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Division of Infectious Diseases, Department of Internal Medicine, The University of Texas Medical Branch, Galveston
Correspondence: Dr. Fujio Suzuki, Department of Internal Medicine, The University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0435. E-mail: fsuzuki{at}utmb.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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) produced MCP-1 in
culture fluids. In response to stimulation with herpesvirus antigen, a
population of T cells isolated from CSF cells from HSM mice (CSF-T
cells) produced IL-4 into their culture fluids, although MCP-1 was not
produced by CSF-T cells stimulated by this antigen. IL-4 production by
CSF-T cells was markedly enhanced when they were stimulated with viral
antigen in the presence of murine recombinant MCP-1 (rMCP-1).
Furthermore, IL-4 was produced in naive splenic T cells cocultured with
CSF-M. These results indicate that the severity of HSM is influenced
by MCP-1, which stimulates Th2 responses.
Key Words: cerebrospinal fluids • herpes simplex virus type 2 • interleukin 4 • MCP-1
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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knockout mice to herpes simplex
encephalitis has been shown to be markedly increased compared with that
of their littermates [2
]. When mice infected with
neurovirulent HSV-2 are treated with a mouse IFN--coding sequence in
an HSV-based amplicon vector, the replication of HSV-2 in brains of
these mice is inhibited [2
]. These facts indicate that
responses of T-helper type-1 (Th1) cells play a role in host defense
against HSV infection in the CNS. Conversely, herpes simplex
encephalitis is enhanced in these mice by Th2 responses
[3
]. Administration of interleukin (IL) 4 to mice with
herpes simplex encephalitis causes an increase in the production of
IL-4 from local CD4+ T cells [3
]. The
severity of encephalitis also is markedly enhanced in encephalitic mice
treated with IL-4 [3
]. These results indicate that
Th1/Th2 responses play a very important role in the development of
postinfectious encephalitis in mice infected with HSV-2. Similar results are shown in mice with HSV-induced encephalomyelitis (HSM). The mortality of HSM mice is increased when they are given a mixture of Th2 cytokines (IL-4 and IL-10) or Th2 cells derived from cerebrospinal fluids (CSFs) of HSM mice [4 ]. Furthermore, survival rates of HSM mice are markedly increased when they are given a mixture of monoclonal antibodies (mAbs) for Th2 cytokines [4 ]. These results indicate that the severity of HSM is enhanced by Th2 responses. Th2 cells that produce Th2 cytokines have previously been demonstrated in the CSF of HSM mice [4 ].
Chemokines released from a variety of cells constitute a superfamily of
small peptides (6–14 kDa) that play a crucial role in trafficking and
recruiting effector leukocytes to primary sites of immune responses and
inflammations [5
6
7
8
]. Members of the ß-chemokine
subfamily regulate the migration of various effector cells, such as
monocytes, T cells, neutrophils, eosinophils, basophils, and natural
killer (NK) cells [5
6
7
8
9
]. Macrophage inflammatory
protein (MIP)-1
; MIP-1ß; regulated on activation, normal T
expressed and secreted (RANTES) protein; and monocyte chemoattractant
protein (MCP)-1 are members of the ß-chemokine subfamily. In the CNS,
the expression of MCP-1 mRNA has been demonstrated under pathological
conditions, such as experimental allergic encephalomyelitis, brain
ischemia, or viral meningitis [10
11
12
]. MCP-1 has also
been detected in CSF of patients with viral meningitis and herpes
simplex encephalitis [13
, 14
]. Recently,
the levels of MCP-1 in CSF and sera from patients with herpes simplex
encephalitis have been correlated with the clinical severity of herpes
simplex encephalitis in these patients [15
]. MCP-1 also
has been described as an initiator of Th2 responses that have a
pathogenic role in the development of diseases in mice exposed to HSV-2
[16
17
18
] or infected with Leishmania major
[19
]. In the present study, therefore, we investigated
the effect of MCP-1 on the severity of HSM induced in mice by footpad
infection with HSV-2. Results demonstrated that, in HSV-2-infected
mice, the severity of HSM is greatly enhanced by MCP-1 released from a
population of macrophages in mouse CSF, leading to the possibility that
MCP-1 increases the severity of HSM through induction of Th2 responses.
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Reagents, viruses, media, and Vero cells
Anti-IL-4 and anti-MCP-1 mAbs for enzyme-linked immunosorbent
assay (ELISA) and enzyme-linked immunospot assay were purchased from
PharMingen, San Diego, CA. Recombinant (r) murine IL-4 (rIL-4), rMCP-1,
polyclonal anti-MCP-1 antibody, and rabbit immunoglobulin (Ig) were
purchased from PeproTech, Rocky Hill, NJ. Vero cells were serially
maintained in minimum essential medium (MEM) supplemented with 10%
heat-inactivated fetal bovine serum (FBS), 2 mM
L-glutamine, and antibiotics. Herpes simplex virus type 2
(HSV-2), propagated in Vero cells, was stored at –70°C until used
for the infection experiments. The titer of the stock virus solution
was 2.0 x 107 plaque-forming units (PFU)/mL, as
assayed by the plaque method on Vero cells cultured in maintenance
medium (MEM supplemented with 2% FBS, 2 mM L-glutamine,
and antibiotics). To prepare HSV-2 antigen (HSV-2 Ag), HSV-2 was
UV-irradiated by exposure to a germicidal lamp (George W. Gates & Co.
Inc., New York, NY) at a distance of 20 cm for 15 min
[3
]. This procedure made a 107-fold
reduction in the titer of HSV-2. RPMI 1640 medium supplemented with
10% FBS, 2 mM L-glutamine, antibiotics, 30 mM HEPES, and
5 x 10-5 M 2-mercaptoethanol was used as the
complete medium for the cultivation of spleen cells and cells isolated
from CSF (CSF cells).
HSM mice
HSM mice were prepared by previously published methods with
minor modifications [20
, 21
]. Mice
anesthetized with pentobarbital [50 mg/kg intraperitoneally (i.p.)]
were given HSV-2 (40 µL/mouse) subcutaneously into the left hind
footpad. In our murine model, 5 x 104 PFU/mouse of
HSV-2 were equal to one 50% lethal dose (LD50)
[4
]. A dramatic ipsilateral monoplegia was observed in
mice 5–7 days after infection with five LD50s of HSV-2.
Seven to 9 days after infection, the disease progressed to urinary
incontinence and flaccid paraplegia of the hind legs with typical
myelitis. Subsequently, disease developed in the brains of these mice,
along with quadriplegia, apathy, weight loss, and epilepsy—typical
symptoms for encephalitis. Thus, 10–14 days after infection, mice
showed typical symptoms for both myelitis and encephalitis. The
neuroinvasiveness of HSV-2 injected into mouse footpads occurs by
efficient viral replication in sciatic nerves, dorsal root ganglia,
spinal cords, and brains [20
, 21
]. Thus,
mouse hind-footpad infection with HSM has become a standard model
for the neuropathogenesis of HSV-2 infection in the CNS
[20
, 21
].
Preparation of spleen cells (splenic T cells) and CSF cells (CSF-T
cells and CSF-M)
Spleens were removed from mice at various days after HSV-2
infection. Single-cell suspensions of spleen cells were made from these
spleens by mincing tissues through a fine-mesh screen. After the
elimination of erythrocytes with 83% NH4Cl solution, cells
were resuspended in complete medium and used as spleen cells. CSF was
obtained from HSM mice by the method of Fleming et al.
[22
] with minor modifications. CSF cells were isolated
by centrifugation (820 g, 5 min). Similar cell populations
also were obtained by the following methods. Thus, after intracardiac
perfusion with 30 mL of phosphate-buffered saline (PBS), vertebral
columns were removed from the cervical region of HSM mice. Vertebral
columns removed from HSM mice 5–7 days after HSV-2 infection at
fivefold the LD50 contained an average of 10 µL of CSF.
CSF cells were obtained by washing these columns with 15 mL of RPMI
1640 medium using a 21-gauge needle attached to a 20-mL syringe
[4
]. CSF cells were harvested by centrifugation (820
g, 5 min) of the rinse solution containing CSF. CSF cell
preparations were suspended in complete medium after the elimination of
erythrocytes with 83% NH4Cl solution. CSF cells, isolated
by Fleming’s methods or prepared by our own procedures, and their
purified cell fractions (T cells and macrophages) showed the same
cytokine- (or chemokine-) producing profiles. Therefore, the cells
mainly used for the experiments were the CSF cells obtained from HSM
mice by our procedure. T cells were purified from CSF cells and spleen
cells by using a mouse T-cell enrichment column (R&D Systems,
Minneapolis, MN) [23
], and these purified T cells were
designated CSF-T and splenic T cells, respectively. Their purity was
>96%, as described previously [23
]. Splenic M and
CSF-M (a macrophage population isolated from CSF cells) were
isolated using fibronectin-coated petri dishes (Protein Polymer, San
Diego, CA) [24
]. Fifteen minutes after the incubation at
37°C, the dishes were washed twice with warmed maintenance medium to
remove nonadherent cells from the dish surfaces. The
macrophage-enriched population (92% pure) was harvested by scraping
these dishes with a rubber policeman [24
]. These cells
were suspended in complete medium and used for respective experiments.
Treatment of HSM mice with anti-MCP-1 antibody
To determine the role of MCP-1 in the severity of HSM, a
polyclonal antibody against MCP-1 (10 µg/mouse) was administered i.p.
to HSM mice every other day beginning 2 h before infection with
2.5 LD50s of HSV-2. The dose of antibody administered to
HSM mice was determined according to our previous studies
[4
]. As a control, HSM mice were treated i.p. with
rabbit Ig. To evaluate the effect of anti-MCP-1 antibody on the HSM
progression, a) morbidity (mean survival time in days), b) mortality
(mortality rates), and c) viral growth in spinal cord tissues in HSM
mice treated with the antibody were compared with those of HSM mice
treated with rabbit Ig. To determine mortality rates and mean survival
days, mice were observed daily for 21 days after infection. For
the viral titration, spinal cord tissues were removed from mice 4 and 7
days after infection. Homogenates of these tissues [20% suspension
(w/v)] were subjected to freezing and thawing three times and then
centrifuged at 820 g for 15 min. The supernatants obtained
were assayed for HSV-2 in Vero cells by the plaque assay
[3
]. Each experiment was performed twice, and results
shown in figures were expressed as the mean values of the data from
these two experiments.
Production and assay of IL-4 and MCP-1
To determine the abilities of cells to produce IL-4, CSF cells
and spleen cells (2.5 x 106 cells/mL) obtained from
mice at various days after infection with 2.5 LD50s of
HSV-2 were stimulated in vitro with HSV-2 antigen (UV-inactivated HSV-2
corresponding to 0.1 multiplicity of infection as measured by
counting live viruses) for 24–72 h at 37°C. To induce the
production of MCP-1, these cells were cultured without stimulation. In
some experiments, splenic T cells (106 cells/mL) from
normal mice or CSF-T cells (106 cells/mL) from HSM mice
were stimulated with rMCP-1 at doses of 0.1–100 ng/mL for the
induction of the IL-4 production. The amounts of IL-4 or MCP-1 in
culture fluids of these cells were measured by ELISA. The lower
detection limits of IL-4 and MCP-1 in each assay were 20 pg/mL and 30
pg/mL, respectively.
ELISPOT assay
CSF-M were cultured with naive T cells in a dual-chamber
transwell culture system. Six-hundred microliters of a cell suspension
for naive T cells (3 x 105 cells/well) were placed
into the bottom chamber of the transwell (0.4-µm-diameter micropores)
(Costar, Corning, NY). One-hundred microliters of the cell suspension
for naive M or CSF-M (6 x 105 cells) were
placed into the upper chamber of the transwell. Forty-eight hours after
cultivation, the upper chambers were removed from the transwells, and
cells in the bottom chambers were examined for IL-4-producing cells.
For enumeration of the number of IL-4-producing cells, the ELISPOT
assay system was used. The ELISPOT assay was performed according to
procedures described in previous papers [25
,
26
]. Briefly described, nitrocellulose-bottomed 96-well
microtiter plates (Milliscreen-HA; Millipore Corp., Bedford, MA) were
coated with 4 µg/mL of anti-IL-4 mAb (diluted in 0.1 M carbonate
buffer, pH 9.6) and incubated overnight at 4°C. After they were
washed three times with PBS, the plates were blocked with 200 µg/mL
of 5% bovine serum albumin solution in PBS for 1 h at 37°C.
After removal of the blocking medium, serially diluted cells harvested
from the bottom chambers of the transwells were added to the wells (100
µL each). The plates were incubated for 20 h at 37°C in a
humidified 5% CO2 incubator. Cells were removed from
plates by washing three times with PBS followed by an additional three
washings with PBS containing 0.05% Tween 20. Then biotinylated
anti-IL-4 mAb was added to each well of the plates, and the plates were
kept for 2 h at room temperature. After washing five times with
PBS containing 0.05% Tween 20, a 1:1,000-dilution of
streptavidine-alkaline phosphatase was added to all wells of the
plates, and the plates were incubated for an additional 1 h at
room temperature. Unbound conjugate was removed from all wells of the
plates by washing thoroughly with PBS, and finally 100 µg/mL of
5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium
substrate solution was added to all wells of the plates. Then these
plates were incubated until spots representing single IL-4-secreting
cells appeared in the wells. These spots were counted using a
dissecting microscope.
Statistical analysis
The survival of mice exposed to HSV-2 was analyzed by log rank
test. Other data were statistically checked by the analysis of variance
test followed by Fisher’s protected least-significant-difference test.
If a P value was <0.05, the result obtained was considered
statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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) was responsible for the production
of MCP-1. CSF-M from CSFs of mice 7 days after infection with 2.5
LD50s of HSV-2 produced MCP-1 into their culture fluids
(Table 1
). However, a population of T cells in CSF cells (CSF-T cells) from
the same HSM mice did not produce MCP-1 into their culture fluids
(P < 0.01).
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on numbers of
IL-4-producing cells in cultures of naive splenic T cells was examined.
CSF-M are a population of macrophages isolated from CSF cells of
mice 7 days after infection with 2.5 LD50s of HSV-2.
Transwell cultures were performed with naive T cells (3 x
105 cells/well) placed into the bottom chamber and CSF-M
(6 x 105 cells/well) placed into the upper chamber.
Forty-eight hours after cultivation, the upper chambers were removed
from the transwells, and the bottom wells were examined for
IL-4-producing cells by ELISPOT assay. The number of IL-4-secreting
cells in transwell cultures of naive T cells and naive M was minimal
(<10 per 106 T cells) (Fig. 5
). However, the number of IL-4-secreting cells markedly increased
when naive T cells were cultured with CSF-M (293 ± 35 cells
per 106 T cells). In addition, when the cocultivation of
naive T cells and CSF-M was conducted in the presence of anti-MCP-1
antibody, the production of IL-4 into their culture fluids was not
demonstrated (Table 2
). These results indicate that, through the production of MCP-1,
M in CSF of HSM mice may stimulate T cells to produce IL-4.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemokines have been shown to induce the specific migration of distinct
leukocyte populations during the period of inflammation in trauma
[35
], autoimmune diseases [36
] and
certain viral diseases [37
, 38
]. Proteins
or mRNAs of C-C chemokines (MCP-1, MIP-1, and RANTES) have been
detected in the trigeminal ganglion of mice with herpes simple
encephalitis [39
] or in CSFs of patients with herpes
simplex encephalitis [15
]. In CSFs, the level of these
parameters for MCP-1 is higher than levels of MIP-1 and RANTES, and
the elevated MCP-1 level has been shown to be correlated with the
clinical severity of herpes simplex encephalitis [15
].
These observations suggest that the pathogenesis of HSV infection in
the CNS is influenced by MCP-1.
In the present study, a pathogenic role of MCP-1 in the severity of HSM
was investigated in HSM mice exposed to hind-footpad inoculation of
HSV-2. The morbidity and mortality of HSM mice were decreased when the
mice were treated with polyclonal antibody for MCP-1. Also, as compared
with HSM mice treated with rabbit Ig, the growth of HSV-2 in spinal
cords of HSM mice decreased after the treatment with anti-MCP-1
antibody. These results indicate that the severity of HSM induced in
mice by footpad infection of HSV-2 is influenced by MCP-1. After
stimulation with HSV-2 Ag, CSF cells from HSM mice produced IL-4 into
their culture fluids. However, a decreased amount of IL-4 was produced
by CSF cells from HSM mice that were previously treated with anti-MCP-1
antibody. CSF cells from HSM mice produced MCP-1 without any
stimulation. A population of macrophages in CSF cells (CSF-M) was
shown to be responsible for the MCP-1 production. A population of CSF-T
cells was shown to be responsible for the IL-4 production. CSF-T cells
from HSM mice did not produce MCP-1 into their culture fluids, even
though they were stimulated with HSV-2 Ag (data not shown). When CSF-T
cells from HSM mice or naive T cells from normal mice were stimulated
with rMCP-1 in vitro, production of IL-4 in their culture fluids was
demonstrated. Also, naive T cells produced IL-4 into their culture
fluids when they were cocultured with CSF-M. These results indicate
that MCP-1 released from CSF-M has a pathogenic role in development
and/or severity of HSM in mice, through the stimulation of CSF-T cells
to produce IL-4.
Recently, we performed two additional experiments. In the first
experiment, the severity of postinfectious encephalomyelitis was
examined in IL-4-/- mice infected with HSV-2. Results
showed that 80% of IL-4+/+ mice (n =10)
exposed to 2.5 LD50s of HSV-2 died within 11 days of the
viral infection, whereas 70% of IL-4-/- mice
(n =10) survived after the infection with the same
amounts of HSV-2. In the second experiment, IL-4-/- mice
(n =7), inoculated with 5 x 106 CSF-T
cells/mouse were infected with 2.5 LD50s of HSV-2. As a
control, IL-4-/- mice (n =8), inoculated
with splenic T cells from normal mice, were exposed to HSV-2. In this
instance, 71% of IL-4-/- mice inoculated with CSF-T
cells died, while 75% of control mice survived. Acute neurological
disorders related to encephalomyelitis were not demonstrated in
IL-4-/- mice regardless of whether they were inoculated
with splenic T cells from normal mice. However, encephalomyelitis
developed in IL-4-/- mice after their inoculation with
CSF-T cells derived from HSM mice, which indicates that Th2 cells share
a pathogenic role in the development of encephalomyelitis induced by
HSV-2 infection. Furthermore, MCP-1 released by CSF-M from HSM mice
was shown to stimulate IL-4 production by CSF-T cells (see Table 2
),
which indicates that, through IL-4 induction by CSF-T cells, MCP-1 can
enhance the severity and/or development of HSM [40]. On the
other hand, MCP-1 has been shown to stimulate macrophages for
prostaglandin E2 and IL-10 production [41
].
Because the killing activity of macrophages and Th1-associated cell
lysis are suppressed by prostaglandin E2 and IL-10
[42
, 43
], MCP-1 may have no beneficial
roles in host defense against HSM.
The contribution of MCP-1 to the induction of granulomatous
inflammation in lungs, dictated by Th2 cells, has been recently
demonstrated [44
, 45
]. IL-4 has been
demonstrated to be produced by CD4+ T cells stimulated with
MCP-1 or cocultured with MCP-1-producing fibroblasts
[46
47
48
]. In contrast, IFN- levels were significantly
enhanced in cocultures of Ag-stimulated CD4+ T cells and
lung fibroblasts after immunoneutralization of MCP-1
[18
]. MCP-1 has been postulated to directly
down-regulate IL-12 production that results in a partial or complete
block of Th1 cell differentiation [49
]. These
descriptions support our findings shown herein that MCP-1 released from
CSF-M of HSM mice is a stimulator for IL-4 production by CSF-T
cells. Further studies are required to elucidate the role of MCP-1 in
the pathogenesis of HSV infection in the CNS.
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ACKNOWLEDGEMENTS |
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Received September 7, 2000; revised April 25, 2001; accepted April 26, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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attenuates neutrophil recruitment in the central nervous system during experimental bacterial meningitis Infect. Immun 67,2590-2601 prevents the development of blinding herpes stromal keratitis J. Viol 72,3705-3710 and IL-4 knockout mice: analysis of local and regional cytokine and chemokine networks J. Immunol 159,3565-3573[Abstract]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) 2 h before and 2 and 4 days
after infection. As a control, a group of 20 HSM mice were treated with
rabbit Ig (10 µg/mouse, •) in the same manner. Survival of mice in
each group was observed daily for 3 weeks after infection.
)
was treated with rabbit Ig (10 µg/mouse) as a control. CSF cells
(2.5 x 106 cells/mL) from these groups of mice at 3,
5 and 7 days after infection were stimulated in vitro with HSV-2 Ag
(0.1 multiplicity of infection as an amount for live HSV-2). Culture
fluids harvested 48 h after the stimulation were assayed for IL-4
by ELISA. Data are means of triplicate cultures.
