Department of Microbiology, University of Tennessee, Knoxville
Correspondence: Dr. Barry T. Rouse, Department of Microbiology, M409, Walters Life Sciences Building, University of Tennessee, Knoxville, TN 37996. E-mail: btr{at}utk.edu
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Key Words: lipopolysaccharide • macrophage • HSK • stress proteins
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Virus and nonspecific stimulants
HSV type 1 (HSV-1; KOS strain) was grown on vero cell
monolayers (American Type Culture Collection, Manassas, VA; CCL81). HSV
mutants ICP4-/-(d120) and E5-supplementing vero cells were provided
by Dr. DeLuca, University of Pittsburgh (PA), and replication-defective
virus AN-1 was generously provided by Dr. Sandra K. Weller, University
of Connecticut Health Center (Farmington). All viruses were stored as
infectious cell preparations at -80°C in aliquots.
Lipopolysaccharide (LPS; Sigma Chemical Co., St. Louis, MO; Cat. No.
L2630, 1 µg/ml) and SAC (fixed Staphylococcus aureus Cowan
strain, Pansorbin, Calbiochem, San Diego, CA; 0.0075% w/v)
were used as positive inducers of IL-12 production. Cells were pulsed
for 90 min with LPS or SAC and were washed and incubated.
Cell lines
J774A1, a macrophage cell line; 2D6 [22
], an
IL-12-dependent cell line (obtained from Dr. H.Fujiwara, Osaka
University Medical School, Japan); and vero and E-5 (complementing cell
line) were used.
Antibodies
Fluorescein isothiocyanate (FITC)-conjugated rabbit anti-HSV-1
(MAC INTYRE) Code No. F 318 Lot 041 and unlabeled anti-HSV
antibody were obtained from Dako Corp., Carpinteria, CA. IL-12-coating
antibody was purchased from Pharmingen, San Diego, CA (Cat. No.
20011D).
Isolation of adherent splenocytes
To obtain adherent cells, single-cell suspensions of splenocytes
in RPMI-10% fetal calf serum were plated onto 150 cm2
tissue-culture bottles for 90 min. Adherent cells were recovered by
scraping after repeated washing to remove nonadherent cells. It
consists of about 60% of macrophages and 25% of dendritic cells, and
the rest included T and B lymphocytes, natural killer, and
polymorphonuclear neutrophils, as evidenced by flow cytometry analysis.
Isolation of peritoneal macrophages
Thioglycollate (TG) was injected intraperitoneally (1 ml/mouse).
Four days later, the peritoneal cavity was aseptically lavaged with 10
ml RPMI-1640 (Gibco, Grand Island, NY) containing 1 U/ml sterile
heparin to obtain peritoneal exudate cells (PECs). PECs from three to
five mice were pooled in each experiment to obtain enough cells for
analysis, and each experiment was performed multiple (three) times.
PECs were plated onto 96-well flat-bottom microtiter plates and were
incubated at 37°C, 5% CO2, 95% air, and 95% humidity
for 6 h to allow the macrophages to adhere to the plate. The
plates were then washed four times with RPMI 1640 to remove all
nonadherent cells, and the macrophage number was quantified. The
macrophages were washed (190 g, 5 min, 4°C) twice, counted, and
stained with trypan blue (>95% viable). The cells were adjusted to a
concentration of 2 x 106 cells/ml in RPMI containing
5% heat-inactivated, low-endotoxin (<0.01 ng/ml) fetal bovine serum
(FBS; Sigma Chemical Co.), 105 M 2-mercaptoethanol,
penicillin (100 U/ml), streptomycin (100 U/ml), and glutamine (20 mM)
for use and flow cytometric analysis. The PECs and postadherent samples
were analyzed by flow cytometry with FITC-conjugated monoclonal
antibodies (mAb) against Mac-3 (clone M3/84; Pharmingen), a surface
glycoprotein found on mature, TG-elicited M but not on lymphocytes,
monocytes, or neutrophils.
In vitro culture of corneal cells
Corneas were excised from naive mice under a dissecting
microscope and freed of scleral tissues and iris. They were suspended
in Hanks’ balanced salt solution containing 60 U/ml collagenase D
(Boehringer Mannheim & Co., Mannheim, Germany) and 1% heat-inactivated
FBS for 30 min at 37°C in a humidified atmosphere of 5%
CO2. The corneas were then minced, gently disrupted on a
stainless steel mesh with a syringe plunger, washed, collected, and
resuspended in RPMI-1640 containing penicillin, streptomycin, and 10%
FBS. The cells were plated in a 12-well plate.
Infection of the cells
Cells were infected with UV-inactivated HSV-1, heat-killed
HSV-1, HSV-1 (KOS), and HSV mutants (ICP4-/- and AN-1) at different
multiplicities of infection (MOI; ranging from 0.1 to 5 MOI) for 1 h in serum-free media, were then washed off, media-containing serum was
added, and the incubation continued.
Inhibition of viral protein synthesis
The protocol using protein synthesis inhibitor cycloheximide
(CHX) described by Trinchieri and co-workers [23
] was
followed. Briefly, cells were pretreated with different doses (10, 50,
or 100 µg/ml) of CHX, 1 h before infection with HSV to prevent
de novo protein synthesis. After thorough washing, the cells were
treated as required.
RNA isolation
The total cellular RNA was isolated from the tri-reagent
cellular lysate by adding chloroform and centrifugation followed by
ethanol/isopropyl alcohol precipitation of the aqueous RNA solution
according to the manufacturer’s instructions. Total cellular RNA thus
obtained was stored as dry pellets or as aqueous solution in aliquots
at -70°C until used.
Reverse transcription (RT)
Total cellular RNA (5–8 µg) was reverse-transcribed using
avian myeloblastosis virus RT (Promega, Madison, WI) and oligo(dt) 18
(301 DNA Synthesizer, Applied Biosystems, Foster, CA). The reaction mix
(5 mM MgCl2, 50 mM KCI, 0.1% Triton X-100, 2
mM dNTP, and 40 U RNase inhibitor; Promega) was incubated at
ambient temperature for 15 min for oligo(dt) priming and was then
incubated at 42°C for 90 min. The RT mix was then heated at 99°C
for 5 min and cooled on ice.
Qualitative polymerase chain reaction (PCR)
Aliquots of cDNA were used in a 25.0 µl PCR for initial
qualitative detection of ß-actin, IL-12 (p40), and IL-12 (p35). The
primer sequences and the expected product size (base pairs) are as
published earlier [7
]. The reaction mix consisted of 2.0
mM MgCl2; 0.01% Triton X-100; 125 µM concentrations each
of dATP, dCTP, dGTP, and dTTP; 50 mM Tris-HCL, pH 8.3; and 1.0 U
Taq-DNA polymerase (Life Technologies, Carlsbad, CA). The conditions
for PCR amplification were 94°C (denaturation) for 90 s,
annealing at 55°C for 60 s, and extension at 72°C for 120 s. For each message, the PCR was conducted for 35 cycles. Approximately
20–30 pmol of each primer was used.
Ab-capture bioassay for IL-12 activity
The system was essentially as described by Gately and Chizzonite
[24
] and modified by Fujiwara and co-workers
[22
]. We have incorporated certain changes to suit our
needs. Briefly, 96-well microculture plates were coated with 4 µg
capture-antibody (Cat. No. 20011D, Pharmingen) for 18 h at 4°C.
After extensive washing, various dilutions of culture supernatants to
be tested or standard murine recombinant IL-12 (Cat. No. 19361V,
Pharmingen) were incubated in wells of mAb-coated plates. The unbound
material was washed, and 0.1 ml containing 105 2D6
(IL-12-responsive cell line) cells was cultured for 48 h and
pulse-labeled with 1 µci/well 3H-thymidine for the final
6–8 h. The cells were harvested, and cpm were recorded using the
automatic cell harvester and reader. The samples were cultured in
triplicate, and the absolute concentrations of IL-12 were determined by
extrapolating from a standard curve obtained using known concentrations
of rIL-12. The assay is able to measure concentration as low as 2–3
pg/ml.
Intracellular staining for HSV and IL-12
The chamber slides (Lab-Tek®, Cat. No. 177380), containing the
adherent cells that were infected or treated with LPS, were processed
for intracellular staining at different timepoints. An intracellular
staining kit supplied by Pharmingen was used as per the manufacturer’s
instruction. In brief, the cells were fixed and permeabilized using
Cytofix for 
30 min, followed by washing with perm wash. The cells
were stained with FITC-labeled anti-Herpes antibody (1:500) and
phycoerythrin (PE)-labeled anti-IL-12 (1 µg) and incubated for 45
min. The controls included untreated cells, isotype antibodies, and
unstained cells. After extensive washing with perm wash and cold
phosphate-buffered saline (PBS) to remove unbound antibodies, the
slides were blot-dried carefully, and a drop of glycerol (75% in PBS)
was added, mounted with a coverslip, and kept in a dark and cold place.
Confocal microscopy
The cells that were to be analyzed for simultaneous presence of
the virus and the IL-12 were stained and immediately screened for
FITC-labeled HSV and PE-labeled IL-12 using a Leica TCS-4D confocal
scanning laser microscope. Multiple sections were analyzed, than
overlayed, and the final projected image was documented.
Transwell plate assay
The inserts of transwell plate were seeded with corneal
epithelial cells that were infected or pulsed with LPS or SAC. Two
hours later, they were washed off and placed into transwells containing
J774A.1 cells. The inserts were removed at 3-h intervals, and the
J774A.1 cells were allowed to continue incubation. At the end of the
24-h incubation period, the conditioned media were collected and
analyzed for IL-12p70 protein. Some of the wells containing the J774A.1
were supplemented with anti-HSV antibody to neutralize any free virus
and polymyxin B to bind the residual LPS. In some instances, J774A.1
cells were replaced with adherent splenocytes to see if they reacted
similarly. The media from control wells were also tested for HSV
presence by plating them on vero cells or were assessed for endotoxin
presence by EtoxateTM assay. Minimal or no virus was detected, and the
endotoxin level was <0.05 EU.
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Figure 1. (a) Corneal epithelial cells do not make IL-12. One million
splenic-adherent cells, TG-elicited macrophages (M ), J774A.1, and
corneal epithelial cells were plated onto a 12-well plate. They were
infected with 1 MOI HSV and incubated for 37°C and 5%
CO2. The supernatents were collected 18-h p.i. and were
analyzed for the presence of IL-12p70 protein by Ab-capture bioassay.
Five separate experiments gave similar pattern of results. The figure
represents the average of three separate experiments. (b) Dose response
and kinetics of IL-12 production on HSV-1 infection. One million
adherent splenocytes were plated onto a 12-well plate. They were then
infected with HSV KOS at 0.1, 1, and 5 MOI or were stimulated with LPS
(1 µg/ml) and SAC (0.0075% w/v) for 90 min and were later washed
and incubated. The cell-free supernatants were collected at 9-,
12-, 18-, and 24-h intervals post-incubation and were analyzed for
IL-12 levels by Ab-capture bioassay.
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Figure 2. IL-12 production needs actively replicating virus. Adherent splenocytes
were infected with heat-inactivated (HI), UV-irradiated, HSV ICP4-/-
(at 1 and 5 MOI), and HSV AN-1 (at 1 and 5 MOI) for 90 min, and later,
they were washed to remove the unbound virus and were incubated. The
cell-free supernatant was collected at 9-, 12-, 18-, and 24-h intervals
and analyzed for IL-12 protein by Ab-capture bioassay. The
table;8> represents the average of the three experiments.
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Figure 3. IL-12 p40 mRNA expression needs de novo protein synthesis.
Splenic-adherent, corneal cells, peritoneal macrophages, and
J774A.1 cells were infected with HSV and incubated in a 12-well plate.
The cells were collected after 18 h, and total cellular RNA was
isolated using Tri-Reagent and was reverse-transcribed. RT-PCR was
performed to analyze for the presence of IL-12p40 mRNA. In some wells,
the splenic-adherent cells were pretreated with cycloheximide at
different doses (10, 50, and 100 µg/ml concentration) to prevent
protein synthesis. As a control, some of the wells were uninfected and
also processed for IL-12p40 mRNA analyses. The figure represents one of
the five separate experiments. M, Marker; +, positive control; S,
splenic-adherent cells; J, J774A.1, 6 h p.i.; P, peritoneal M,
6 h p.i.; C, corneal cells; Cx, cycloheximide (10, 50, and 100
µg/ml); Ui, uninfected M.
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Table 1. Consequences of HSV Infection of Adherent Splenocytes
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Figure 4. Confocal microscopic studies for dual staining of HSV and IL-12p70.
Splenic-adherent cells were allowed to attach on slides and were later
infected with 1 MOI of HSV and incubated. One hour later, 10 µg/ml
brefeldin A was added to the culture. The slides were removed from
incubation at various timepoints starting from 3 h p.i.. The
slides were stained intracellularly with FITC-labeled, anti-HSV
antibodies and PE-labeled, anti-IL-12 antibody or isotype-control
antibodies. The slides were visualized using a Leica TCS-4D confocal
scanning microscope. Leica’s 3-D volume-rendering software was used to
produce reconstructions of confocal stacks. The figure shows the cells
processed at (A) 3-, (B) 9-, (C) 12-, and (D) 24-h p.i. The arrows
indicate double-stained cells (IL-12+ HSV+).
This was observed only in samples collected 9-h p.i.
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To further evaluate this idea, corneal epithelial cells (incapable of producing IL-12) were infected at high multiplicity with HSV. After 2 h, cells were repeatedly washed in medium containing anti-HSV serum and added to J774A.1 cells and incubated for a further 18 h. Such cocultures produced IL-12 (561±98 pg/ml), whereas coculture with mock-infected corneal cells and J774A.1 cells produced only 28 ± 17 pg/ml IL-12 over the same period. As in this coculture experiment, it was not possible to exclude direct cell to cell transfer of virus, additional experiments were performed. In the first, corneal epithelial cells were infected for various times with HSV, and culture supernates were collected. After addition of anti-HSV antibody to neutralize virus, supernates were added to J774A.1 or adherent splenocyte cultures, and levels of IL-12p70 were measured after an additional 18-h culture period. As shown in Figure 5 , IL-12 was induced in J774A.1 and adherent cells as long as the corneal epithelial cells were infected for 6 h or more.
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Figure 5. Effect on J774A.1 and adherent splenocytes IL-12 production by mouse
corneal cell culture-conditioned medium. One million corneal epithelial
cell were infected with UV-irradiated HSV KOS or HSV KOS at 1 MOI or
were stimulated with SAC (0.0075% w/v) and LPS (1 µg/ml) for 90 min;
later, they were washed and incubated for 15 h. The cell-free
culture supernatants collected at different timepoints during the
incubation were processed for removal of LPS activity by adding
polymyxin B (0.05 EU as measured by Etoxate assay), and free viral
particles were neutralized with 10 µg/ml anti-HSV antibody at 37°C
for 30–45 min. (Isotype-control for antibody was also used.) These
were then added to the J774A.1 cells or adherent splenocytes to analyze
their effect on IL-12 induction as measured by the Ab-capture bioassay.
The data not shown in the figure but carried out in parallel include
HSV-infected corneal cells collected at various timepoints p.i., which
were formalin-fixed and were also tested for their ability to induce
IL-12 but were not capable of inducing IL-12. The corneal cell culture
stimulated with LPS (for 3–6 h) was able to induce the highest
level (735±163.6) recorded. The antibody dilutions and polymixin
concentration were based on dose-response studies. The figure
represents an average of three separate experiments.
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In separate experiments, corneal epithelial cells were infected at high MOI with HSV for various times, were then fixed, and were extensively washed. Such cells failed to induce IL-12 when cocultured with J774A.1 cells (data not shown). Taken together, these results support the idea that a soluble factor produced by infected cells was capable of inducing IL-12 production by responder cultures and that fixed, virus-expressing cells were unable to cause IL-12 induction.
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Several intracellular pathogens are well-known to induce IL-12 expression [9 10 11 , 14 ]. Such pathogens usually infect macrophages, a major source of IL-12 production [9 , 25 , 26 ]. However, in instances where virus infection results in IL-12 production, it is not clear if the IL-12 producer cells are infected with the virus [12 , 16 17 18 , 20 ]. With some viral infections, such as measles, infected cells are selectively compromised in their ability to produce IL-12 upon stimulation [27 , 28 ]. This could represent a mechanism by which the measles virus mediates its well-known immunosuppression [27 , 29 ]. In the case of HSV, IL-12 expression by infected cells was not expected to occur. Thus, productively infected cells curtail most host-cell protein expression, and cellular mRNA species may undergo degradation [21 ]. The findings of this report are consistent with such expectation. Accordingly, it was shown that with infection of cells capable of producing IL-12, such as adherent splenocytes or J774A.1 cells, maximal IL-12 protein occurred under conditions in which only a minority of the cell population was infected. Moreover, upon analysis by confocal microscopy of adherent cells similarly infected, initially, cells could be demonstrated that were double-positive for IL-12 and viral antigen, and later on, IL-12-expressing cells lacked viral antigen expression. We interpret these findings to mean that the major source of IL-12 production in an HSV-infected culture may be uninfected cells responding to something generated by dying, infected cells. This idea received further support from experiments using transwells in which infected epithelial cells (shown unable to produce IL-12) were physically separated from macrophages or adherent cell cultures with the medium containing anti-HSV Ab to negate their infection. Such cultures produced impressive levels of IL-12. In other experiments, virus-neutralized supernates taken from infected epithelial cells also caused J774A.1 cells to produce IL-12.
Others have advocated that HSV-2 infection can cause infected peritoneal macrophage to produce IL-12 [19 ]. However, it was not clear from these results if the principal source of IL-12 was the infected cells themselves or uninfected cells, such as we indicate to be the case in the present study. Moreover, it is worth considering that in vivo HSV infection likely infects few if any inflammatory cells. For example, following ocular infection with HSV, antigen expression appears confined to epithelial cells with no demonstrable, viral antigen within recruited, inflammatory cells [30 ].
Our observation that uninfected cells appear to be the major source of IL-12 in response to HSV infection begs the question as to the nature of intermediary molecules generated from infected cells that cause IL-12 expression. Currently, answers to this question are not at hand, but several possibilities are under consideration. As shown by our group [31 ] and recently confirmed by others [32 ], HSV-infected cells do express IL-6, but this cytokine is not known as an inducer of IL-12. An unconfirmed report [33 ] also indicates IL-1 expression could occur in HSV-infected inflammatory cells, and this cytokine can induce IL-12 [34 ]. Other possible mediators under consideration include chemokines, stress proteins, and viral products released from infected cells. With regard to the latter, virions themselves would seem to be unlikely candidates, as UV-inactivated virus and fixed cells expressing viral proteins caused only minimal IL-12 induction. Another candidate could be viral DNA, a source of DNA rich in potentially bioactive CpG motifs [35 ] known to be potent IL-12 inducers [36 , 37 ]. We are currently evaluating the potential role of many of these factors as signals for IL-12 and other cytokines involved in initial responses to HSV infection.
We thank Dr. Hiromi Fujiwara, Department of Oncology, Biomedical Research Center, Osaka University Graduate School of Medicine (Japan), for providing us the IL-12-dependent cell line (2D6).
Received February 28, 2002; revised April 18, 2002; accepted April 24, 2002.
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