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(Journal of Leukocyte Biology. 2002;72:564-570.)
© 2002 by Society for Leukocyte Biology

The IL-12 response to herpes simplex virus is mainly a paracrine response of reactive inflammatory cells

Udayasankar Kumaraguru and B. T. Rouse

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Herpes simplex virus (HSV) infection results in rapid and sustained up-regulation of interleukin (IL)-12, but the primary cellular source of IL-12 after HSV infection is unknown. We demonstrate that this cytokine largely derives from inflammatory cells rather than from productively infected epithelial cells. For optimal IL-12 induction, epithelial cells needed to be infected with replication-competent virus, and cells needed to be able to synthesize proteins. Our results also indicate that HSV-infected cells generate intermediary products that signal recruited inflammatory cells, which themselves were not HSV-infected, to generate IL-12. Possible mechanisms by which infected cells communicate with inflammatory cells to cause IL-12 production are discussed.

Key Words: lipopolysaccharide • macrophage • HSK • stress proteins


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Immediate reactions to invasion by pathogens involve humoral and cellular events, some of which serve to shape the nature and efficacy of the ensuing adaptive-immune response [1 2 3 4 ]. One such event is interleukin (IL)-12 production. This cytokine mainly originates from dendritic cells and macrophages [5 6 7 8 ], but the exact mechanism by which infection results in IL-12 induction remains poorly understood. In the case of bacterial and parasitic infections, IL-12 production may be the direct consequence of infection of producer cells or exposure of such cells to conserved, molecular patterns expressed by microbial surfaces [9 10 11 ]. Certain virus infections may also induce prominent IL-12 responses [12 13 14 ]. Examples include cytomegalovirus [15 , 16 ], influenza virus [17 ], and herpes simplex virus (HSV) [18 , 19 ]. It is unclear how such virus infections result in IL-12 production. In the case of HSV infection, which induces a prominent IL-12 response after ocular infection [20 ], the primary source of the cytokine is unlikely to be virus-infected cells themselves. Thus, productive infection with HSV results in the shut-down of most host gene expression as well as the degradation of host mRNA in infected cells [21 ]. Moreover, HSV mainly infects cells in vivo, such as epithelial cells, keratinocytes, and mucosal endothelial cells, which are not considered as primary sources of IL-12. In this report, we infected various cell types with HSV to determine which can act as a source of IL-12. Our results show that infected, inflammatory cells produce IL-12 in response to replication-competent HSV. However, our results are consistent with the notion that the major sources of IL-12 production in such cultures are uninfected cells, which respond to something released from dying, infected cells. Possible mechanisms by which infected cells communicate with inflammatory cells to cause IL-12 production are discussed.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
Balb/c mice (Harlan Sprague Dawley, Indianapolis, IN) were used as the source of splenocytes and corneal epithelial cells. All procedures were conducted in compliance with the Guide for the Care and Use of Laboratory Animals, as proposed by the Committee on Care of Laboratory Animal Resources, Commission on Life Sciences, National Research Council. The facility used was fully accredited by the American Association for Accreditation of Laboratory Animal Care.

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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cell types, kinetics, and dose response
Initially, various cell types were tested for their capacity to produce IL-12 upon infection with HSV-1 KOS. Cells tested included adherent splenocytes, TG-elicited macrophages, the J774A.1 macrophage cell line, and corneal epithelial cells. Six hours after infection, all of the above cells except the corneal epithelial cells were positive for IL-12 p40 mRNA as detected by RT-PCR (data not shown). To determine if bioactive IL-12 p70 was produced, culture supernatants were collected at different timepoints, and Ab-capture bioassays were performed. As observed in Figure 1 a , except for the corneal epithelial cells, all cells produced the bioactive IL-12 p70 cytokine. Peritoneal macrophages were the best producers, followed by adherent splenocytes. Infection of adherent splenocytes with lower dose of virus (0.1 and 1 MOI) resulted in IL-12 levels that elevated with increase in time, the peak being observed between 18 and 24 h post-infection (p.i.). However, when the dose of virus was increased to 5 MOI, IL-12 production levels were markedly less and did not increase with time (Fig. 1b) . These results could indicate that when all cells in a culture are successfully infected with virus, IL-12 gene expression is shut down. Conceivably, IL-12 induction occurs mainly in uninfected cells, perhaps in response to products released from infected cells.



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  		Figure 1. (a) Corneal epithelial cells do not make IL-12. One million splenic-adherent cells, TG-elicited macrophages (M{phi}), 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.

 
Need for replication-competent virus and de novo protein synthesis
To investigate if IL-12 production was dependent on the replication competence of the virus, adherent splenocytes from Balb/c mice were infected with HSV-1 KOS, UV-HSV, or replication-defective mutants (ICP4-/- early protein mutant and AN-1 late protein mutant) at 1 or 5 MOI. LPS and SAC were used as controls. The supernatants from stimulated cultures collected at various times were tested for bioactive IL-12 p70 levels. As shown in Figure 2 , heat- and UV-inactivated HSV induced IL-12, but levels were barely above the basal level and then only at the highest dose investigated. With the mutant viruses, minimal IL-12 levels were also induced with the response to AN-1 exceeding that to the ICP4-/- mutant. In the same experiment, responses to wild-type HSV-1 KOS, as well to LPS or SAC, were high, peaking at 18–24 h p.i. Furthermore, if the splenic-adherent cells were pretreated with cycloheximide prior to infection with wild-type HSV, the IL-12 production was abrogated (Fig. 3 ). Thus, it appears that the IL-12 production is dependent on de novo protein synthesis and that molecules derived from dying cells infected with replication-competent virus may be the principal stimulus for IL-12 production.



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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{phi}, 6 h p.i.; C, corneal cells; Cx, cycloheximide (10, 50, and 100 µg/ml); Ui, uninfected M{phi}.

 
Cellular source of IL-12 after HSV infection
To measure if virus-infected cells themselves were the major source of IL-12, splenic-adherent cells were infected with wild-type HSV (1 MOI) and examined by confocal microscope at different timepoints for the intracellular expression of viral antigens and IL-12. Cells expressing HSV antigens were present at all times tested between 6 and 24 h (see summary, Table 1 ). However, IL-12 expression was not evident until 9 h p.i. At this time, most IL-12-expressing cells additionally contained viral antigens (double-positive; Fig. 4 ). In the 12- and 18-h samples, fluorescing cells were mainly singly positive for viral antigen, but by 24 h, IL-12-expressing cells were again evident. However, at this timepoint, IL-12-expressing cells were mainly single-positive and failed to express viral antigens (Fig. 4) .


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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.

 
Taken together, these results indicate that initially, virus-infected, inflammatory cells may express IL-12, but after 12 h or more infection, such protein expression in infected cells became undetectable. However, dying, infected cells may release something that causes uninfected cells in the culture to express IL-12 as was observed in 24-h samples.

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.

 
In the second experimental approach, corneal epithelial cells grown on the membrane insert (0.2 µm) of a transwell plate were infected at high multiplicity with virus for 2 h. Washed inserts were then placed in transwell plates of J774A.1 or adherent splenocyte in media containing 10 µg/ml anti-HSV and 5 µg/ml polymyxin B (to inactivate any LPS). Inserts containing the HSV-infected corneal cells were removed at 3-h intervals, and the transwell cultures continued to incubate for a further 24 h after insert removal. In such experiments, IL-12 production occurred in J774A.1 and adherent splenocytes (as observed in the previous experiment), as long as the corneal cells were virus-infected for at least 6 h prior to removal.

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.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
It is becoming increasingly evident that early innate-immune responses to pathogens such as IL-12 production strongly influence the subsequent pattern and efficacy of T cell-mediated immunity [1 2 3 ]. The primary source of IL-12 in vivo is usually macrophages and dendritic cells [5 6 7 8 ]. In most studies, however, it is not clear if the response of such cells is the consequence of direct infection or is the indirect outcome of cell stimulation by intermediary products from infected cells or even recognition of surface patterns on pathogens that trigger cognate receptors on IL-12-producing cells. As first shown by our group, IL-12 production is a prominent, early response to HSV infection [18 ]. In the present report, we demonstrate that this cytokine largely derives from inflammatory cells rather than from productively infected epithelial cells. For optimal IL-12 induction, epithelial cells needed to be infected with replication-competent virus and capable of synthesizing proteins. Our results indicate that HSV-infected cells generate intermediary products that signal recruited inflammatory cells, which themselves were not HSV-infected, to generate IL-12. Suggestions are made as to the possible identity of the intermediary products.

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.


   ACKNOWLEDGEMENTS
 
The work was supported by National Institutes of Health grants EY05093 and AI 14981.

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.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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K. Banerjee, P. S. Biswas, B. Kim, S. Lee, and B. T. Rouse
CXCR2-/- Mice Show Enhanced Susceptibility to Herpetic Stromal Keratitis: A Role for IL-6-Induced Neovascularization
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