicate that DiNAC is 100 to 1000 times more potent than NAC and twice as effective at the highest examined comparable dose.
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) is not enhanced in animals treated
with D7193 (results not shown).
Dose-response assessments of the effects of D7193 after oral, i.p.,
i.v., or intratracheal administration (Fig.
2) reveal that 0.03 µmol/kg as well as
3 µmol/kg of the compound stimulated the CS reaction to a similar
degree irrespective of route of administration whether the reactions
were read 24 h or 48 h (not shown) after challenge. In these
experiments, even the 0.0003 µmol/kg dose of D7193 induced a
significant enhancement of the CS reaction. Mice given D7042 in the
drinking water displayed increased CS reactivity of the same degree as
those given the compound by gavage treatment once daily (results not
shown).
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), or carries an autoimmune phenotype (MRL-lpr/lpr, MRL-Mp or NOD).
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Modulation of Effects of D7193 on CS Skin Reactions to Oxazolone in
Mice by Selected Agents.
To assess the influence of NAC on the
effect of D7193, CS reactions in BALB/c were assessed in mice given
either D7193 at the indicated doses, freshly prepared NAC at
the indicated doses, or both agents (administered separately by gavage
once daily according to the standard schedule treatment). The results
show that simultaneous treatment with NAC reduces the enhancement of
the D7193-induced CS reaction to levels that are even lower than those
recorded in animals treated with NAC alone (Fig.
5A). Oxazolone-sensitized mice treated
with the glutathione-depleting agent BSO (2 mmol/kg twice a day i.p.
and 20 mM in drinking water (Leeuwenburgh and Ji, 1995) express
markedly increased CS reactions, which are even further augmented into
very large reactions by simultaneous D7193 treatment (Fig. 5B).
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) from days 0 to 6 do
not display significantly reduced CS reactions. Simultaneous treatment with rapamycin and D7193 leads to effects similar to those seen with
treatment with CSA; rapamycin blocks the enhancement recorded with the
low dose of D7193 and partly blocks that induced by treatment with 3 µmol/kg D7193 (results not shown). Oxazolone-sensitized mice treated
with the nitric oxide (NO) synthase inhibitor AMG (Tracey et
al., 1995; Brenner et al., 1997) display slightly reduced CS reactions.
Still, the CS reactions are enhanced in mice simultaneously treated
with AMG and D7193 (Fig. 6B). In one of two experiments, animals
simultaneously treated with AMG and D7193 expressed a significantly
enhanced CS reaction compared with that in animals treated with D7193
alone (data not shown). Oxazolone-sensitized mice treated orally with
another NO synthase inhibitor, L-NAME, at a dose which is
effective in colonic inflammation in rats (Kiss et al., 1997)-a
species less sensitive to L-NAME than mice (Tracey et al.,
1995)-displayed slightly increased CS reactions; despite this,
simultaneous treatment with L-NAME tended to reduce the enhancement of the CS reaction brought about by D7193 treatment (Fig.
6C).
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Histological and Immunohistochemical Assessment of the Enhancement of the Skin CS Reaction Induced by D7193 in BALB/c Mice. Histological and immunohistochemical examinations were performed on ear specimens from six normal nontreated mice, six mice sensitized to and challenged with oxazolone, and eight mice sensitized to and challenged with oxazolone that were also treated with 3.0 µmol/kg D7193 orally for 7 days. Hematoxylin and eosin staining of ear sections showed that in three animals treated with D7193 there was marked cell infiltration and edema. In the rest of the animals in this group, moderate to low levels of cell infiltration were observed; this effect was also seen in sensitized, challenged, but nontreated animals (Fig. 7A). In D7193-treated mice, the number of CD8+ cells was increased compared with similarly sensitized and challenged, but untreated mice, with the most pronounced difference in epidermis compared to dermis (Fig. 7, B and C, respectively). Representative examples showing increases in epidermal CD8+ cells in D7193-treated versus nontreated animals are shown in Fig. 8, A and B. No marked differences in numbers of CD4+ cells in epidermis or in dermis were observed between the animals treated or not treated with D7193. MHC class II+ and ICAM-1+ keratinocytes were more frequently observed in D7193-treated mice compared with sensitized, challenged, and untreated animals (data not shown). There was a slight increase of LFA-1+, ICAM-1+, and VCAM-1+ cells in dermis in the D7193-treated compared with untreated animals. Other examined cell surface markers were not affected by the D7193 treatment as revealed by immunohistochemical assessment.
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RPA Analysis of Chemokine/Cytokine Profiles in CS Reaction Sites
24 h after Challenge in Ears from BALB/c Mice Sensitized to
Oxazolone
In ear tissue obtained from BALB/c mice
sensitized to oxazolone, several chemokines/cytokines were found to be
induced 24 h after challenge (Fig. 9
A and B). Thus, IL-4, IL-10, IL-13, IL-6, lymphotactin, RANTES,
MIP-1, MIP-1, MIP-2, MCP-1, and low levels of IFN- can be
detected in the two groups of mice sensitized to and challenged with
oxazolone. IL-5, IL-9, and TCA-3 could not be detected. Expression of
eotaxin, IL-2, and IL-15 was detected in tissue from control animals as
well as in tissue from sensitized animals. There were no significant
changes in chemokine/cytokine expression pattern between mice that were
treated with D7193 or with vehicle whether or not they were sensitized to and challenged with oxazolone.
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Structure Activity Studies and Effects of Reference Compounds.
The effects of the disulfides ADA 202-718 and HEDS, previously
reported to act as immunostimulants (Hiestand and Strasser, 1985a,b)
and that of the immunomodulator DDTC, as well as those of some other
compounds closely related in structure to DiNAC, were also examined in
the oxazolone-induced CS reaction in BALB/c mice. Effects were
expressed relative to those of DiNAC determined on the same test
occasions. The data for the three compounds mentioned (Table
1) show that DiNAC is as effective as
DDTC and ADA 202-718 and slightly more effective than HEDS. Effects
similar to those of DDTC were also recorded with its disulfide dimer
disulfiram (data not shown). A lack of effect (when examined at 0.03 or
3 µmol/kg/day) was recorded for some compounds closely related to DiNAC, e.g., L-homocystine,
N,N'-diacetyl-L-homocystine,
cystamine, N,N-diacetyl-L-cystathionine,
and N,N'-diacetyl lanthionine, whereas a
borderline activity was recorded for
N,N'-diacetyl-L-cystamine. A low, albeit significant, effect was recorded for the
D-form of the DiNAC, i.e.,
N,N'-diacetyl-D-cystine
(efficacy reduced to 50% of that of D7193 at 0.03 as well as 3 µmol/kg/day).
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Effects of D7193 on CS Reactions to Oxazolone in Rats and Rabbits. The effect of D7193 treatment on oxazolone-induced CS reactions was also examined in Sprague-Dawley rats and in New Zealand White rabbits. CS responses in Sprague-Dawley rats were augmented by treatment with DiNAC according to a similar dose-response relationship as that recorded in mice (results not shown). A similar situation was observed in outbred New Zealand White rabbits, although a tendency to a bell-shaped dose-response curve with slightly reduced responses at the highest dose when compared to those at lower doses was observed in this species (results not shown).
Effects of D7193 on CS Reactions Induced by FITC and DNFB in BALB/c
Mice.
In BALB/c mice, FITC induces a TH2 type response, whereas
DNFB induces a TH1 type response (Tang et al., 1996). Mice were sensitized to FITC and to DNFB, and challenges were performed with the
corresponding agent in groups of animals treated with different daily
oral doses of D7193 or the corresponding vehicle. Interestingly, in
three of three experiments, D7193 enhanced the response to FITC in
FITC-sensitized animals in a dose-dependent manner but markedly reduced
the response to DNFB in DNFB-sensitized animals (results from
representative experiments are shown in Fig.
10, A and B, respectively). DiNAC is
less potent in augmenting the CS reaction in the FITC system (Fig. 10A)
than in the oxazolone system (Fig. 1). Similar effects were seen when
Sprague-Dawley rats were sensitized to and challenged with DNFB or FITC
with D7193 (results not shown).
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Effects of D7193 on Footpads DTH Reactions Induced by mBSA in Mice. Treatment with D7193 reduces footpads' DTH reactions induced by mBSA in mBSA-sensitized mice in a dose-dependent manner (Fig. 11).
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Effects of D7193 Treatment on a DTH Granuloma Reaction Induced by
mBSA.
A DTH reaction to mBSA induced by the soaked filter paper
method (Dunn et al., 1989) results in a quantifiable chronic
granulomatous lesion. Treatment with 3 µmol/kg D7193 from days 0 to
21 reduced the development of this chronic cell-mediated inflammatory
reaction (Fig. 12); treatment with 0.03 µmol/kg did not have a effect (data not shown). Treatment during days
0 to 6 did not influence the DTH granuloma reaction, whereas treatment
during days 21 to 28 was as effective as treatment during days 0 to 21 (results not shown).
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The present data indicate that DiNAC modulates CS and DTH responses in rodents. The cellular and molecular targets of DiNAC are not defined. Their identification is hampered by the lack of effect of the compound at relevant concentrations in in vitro systems hitherto examined (results not shown). The reason for this is not clarified; possible explanations include: 1) that the appropriate in vitro system has not yet been examined or 2) that D7193 acts as an oxidant (see below) and that the high oxygen tension in vitro compared to that in vivo perhaps masks effects of DiNAC in the former situation.
Does DiNAC Act as an Immunomodulator?
A CS reaction resulting
from challenge of appropriately sensitized rodents reflects a response
mediated by antigen-presenting cells and T cell subsets (Askenase,
1992; Grabbe and Schwarz, 1998). Antigen presentation in the skin is
largely performed by Langerhans cells; early-acting CS-initiating T
cells and mast cells are also required in the afferent phase of the
response. Removal of epidermal Langerhans cells by steroid application
enhances the effector phase, suggesting that these cells
may exert a down-regulatory rather than a stimulatory role in the
efferent phase (see Grabbe and Schwarz, 1998). A compound that affects
only Langerhans cells would thus be expected to influence early and
late phases of the CS reaction in opposite directions. The stimulatory
effect observed with D7193, whether treatment is performed early or
late, would suggest that it does not act solely on Langerhans cells.
). The present observation that D7193 increases the number of
CD8+ cells (but does not affect
CD4+ cells) in oxazolone-induced CS reactions
suggests that CD8+ T cells are direct or indirect
targets of the compound.
The response to oxazolone in BALB/c mice is characterized by 1) IgG2a
but not IgE antibody production and 2) IL-2 and IFN- production but
low levels of IL-4 and IL-10 generation by mitogen-stimulated lymph
node cells (Dearman et al., 1995; Dieli et al., 1997) suggesting a TH1
type of response to this sensitizer. In contrast, with respect to its
response to Leishmania, BALB/c mice are considered to have a
TH2 phenotype (Bogdan et al., 1993). The relative effect of D7193 in
oxazolone-sensitized mice is neither more nor less expressed in the
BALB/c strain than in other strains, which, in contrast to BALB/c based
on their responses to Leishmania, represent primarily the
TH1 phenotype mice (Bogdan et al., 1993). However, using the observation that FITC induces a TH2 response in BALB/c mice whereas DNFB induces a TH1 response (Tang et al., 1996), we observed that D7193
augments the CS reaction in the former system, but reduces it in the
latter. This finding suggests that the compound may influence TH1- and
TH2-mediated responses differentially in different systems. Although
additional experiments are needed to clarify the effect of D7193 in the
context of a TH1/TH2 concept, the contrasting effects of D7193 in the
FITC and DNFB systems are unlikely to be explained by nonimmunological effects.
Does D7193 Act on Cellular Adhesion or Homing?
The development
of optimal CS responses relies on several types of adhesion molecules
(see Grabbe and Schwarz, 1998). For example, blocking LFA-1 - ICAM-1
interactions during the afferent phase induces a state of
antigen-specific nonresponsiveness (Scheynius et al., 1996), and CS
reactions to DNCB are impaired in mice deficient in ICAM-1 (Sligh et
al., 1993). Although expression of selectins was not examined in the
present experiments, there was only a slight increase in
ICAM-1+ and LFA-1+ cells in ear specimens from
D7193 versus vehicle-treated animals. This suggests that D7193 does not
primarily influence the LFA-1 - ICAM-1 interaction.
). The present data do not
reveal any change in CD44 expression with D7193 treatment.
Does D7193 Treatment Influence the Expression of
Chemokines/Cytokines?
Although there is a nonspecific
up-regulation of TNF- and IFN- in tissue exposed to irritants as
compared with a more specific increase in IL-1, MIP-2, IP-10, and
MHC class II signals early in the afferent phase of allergen-specific
CS reactions (Enk and Katz, 1992), TNF- and IFN- are main
effector cytokines in the latter (Gautam et al., 1994; Grabbe and
Schwarz, 1998). Previous data also suggest that IL-12 drives the CS
reaction, whereas IL-10 reduces it (see Grabbe and Schwarz, 1998). The
role of IL-4 apparently differs with the CS system examined; IL-4 does
not influence the CS response to oxazolone but apparently augments that
to picryl chloride (see Thomson et al., 1993; Asherson et al., 1996;
Grabbe and Schwarz, 1998). The present experiments detected expression of RANTES, MIP-1, MIP-1, MIP-2, MCP-1, IL-4, IL-6, and IL-10 in
ear tissue from animals sensitized to and challenged with oxazolone. However, mRNA expression levels for none of the examined
chemokine/cytokines were altered by D7193 treatment.
Precedents for the Activity of D7193?
Low molecular thiols
enhance various forms of the immune response in vivo and in vitro but
may also reduce such responses. Disulfides like HEDS and ADA 202-718
augment allogenic responses and IFN- production in mixed lymphocyte
reactions, and potentiate primary and secondary humoral immune
responses in vivo (Hiestand and Strasser, 1985a,b; Kinscherf et al.,
1994). Bell-shaped concentration-response relations in some tests for
ADA 202-718 suggest a complex mode of action (Hiestand and Strasser,
1985b). Thus, the effect of DiNAC in the CS reaction has functional
precedents. A stringent structure-activity relation was disclosed in
the oxazolone system as exemplified by 1) a lack of effect recorded for
some compounds closely related to DiNAC, e.g.,
L-homocystine,
N,N'-diacetyl-L-homocystine, cystamine,
N,N'-diacetyl-L-cystathionine,
and N,N'-diacetyl lanthionine, 2) a
borderline activity recorded for
N,N'-diacetyl-L-cystamine, and 3) a low effect recorded for the D form of the DiNAC. These results, as well as the dose relation for D7193 compared with that of
NAC (Fig. 1) showing that D7193 hardly acts as a prodrug of the latter,
and the potency of D7193, together suggest that DiNAC acts as an
oxidant at stereochemically defined site(s) of some specific target protein(s).
Possible Molecular Targets of D7193?
There are several
possible targets of DiNAC, some of which are presently being examined.
Thus, DiNAC may interfere with oxidoreductases like thioredoxin,
glutaredoxin, or protein disulfide isomerase or their corresponding
oxidoreductase reductases. The importance of these systems for immune
responses in general is underlined by findings that thioredoxin
modulates the production of a number of cytokines in vitro (Schenk et
al., 1996), is a growth factor for T cells inducing expression of the
-chain of the IL-2 receptor (Tagaya et al., 1989), and modulates
activities of transcription factors such as AP-1, NF-B, and TCF-1
(Schreck et al., 1991; Dröge et al., 1994; Schenk et al., 1996).
In this context, it is interesting to note that the contact sensitizer
1-chloro-2,4-dinitrobenzene (DNCB), a glutathione-depleting agent which
also affects T cell signal-transduction pathways (Kavanagh et al.,
1993), is an effective inhibitor of thioredoxin reductase (Arner et
al., 1995). However, the contrasting effects of D7193 on the
DNFB-induced CS reaction (inhibition) and on the BSO-enhanced CS
reactivity to oxazolone (further enhancement) suggest that influence on
glutathione levels is not the single major effect mechanism of D7193.
) reduces
autoimmune manifestations in an EAE model and induces a shift in
cytokine profiles from TH1 dominance to a TH2 type (Brenner et al.,
1997). The reduction of the oxazolone-induced CS reaction resulting
from AMG treatment, and its reversal by simultaneous treatment with
D7193, would suggest that D7193 may mimic the action of NO, i.e., by
acting as an oxidizing agent. A nonselective inhibitor of NO synthase,
L-NAME, did not affect the oxazolone-induced reaction and
did not affect the D7193-induced enhancement of the oxazolone CS
reaction. More detailed studies are needed to clarify the basis for
these findings.
Disulfides like HEDS and ADA 202-718 restore a depressed DTH response
in CSA-treated mice (Hiestand and Strasser, 1985b; St Georgiev, 1988).
We observed that the D7193-induced enhancement of the CS reaction to
oxazolone was reduced by simultaneous CSA or rapamycin treatment.
Because the CSA-induced inhibition of calcineurin leads to a block in T
cell IL-2 production whereas rapamycin-induced immune suppression
targets IL-2-induced T cell activation, the present findings suggest
that neither IL-2 production nor IL-2 action are sole targets of D7193.
As will be reported elsewhere, D7193 at appropriate doses in vivo also
markedly prolongs the lifespan of MRL-lpr/lpr mice (B.S. et
al., unpublished data), reduces development of atherosclerosis in
rabbit and mice model systems (K. Pettersson et al., unpublished data),
and reduces early and blocks late airway reactions in
allergen-challenged sheep (B. Abraham et al., unpublished data).
The immunomodulatory activity of D7193 in vivo is thus not restricted
to the CS reaction.
In conclusion, DiNAC is a potent and effective enhancer of the CS
reaction to oxazolone in rodents. In contrast, it reduces the CS
reaction to DNFB and the DTH reaction to mBSA. From the present data,
it may also be relevant to ask whether the immunological effects
attributed to NAC in vivo may be due, at least partly, to the activity
of a contaminating disulfide dimer molecule, DiNAC, with oxidant activity.
| |
Acknowledgments |
|---|
We thank Maria Grylling for technical assistance with immunohistochemical stainings.
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Footnotes |
|---|
Accepted for publication October 16, 1998.
Received for publication May 22, 1998.
1 This work was supported in part by Grant 7924 from the Swedish Medical Research Council, and grants from the Swedish Council for Work Life Sciences and the Swedish Foundation for Health Care Sciences and Allergy Research.
2 Current address: Department of Human Pharmacology, Box 34, S 22100 Lund, Sweden.
3 Current address: Department of Laboratory Medicine, Division of Clinical Immunology, Karolinska Hospital and Institute, S 171 76, Stockholm, Sweden.
4 Current address: Department of Cell & Molecular Biology, Astra Draco AB, Box 34, S 221 00 Lund, Sweden.
Send reprint requests to: Håkan Bergstrand, Department of Cell & Molecular Biology, Astra Draco, P.O. Box 34, S-22100, Lund, Sweden. E-mail: hakan.bergstrand{at}draco.se.astra.com
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Abbreviations |
|---|
ADA 202-718, ethylene-2,2'-bis(dithio)-bis(ethanol);
AMG, aminoguanidine;
BSO, L-buthionine-[S, R]-sulfoximine;
CS, contact
(hyper)sensitivity;
CSA, cyclosporin A;
DDTC, diethyldithiocarbamate;
DiNAC, N,N'-diacetyl-L-cystine;
DNFB, 2,4-dinitrofluorobenzene;
DTH, delayed type hypersensitivity;
FITC, fluorescein isothiocyanate;
HEDS, bis-(2-hydroxyethyl)-disulfide;
mAbs, monoclonal antibodies;
mBSA, methylated BSA;
L-NAME, N-nitro-L-arginine methyl
ester;
NAC, N-acetyl-L-cysteine;
NO, nitric
oxide;
Oxazolone, 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one;
RPA, RNase protection assay.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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-producing CD8+
+ T lymphocytes and IL-2-producing CD4++ T lymphocytes during contact sensitivity.
J Immunol
158:
2567-2575[Abstract].B transcription factor and HIV-1.
EMBO J
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2247-2258[Medline]. and interleukin-4 in oxazolone-specific Ig and T-cell responses.
Immunology
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185-192[Medline].This article has been cited by other articles:
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) BALB/c mice
(n = 6) and mice sensitized to and challenged with
oxazolone with (
, n = 8) or without (
,
n = 6) treatment with D7193 3.0 µmol/kg orally
for 7 days. Hematoxylin and eosin staining (A), presence of epidermal
CD8+ cells (B), and presence of CD8+ cells (C)
in dermis. For explanation of evaluation scores see Materials
and Methods.
