CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, Jan. 2004, p. 203–210
1071-412X/04/$08.00?0 DOI: 10.1128/CDLI.11.1.203–210.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 11, No. 1
Induction of Nitric Oxide Production Mediated by Tumor Necrosis
Factor Alpha on Staphylococcal Enterotoxin C-Stimulated
Bovine Mammary Gland Cells
Ken-ichi Komine,1* Toshinobu Kuroishi,1Yumiko Komine,1Kouichi Watanabe,2
Jin Kobayashi,3Takahiro Yamaguchi,2Shin-ichi Kamata,4
and Katsuo Kumagai1
T-Cell Research Institute, Minami-Yoshinari, Aobaku,1Graduate School of Agricultural Science, Tohoku University,2
and Department of Animal Reproduction, Miyagi Agricultural College Research Farm,3Sendai,
and Department of Veterinary Science, Nippon Veterinary and Animal Science University,
Received 5 May 2003/Returned for modification 22 July 2003/Accepted 21 September 2003
Mammary gland (MG) secretions (MGS) derived from secretory cows infected with coagulase-negative
staphylococci (CoNS) showed somatic cell counts and lactoferrin similar to levels found in the MGS of
secretory cows infected with Staphylococcus aureus. However, nitrite and nitrate (NOx) and staphylococcal
enterotoxin C (SEC) were found in MGS infected with S. aureus at much higher levels than in cows infected
with CoNS. These results suggested that NOx could be intimately correlated with the production of SEC in
secretory cows infected with S. aureus. Therefore, we examined the production of NOx and the expression of
proinflammatory cytokines and microsomal cytochrome P450 (CYP450) after injection of SEC into the MGS
of secretory cows. We were able to detect NOx and the proinflammatory cytokine tumor necrosis factor alpha
(TNF-?) on MG cells of SEC-injected MGS. It was also found that CYP450 in the MG cells from SEC-injected
MGS was down-regulated by approximately one-third in comparison with the cells from phosphate-buffered
saline-injected MGS. This in vitro system also showed that NOx could be induced in the culture of bovine
macrophage-lined cells (FBM-17) with the supernatants of SEC-stimulated bovine peripheral blood lympho-
cytes (BoPBLs) but not in the culture of peripheral mononuclear cells with SEC-stimulated BoPBLs. The
expression of the mRNA for both inducible nitric oxide synthase and TNF-? in FBM-17 was enhanced by
culturing with the supernatant of SEC-stimulated BoPBLs, although CYP450 was down-regulated. These
results indicate that the down-regulation of CYP450 was caused by the production of TNF-? in SEC-stimu-
lating MG cells containing macrophages and via NOx production. Therefore, we suggest that NOx released
from activated MG cells via the superantigenic activity of SEC caused oxidative damage to the MG in S.
Staphylococcus aureus is the major causative bacteria of bo-
vine mastitis. S. aureus infections result in changes of T-cell
subpopulations and proinflammatory cytokine and chemokine
production (27, 35). Moreover, lymphocytes and leukocytes
stimulated with staphylococcal enterotoxins produce proin-
flammatory cytokines (36) and nitric oxide (17), which show
inflammatory effects. In ruminants, many strains of S. aureus
isolated from mammary gland (MG) secretions (MGSs) pro-
duce staphylococcal enterotoxin C (SEC) (16, 20, 33). It was
reported that SEC induced clinical signs of bovine mastitis
such as an increase in the number of leukocytes in the MG
(16). SEC binds to the specific V? chain of the T-cell receptor
via the major histocompatibility complex class II molecules of
antigen-presenting cells such as macrophages and activates
bovine T lymphocytes (5, 18, 36). Then, the activated T lym-
phocytes and macrophages produce proinflammatory cyto-
kines such as interleukin-1 (IL-1), IL-6, gamma interferon, and
tumor necrosis factor alpha (TNF-?) (35, 36). Therefore, it is
suggested that the superantigenic activity of SEC induces bo-
vine mastitis (5, 36). Thus, it is indicated that inflammatory
cytokines such as IL-1, IL-6, and TNF-? are produced by
activated leukocytes and lymphocytes in the early stages of
Down-regulation of intracellular cytochrome P450 (CYP450)
expression is caused by IL-1, IL-6, and TNF-? (24, 25), which
are produced in response to certain toxins such as staphylo-
coccal enterotoxin B (31), aflatoxin B1 (15), and Escherichia
coli lipopolysaccharide (LPS) (19). These cytokines also induce
the expression of the inducible isoform of nitric oxide (NO)
synthetase (iNOS), which results in the production of NO in
certain cells including hepatocytes, macrophages, endothelial
cells, and leukocytes during the cellular response. Several stud-
ies have suggested that NO production is mediated by the
down-regulation of CYP450 expression in cytokine and LPS
models of inflammation, based on the ability of exogenously
administered NO to down-regulate CYP450 gene expres-
sion and attenuate the down-regulation by NO inhibitors (13,
14). Then, the production of NO is regulated by the down-
regulation of CYP450 and causes oxidative damage to tissues
In this study, we examined whether leukocytes stimulated
* Corresponding author. Mailing address: T-Cell Research Institute,
Bldg. ICR 6-6-3, Minami-Yoshinari, Aobaku, Sendai 989-3204, Japan.
Phone: 81 22 279 9476. Fax: 81 22 279 9548. E-mail: komi502
with SEC induce the down-regulation of CYP450 expression
mediated by TNF-? and production of NO.
MATERIALS AND METHODS
Milk samples. A total of 10 milk samples from 10 cows were collected: 3 from
healthy MGs of 3 cows, 5 from chronic mastitic MGs of 5 cows, and 2 from acute
mastitic MGs of 2 cows. For all samples, we examined the inflammatory signs of
MGs with a PL tester (Nippon Zenyaku Kogyou, Fukushima, Japan) to perform
the modified Californian mastitis test (MCMT) and detect clots. The somatic cell
counts (SCC) of MGSs were made with a flow cytometer (FACSCalibur; Becton
Dickinson, Co., Ltd., San Jose, Calif.) and based on a cell count due to the
specific binding of propidium iodide to DNA (11). The concentration of lacto-
ferrin (Lf) was measured by the single radial immunodiffusion method (Eco-
Biosystem Institute, Co., Furukawa, Japan) (12). The causative staphylococci in
mastitic MGSs were isolated with staphylococcus no. 110 agar medium (Nissui
Pharmaceutical Co., Ltd., Tokyo, Japan), and the staphylococcus counts were
measured as described by Kai et al. (9, 10). Coagulase production was deter-
mined by tube test of rabbit plasma (Eiken Chemical Co., Ltd., Tokyo, Japan).
Isolated staphylococci were identified with a commercial kit (Api staph system;
bioMe ´rieux sa., Marcy l’Etoile, France). The SEC concentration in MGSs was
measured by using the sandwich enzyme-linked immunosorbent assay with anti-
SEC sheep immunoglobulin G (IgG) and horseradish peroxidase-conjugated
anti-SEC sheep IgG (Toxin Technology, Inc., Sarasota, Fla.) (16).
SEC injection into the mammary glands of lactating cows. The tested cows
were bred in our laboratory, and 7 MGs of 3 Holstein cows were used. The cows
were 3 to 5 years old and more than 6 months parturient. In this experiment, we
used MGs for which there were no clinical signs of mastitis, the MCMT was
negative, total bacteria counts in MGSs were less than 103CFU/ml, S. aureus and
E. coli were not detected, and SCC in MGSs were less than 3 ? 105/ml. One
hundred micrograms of SEC (Toxin Technology, Inc.) was dissolved in 10 ml of
phosphate-buffered saline (PBS) and sterilized by filtration through a membrane
filter (pore size, 0.45 ?m). Before the experiment, these cows were injected with
antibiotics (cefazolin sodium; Mitaka Pharmaceutical Co., Ltd., Tokyo, Japan)
every day for 3 days. After the antibiotic treatment, SEC (4 udders) and/or PBS
(3 udders) was injected into each MG after milking by using a cannula (9, 10, 16).
Measurement of MGSs. We examined the MG for inflammatory signs by using
a PL tester to perform the MCMT and SCC and detect clots in MGSs. Milk
samples were obtained aseptically from each injected MG. Polymorphonuclear
cells (PMNs) and mononuclear cells (MNs) were collected with a cytospin (Shan-
don Scientific Ltd., Runcorn, Cheshire, England) and stained with May-Gu ¨n-
wald-Giemsa reagent. After drying, a minimum of 300 cells were identified and
the proportions of various cell types were determined. MG cells were classified
as PMNs, MNs, and/or other type cells. The proportions of PMNs and MNs were
calculated by division by the total cell count (11).
Cells. Cells of SEC-injected MGs were separated as described previously (9).
MGS was centrifuged at 1,000 ? g for 20 min at 4°C to remove fat and whey and
then washed three times with PBS containing 20 mM EDTA. After the wash,
MG cells were suspended in RPMI 1640 medium (Nissui Pharmaceutical Co.,
Ltd.) containing 5% fetal calf serum (FCS; GIBCO BRL, Life Technology, Inc.,
Gaithersburg, Md.). The bovine macrophage cell line used was FBM-17 (ob-
tained from K. Yoshihara, National Institute of Animal Health) (37). The
FBM-17 line was established from a fetal bovine thymus and expresses major
histocompatibility complex class I and II antigens on the cell surface. The culture
medium used for the FBM-17 cells was RPMI 1640 supplemented with 15% FCS
including 10?5M 2-mercaptoethanol. Both bovine peripheral blood lymphocytes
(BoPBLs) and PMNs were collected from the peripheral blood of healthy Hol-
stein cows. Heparinized peripheral blood was separated with Lympholight H
(Sederan, Hornby, Canada), and BoPBLs were collected as the lymphocyte
fraction. The precipitated cells were incubated at 37°C for 4 h, and PMNs were
collected as cells that floated on the culture medium. The culture medium for
PMNs was RPMI 1640 containing 10% FCS. BoPBLs (2 ? 105cells/ml) were
incubated with SEC (1 ?g/ml) at 37°C for 18 h. FBM-17 cells and PMNs were
cultured with the culture supernatants of SEC-stimulated BoPBLs for 18 h, and
the concentration of NO in the supernatants was measured. The expression of
iNOS and TNF-? was stimulated in both FBM-17 cells and PMNs with the
supernatants of SEC-stimulated BoPBLs in RPMI 1640 medium containing 1%
FCS, and samples were collected at 4 h after cultivation. For the measurement
of CYP450 content, FMB-17 was incubated with the supernatant of SEC-stim-
ulated BoPBLs for 3 days at 37°C in a CO2incubator.
NOx measurement of MGSs and culture supernatant. MGSs were treated
with cold 0.1 M sodium acetate solutions (pH 4.0) to precipitate caseins (2). The
sodium acetate-treated MGSs were centrifuged at 4,000 ? g for 10 min at 4°C to
TABLE 1. Concentrations of SEC and NOx in staphylococcal mastitic MGSsa
Staphylococcus lentus, Staphylococcus xylosus,
Staphylococcus sciuri (n ? 3)
(3.6 ? 2.8) ? 102
5.3 ? 0.01
131.8 ? 21.5
0.002 ? 0.001b
2.02 ? 0.47
Staphylococcus xylosus, Staphylococcus sciuri,
Staphylococcus warneri (n ? 3)
(6.7 ? 4.8) ? 104
801.9 ? 799.1
793.5 ? 479.3
0.077 ? 0.06
4.31 ? 1.53
Staphylococcus aureus (n ? 2)
(7.8 ? 1.2) ? 102
1,195.0 ? 505.0
908.3 ? 119.7
11.20 ? 2.29b
11.72 ? 1.81b
Staphylococcus aureus (n ? 2)
(2.5 ? 0.4) ? 106b
1,025.0 ? 345.0
558.1 ? 230.5
11.14 ? 2.02b
11.15 ? 1.02b
aResults are means ? standard deviations.
bSignificant difference compared with CoNS-infected chronic MGSs (P ? 0.01).
204 KOMINE ET AL.CLIN. DIAGN. LAB. IMMUNOL.
remove casein and fat, and the supernatants were collected. FBM-17 cells and/or
PMNs were incubated with the supernatants of SEC (1 ?g/ml)-stimulated
BoPBLs for 18 h at 37°C in a CO2incubator. The cultures of both cells were
centrifuged at 1,000 ? g for 15 min, and the supernatants were collected. The
nitrate and nitrite (NOx) concentrations of the supernatants of sodium acetate-
treated MGSs and culture supernatants of both FBM-17 cells and PMNs were
measured by adding 100 ?l of Griess reagent [2% sulfanilamide in 5% phospho-
ric acid mixed with 0.2% N-(1-naphtyl)-ethylenediamine (1:1, vol/vol)] and mon-
itoring the absorbance at 550 nm (2, 3).
Immunohistochemistry. Mammary gland parenchymal tissue was taken from
the central area of the upper body of the gland and from the area surrounding
the gland cistern. Tissue samples were cut into cubes of less than 1 cm3, quickly
frozen in acetone dry ice at ?70°C, and stored below ?70°C. Cryostat sections,
5 ?m thick, were prepared from the frozen tissues. The sections were incubated
with monoclonal antibody to ovine IL-1?, IL-6, and TNF-? (Chemicon Interna-
tional, Inc., Temecula, Calif.) at a 1:100 dilution for 14 h at 4°C. After three
washes with PBS, fluorescein isothiocyanate-conjugated goat F(ab?)2anti-mouse
IgG1 and/or goat F(ab?)2anti-mouse IgG2a diluted at 1:500 was added and the
sections were incubated at room temperature (20°C) for 45 min. The immuno-
reactive cells were observed by using a confocal laser microscope (MRC-1024;
Bio-Rad, Richmond, Calif.) (34).
Analysis of mRNA of IL-6, TNF-?, and iNOS by RT-PCR. In MG cells, PMNs,
and FBM-17 cells, the mRNA expression of IL-6, TNF-?, iNOS, and glyceral-
dehyde-3-phosphate dehydrogenase (GAPDH) was analyzed by reverse tran-
scription-PCR (RT-PCR). Total RNA was extracted from the cells of SEC-
and/or PBS-injected MGs, PMNs, and FBM-17 cells by using guanidinium thio-
cyanate and isolated with oligo(dT)-cellulose (QuickPrep micro mRNA purifi-
cation kit; Amersham Biosciences UK Ltd., Little Chalfont, England). The
mRNA sample (20 ?l) was heated at 65°C for 10 min and cooled on ice. The first
standard cDNA synthesis was performed with a first standard cDNA synthesis kit
(Amersham Biosciences UK Ltd.). The samples were heated at 90°C for 5 min.
For each PCR, 0.75 ?l of input first standard cDNA was used in a final reaction
volume of 50 ?l containing 200 ?M deoxynucleoside triphosphate, 0.4 ?M
specific forward primer, 0.4 ?M specific reverse primer, 10 mM Tris-HCl (pH
8.3), 50 mM KCl, and 2.5 U of Taq DNA polymerase (Takara, Kyoto, Japan).
The specific forward and reverse primers for GAPDH, IL-6, TNF-?, and iNOS
were based on previous reports (3, 4). Thermocycling was accomplished by using
a program with an initial denaturing step of 95°C for 10 min followed by 40 cycles
of 94°C for 30 s, 55°C for 90 s, and 72°C for 60 s in a Perkin Elmer 9600
thermocycler (Perkin Elmer, Palo Alto, Calif.). The mRNA expression levels for
IL-6, TNF-?, and iNOS are presented as relative units after normalization to the
observed GADPH level.
Measurement of microsomal CYP450 concentration. SEC-exposed MG cells
and FBM-17 cells incubated with the culture supernatant of SEC (1 ?g/ml)-
stimulated BoPBLs for 15 h at 37°C were removed and homogenized in 10
volumes of 1.15% KCl containing 1 mM EDTA (pH 7.4). The homogenates were
centrifuged at 10,000 ? g for 20 min. The supernatants were again centrifuged at
105,000 ? g for 90 min. Pellets were resuspended in 10 mM phosphate buffer
containing 1.15% KCl and 1 mM EDTA, pH 7.4 (26). Concentrations of CYP450
were obtained from the molar absorptivity, 1.20 ? 105M?1cm?1at 450 nm, of
the CO-reduced form (8).
Statistical analysis. The results are shown as the means ? standard devations
for these experiments. Data were compared by using the two-tailed Student’s t
test to determine significance.
NOx and SEC concentrations in staphylococcal mastitic
MGSs. We isolated coagulase-negative staphylococci (CoNS)
in clinically normal MGSs, and these MGSs showed low levels
of staphylococcus counts, concentrations of SEC and NOx, and
other clinical mastitic markers such as SCC and the Lf con-
centration in MGSs. Chronic mastitic MGSs were classified
into two groups: CoNS-infected MGSs and S. aureus-infected
MGSs. In acute mastitic MGSs, we found that only S. aureus-
infected MGSs were confirmed. In CoNS-infected chronic
MGSs, we detected the mastitis value of staphylococcus counts
(6.7 ? 104? 4.8 ? 104CFU/ml), SCC (801.9 ? 104? 799.1 ?
104/ml), and Lf concentration (793.5 ? 479.3 ?g/ml) as the
same as those in S. aureus-infected chronic (staphylococcus
counts, 7.8 ? 102? 1.2 ? 102CFU/ml; SCC, 1,195.0 ? 104?
505 ? 104/ml; Lf concentration, 908.3 ? 119.7 ?g/ml) and
acute (staphylococcus counts: 2.5 ? 106? 0.4 ? 106CFU/ml;
SCC, 1,025.0 ? 104? 345.0 ? 104/ml; Lf concentration, 558.1
? 230.5 ?g/ml) mastitic MGSs. However, the concentrations of
both SEC (chronic mastitic MGSs, 11.20 ? 2.29 ng/ml; acute
mastitic MGSs, 11.14 ? 2.02 ng/ml) and NOx (chronic mastitic
MGSs, 11.72 ? 1.81 ?M/ml; acute mastitic MGSs, 11.15 ? 1.02
FIG. 1. Clinical signs of MGSs after SEC injection. The inflammation of quarters was examined with a PL tester to perform the MCMT (pH,
dark gray bars; agglutination, light gray bars) and to measure clots (white bars) of MGS.
VOL. 11, 2004TNF-? INDUCES NO PRODUCTION BY SEC 205
?M/ml) in S. aureus-infected MGSs were higher than those in
CoNS-infected chronic mastitic MGSs (SEC concentration,
0.077 ? 0.06 ng/ml; NOx concentration, 4.31 ? 1.53 ?M/ml)
(Table 1). The differences in the concentrations of SEC and
NOx in MGSs between CoNS-infected MGSs and S. aureus-
infected MGSs was significant (P ? 0.01).
Clinical signs and cell concentrations in SEC-injected MGs.
A positive result for the MCMT and the presence of clots in
MGSs from SEC-injected MGs were confirmed at 17 h postin-
jection (hpi). However, we could not detect clinical signs in
PBS-injected MGs (Fig. 1). SCC increased to 1,735.0 ? 104?
994.6 ? 104/ml in MGSs of SEC-injected MGs at 17 hpi (Fig.
2A), and PMNs accounted for approximately 80% (1,465.6 ?
104? 753.8 ? 104/ml) of the SCC of SEC-injected MGs (Fig.
2B). Moreover, the number of MNs from SEC-injected MGs
(236.0 ? 104? 216.2 ? 104/ml) had increased over 15-fold in
comparison with MNs from PBS-injected MGs at 17 hpi (Fig.
2C). The difference in the number of these cells in MGs be-
tween SEC-injected and PBS-injected MGs was significant (P
NOx of MGSs and culture supernatant. In MGSs from SEC-
injected MGs, the concentration of NOx increased to 13.52 ?
3.45 ?M/ml at 15 hpi. However, NO production did not occur
in MGS from PBS-injected MGs (Fig. 3A). We were able to
confirm that the NOx concentration in the MG differed signif-
icantly between SEC-treated cows and PBS-treated cows (P ?
0.01). Moreover, we found that supernatants of the culture of
FBM-17 cells with the supernatants of SEC-stimulated
BoPBLs showed higher levels (2.10 ? 0.51 ?M/ml) than the
culture supernatants of unstimulated FBM-17 and the culture
of PMNs with the supernatants of SEC-stimulated BoPBLs (P
? 0.01) (Fig. 3B).
Immunohistochemistry of SEC-injected MG and the mRNA
expression of IL-6, TNF-?, and iNOS on SEC-stimulated MG
cells and bovine macrophages. More infiltrating cells positive
for ovine TNF-? were found in the cistern area of SEC-in-
FIG. 2. Changes of cell counts in MGSs from SEC-injected MGs. SCC (A) of each quarter were also measured by flow cytometry with
propidium iodide (10). Both PMNs and MNs of MGSs were made to adhere to slides by using cytospin. Then the slides were stained with
May-Gru ¨nwald-Giemsa reagents, and cells were classified as PMNs (B), MNs (C), and other type cells (D). F, cell counts of SEC-injected MGs;
E, cell counts of PBS-injected MGs; *, significant difference compared with PBS-injected MGs. All cell counts are given as 104cells per milliliter.
206KOMINE ET AL.CLIN. DIAGN. LAB. IMMUNOL.
jected MGs than in PBS-injected MGs. However, the reaction
for both IL-1? and IL-6 did not differ between SEC and PBS
injection (Fig. 4). The expression of mRNA for TNF-? (4.2 ?
0.85 U) and iNOS (15.4 ? 3.21 U) was confirmed on SEC-
injected MG cells. However, the mRNA of TNF-? and iNOS
could not be detected in PBS-injected MG cells. No expression
of mRNA for IL-6 was detected on either SEC- or PBS-in-
jected MG cells. In the culture of FBM-17 cells with the su-
pernatants of SEC-stimulated BoPBLs, the mRNA expressions
of IL-6 (41.9 ? 3.16 U), TNF-? (29.4 ? 0.58 U), and iNOS (7.2
? 1.24 U) were stronger than that in unstimulated FBM-17
cells (Fig. 5). We were able to confirm that the mRNA expres-
sions of TNF-? and iNOS differed significantly (P ? 0.01).
CYP450 concentrations in SEC-stimulated MGSs and
FBM-17 cells. Microsomal CYP450 concentrations in MG cells
from SEC-injected MGs (average, 0.38 ? 0.31 nmol/106cells)
were approximately one-third times lower than those in MG
cells of PBS-injected MGs (1.55 ? 0.31 nmol/106cells) at 15
hpi. Moreover, microsomal CYP450 concentrations of the cul-
ture of FBM-17 cells with SEC-stimulated BoPBLs (0.24 ?
0.09 nmol/106cells) were approximately half of those in un-
stimulated FBM-17 cells (0.56 ? 0.16 nmol/106cells) and cells
of SEC-injected MGs (Fig. 6). In the case of the MG cells and
FBM-17 cells, we confirmed the significance of the difference
between SEC stimulation and no stimulation (P ? 0.01).
In staphylococcal mastitic MGSs, some inflammatory mark-
ers, including SCC and Lf concentration in CoNS-infected
chronic mastitic MGSs, were confirmed at the same levels of S.
aureus-infected MGSs. However, the NOx and SEC concen-
trations in S. aureus-infected MGSs were significantly (P ?
0.01) higher than those in CoNS-infected chronic mastitic
MGSs. These results suggest that the NOx concentration in
mastitic MGSs correlates with the SEC concentration in
MGSs. Thus, we examined the production of NO in MGs via
SEC injection into healthy bovine MGs. Kuroishi et al. (16)
reported that the injection of SEC into healthy mammary
glands resulted in symptoms of mastitis and an increase in
SCC. In this study, the same results were obtained for the
kinetics of SCC. The PMNs among MG cells increased after
SEC injection to over 90%. Moreover, MNs had increased
approximately 20-fold at 15 hpi, with a peak increase of ap-
proximately 120-fold after SEC injection in comparison with
MN counts from PBS-injected MGs. In addition, NO was de-
tected in the MGSs from SEC-injected cows at 15 hpi. More-
over, we detected the production of NO in the culture of
FBM-17 cells with the supernatants of SEC-stimulated
BoPBLs but not in the culture of PMNs with the supernatants
of SEC-stimulated BoPBLs. In addition, NO production in the
SEC-stimulated FBM-17 cells was less than that in the culture
of FBM-17 cells with the supernatants of SEC-stimulated
BoPBLs (data not shown). These results suggest that SEC
caused the symptoms of mastitis and oxidative damage by NO
from activated MNs, including macrophages, via superantigen
We detected an increase in the mRNA expression for
TNF-? and iNOS in MG cells from SEC-injected MGs in
comparison with MG cells from PBS-injected MGs. Moreover,
we confirmed the expression of TNF-?, but not IL-6, in the
cistern area of the SEC-injected mammary gland. NO produc-
tion was detected in the culture of FBM-17 cells with the
supernatants of SEC-stimulated BoPBLs. In bovine mastitis,
Bouchard et al. (2) reported that NO is released from MG cells
during endotoxin-induced mastitis. However, neutrophils, the
major component in mastitic SCC, express the mRNA of iNOS
and cannot produce NO (3). Therefore, it is suggested that NO
in MGSs from SEC-injected cows was produced by activated
macrophages via the superantigen activity of SEC.
In acute mastitis with E. coli, TNF-? is a major mediator of
endotoxin shock, and elevated TNF-? concentrations in milk
were found in cows that had died from acute E. coli mastitis
during the periparturient period (32). In addition, Blum et al.
(1) reported intramammary production of TNF-? and NOx in
quarters challenged by E. coli and LPS administration but not
FIG. 3. NO production of MGSs from SEC-injected MG and SEC-
stimulated FBM-17 cells and PMNs. MGSs were treated with 0.1 M
sodium acetate (pH 4.0) and dissolved in casein. (A) The NO concen-
trations of these supernatants were measured with Griess reagent. F,
cell counts of SEC-injected MGs; E, cell counts of PBS-injected MGs.
(B) The NO production of the supernatant (Sup) on PMNs and
FBM-17 cells was measured with Griess reagent after SEC stimulation
for 3 days. *, significant difference compared with PBS-injected MGs
or untreated cultures.
VOL. 11, 2004 TNF-? INDUCES NO PRODUCTION BY SEC 207
in unchallenged quarters. The enhanced TNF-? production
was likely responsible for the increased intramammary NOx
production and probably caused enhanced systemic NOx for-
mation, which may contribute to the severity of clinical signs.
In a recent report, staphylococcal enterotoxin B stimulation
caused a down-regulation of CYP450 expression (31). The
CYP450 gene is down-regulated by proinflammatory cytokines,
including TNF-?. In the acute-phase reaction of an inflamma-
FIG. 4. Expression of proinflammatory cytokines (IL-1?, IL-6, and TNF-?) in SEC-injected bovine MGs. Cryosections of the cistern area of
PBS-injected MGs and SEC-injected MGs are shown. Tissues were stained with anti-ovine IL-1?, IL-6, and TNF-? (green). Cell nuclei were
counterstained with propidium iodide (red).
FIG. 5. mRNAexpressionofIL-6,TNF-?,andiNOSinbothSEC-stimulatedMGcellsandtheculturesupernatant(Sup)ofSEC-stimulatedBoPBLs
stimulated with FBM-17 cells. MG cells were collected from PBS- and SEC-injected MG 15 h after SEC injection. The culture of FBM-17 cells with the
supernatant of SEC (2 ?g/ml)-stimulated BoPBLs was incubated at 37°C for 4 h. The mRNA expression of GAPDH, IL-6, TNF-?, and iNOS was
analyzed by RT-PCR. The number under each band represents the expression level relative to the gene for GAPDH. ND, not detected.
208KOMINE ET AL.CLIN. DIAGN. LAB. IMMUNOL.
tory disease, previous reports indicated that down-regulation
of CYP450 induced the production of NO and superoxide (7,
23, 29) and caused oxidative damage to tissues (25). Several
studies have suggested that NO production is mediated by the
down-regulation of CYP450 expression in cytokine and LPS
models of inflammation, based on the ability of exogenously
administered NO to down-regulate CYP450 gene expression
and an attenuation of the down-regulation by NO inhibi-
tors (13, 14). In addition, Minamiyama et al. reported that
NO dose-dependently inhibits the hydroxylation activity of
CYP450 (22). In this study, the microsomal CYP450 concen-
tration of MG cells and FBM-17 cells decreased after stimu-
lation with the supernatants of SEC-stimulated BoPBLs.
Moreover, we detected the expression of iNOS in SEC-stimu-
lated MG cells and FBM-17, a macrophage cell line. There-
fore, it is suggested that SEC stimulation caused the expression
of TNF-? and iNOS on MG cells, and this reaction induced the
down-regulation of NO inhibitors mediated by the down-reg-
ulation of CYP450. These results indicate that SEC stimula-
tion caused the production of NO mediated by TNF-? expres-
sion and down-regulation of CYP450 in bovine MG cells.
The expression of TNF-? activates the transcriptional path-
way of nuclear factor ?B (NF-?B), and this activation induces
the expression of iNOS, which results in the production of NO
in leukocytes (24). Therefore, TNF-? expression is a marker of
NF-?B activation. The results of the present study suggest that
after stimulation with SEC, mononuclear cells and macro-
phages expressed iNOS mRNA and activated the NF-?B path-
way. In inflammatory disease, some intracellular enzymes and
factors of the transcriptional pathway show down-regulation
and/or activation, including CYP450 and NF-?B. CYP450 and
the transcriptional pathway of NF-?B play an important role in
the MG. CYP450 has aromatase activity for the differentiation
of adipose fibroblasts into mature adipocytes mediated by per-
oxisome proliferator-activated receptor gamma. TNF-?, which
is expressed in adipose, also inhibits adipocyte differentiation
(21, 28). NF-?B activation is a negative regulator of ?-casein
gene expression in normal mouse MGs (6), and NF-?B acti-
vation and down-regulation of ?-casein expression are medi-
ated by TNF-? (30). Thus, it is suggested that SEC inhibits the
differentiation of adipose fibroblasts into mature adipocytes
and causes the down-regulation of milk protein production.
These results indicate that increases in these oxidative fac-
tors correlated with the down-regulation of CYP450 and were
caused by TNF-?, and NO induced the oxidative damage to
MGs of cows with mastitis. Moreover, it is suggested that SEC
is an important virulence factor in staphylococcal bovine mas-
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FIG. 6. Concentration of microsomal CYP450 in MG cells (A) and FBM-17 cells (B) incubated with the supernatants of SEC-stimulated
BoPBLs. White bars, PBS-injected MG cells and unstimulated FBM-17 cells; black bars, SEC-injected MG cells and SEC-stimulated FBM-17 cells;
*, significant difference compared with PBS-injected MGs or untreated cultures.
VOL. 11, 2004 TNF-? INDUCES NO PRODUCTION BY SEC209
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210KOMINE ET AL.CLIN. DIAGN. LAB. IMMUNOL.