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(Investigative Ophthalmology and Visual Science. 2000;41:1402-1409.)
© 2000 by The Association for Research in Vision and Ophthalmology, Inc.

Role of Macrophages in Restricting Herpes Simplex Virus Type 1 Growth after Ocular Infection

Hao Cheng1, Terrence M. Tumpey1, Herman F. Staats2, Nico van Rooijen3, John E. Oakes1 and Robert N. Lausch1

1 From the Department of Microbiology and Immunology, College of Medicine, University of South Alabama, Mobile; 2 Department of Medicine, Duke University Medical Center, Durham, North Carolina; and 3 Department of Cell Biology and Immunology, Free University, Amsterdam, The Netherlands.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To investigate the role macrophages play in controlling herpes simplex virus (HSV)-1 replication after infection of the murine cornea.

METHODS. Macrophage depletion in selected tissues before or after virus infection was achieved by repeated subconjunctival (SCJ) and/or intravenous (IV) injection of liposomes containing dichloromethylene diphosphonate (L-Cl2MDP). Controls received liposomes containing phosphate-buffered saline (L-PBS). The efficiency of depletion was evaluated by histologic examination. Virus content in infected tissues was determined by standard plaque assay. Delayed-type hypersensitivity (DTH) responsiveness was assessed using the ear-swelling assay. Antibody isotype responses to virus antigens and cytokine production were monitored by enzyme-linked immunosorbent assay.

RESULTS. Balb/c mice given SCJ injection of L-Cl2MDP 4 and 2 days before HSV-1 corneal infection were found to have ocular virus titers as much as 105-fold higher than that seen in the L-PBS–treated controls 8 days after infection. When L-Cl2MDP treatment was delayed until 2 and 4 days after infection, virus titers in the eye were analogous to those in the control animals. Subconjunctival and submandibular lymph node macrophages in mice given local (SCJ) L-Cl2MDP pretreatment were profoundly reduced, whereas the number of corneal Langerhans’ cells and lymph node dendritic cells remained unchanged. Local L-Cl2MDP pretreatment was associated with significantly reduced DTH responsiveness to HSV-1 antigen, and an alteration in selected antibody isotype production. Depletion of macrophages in the subconjunctival tissue before corneal infection was not accompanied by enhanced virus growth at early times (2 or 4 days) after infection.

CONCLUSIONS. Macrophages play an important role in restricting HSV-1 growth after corneal infection. These cells appear to be required for the development of an acquired immune response, presumably by functioning in antigen processing and presentation. The hypothesis that macrophages are major participants in innate immunity to HSV-1 corneal infection was not supported.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Herpes simplex virus (HSV)-1 infection on the scarified murine cornea results in virus replication in ocular tissue and in the trigeminal ganglion. At a high infectious dose, the virus may spread to the brain and cause fatal encephalitis. After a moderate challenge dose in immunocompetent Balb/c mice, the virus is usually eliminated by days 7 through 9 in the cornea1 and by days 9 through 12 in the trigeminal ganglion (Cheng and Lausch, unpublished observations, May 1997). Studies have shown that interferon (IFN)-{alpha}/ß and neutrophils, components of the innate immune response, play a critical role in containing HSV-1 growth. Thus, in mice treated with neutralizing antibody to IFN-{alpha}/ß or without the IFN-{alpha}/ß receptor, virus titers were substantially elevated, and corneal opacity scores were increased.2 3 4 Depletion of neutrophils was associated with elevated virus titers in the eye 3 days after infection, and fatal encephalitis subsequently developed in the majority of animals.1

Although innate immunity is important, an acquired immune response is usually needed to terminate HSV-1 replication. Consequently, humans and animals with impaired T-cell–mediated immunity can experience severe and even life-threatening infections.1 5 In the mouse, the CD4+ T cell subset is dominant in effecting cessation of virus growth after ocular infection.6 Antibodies can also help to suppress HSV-1 replication in the eye of immunized hosts7 and can suppress the immunopathologic response to ocular infection.8 9 Macrophages have been reported to be active participants in host resistance to HSV-1 infection.10 11 Two general types of macrophage-mediated resistance mechanisms have been described.12 One is called intrinsic resistance and refers to the capacity of the macrophage to inhibit virus growth within itself. The second is called extrinsic resistance and relates to the macrophage’s ability to inactivate extracellular virus, suppress virus replication in adjacent cells, and destroy infected cells. Activated macrophages can produce and release antiviral factors such as IFN-{alpha}/ß and tumor necrosis factor (TNF)-{alpha}. The latter has been shown on passive transfer to protect mice from intraperitoneal infection.13 TNF-{alpha} also can synergize with IFN-{gamma} to induce IFN-ß, which in turn suppresses HSV-1 growth in cultured human corneal fibroblasts and epithelial cells.14 15 Macrophages also have the potential to produce chemokines, which can recruit and activate additional cell types to combat virus infection.16 In addition to contributing to innate immunity, macrophages are able to process and present antigen to T lymphocytes. After phagocytosis of particulate antigen, B7 and major histocompatibility complex class II molecules are expressed that serve as costimulatory signals to activate naive T cells.17 18 19

Macrophages can be selectively depleted through administration of liposomes containing dichloromethylene diphosphonate (L-Cl2MDP). Treatment with this agent has been shown to be selective, because only macrophages take up the liposomes.20 21 22 23 Release of the drug from the liposomes leads to macrophage apoptosis by an unknown mechanism.24 25 After their elimination repopulation of macrophages is highly variable, ranging from 1 week to 5 months depending on the site of depletion.21

It is presently unclear what role macrophages play in suppressing HSV-1 replication after corneal infection. To investigate this question, Balb/c mice were treated with L-Cl2MDP before infection. Studies were then conducted to assess what effect macrophage depletion had on the expression of innate and acquired immunity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Three to 4-week-old female Balb/c mice that weighed 15 to 17 g were purchased from Charles River Breeding Laboratories (Wilmington, MA). The animals were maintained in plastic cages in a 12-hour light–12-hour dark cycle. All experiments complied with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Virus
HSV type 1 strain RE was used in these studies. Virus stocks were grown by infecting Vero cells with HSV-1 RE at a multiplicity of infection of three. After 48 hours when maximal cytopathic effect developed, the infected cells were scraped from the flask. After centrifugation, the cell pellet was resuspended in 1 ml RPMI 1640 with 2% fetal bovine serum and sonicated to release intracellular virus. The cell lysate was clarified by centrifugation at 200g (IEC HN-S centrifuge; Damon/IEC Division, Needham Heights, MA). The clarified cell lysate was then aliquoted and stored at -70 °C. The virus titer was determined by a standard 48-hour plaque assay, as described elsewhere.26

Virus Infection
Female Balb/c mice were anesthetized with 1.0 mg phenobarbital sodium in 0.2 ml PBS administered intraperitoneally. The right cornea was scarified by three twists of a 2-mm corneal trephine. A 2-µl volume of RPMI containing 1 to 4 x104 plaque-forming units (PFU) HSV-1 RE was then dropped onto the corneal surface.

Delayed-Type Hypersensitivity Assay
DTH responsiveness in HSV-1 ocular-infected mice was determined using the ear-swelling assay. The test antigen, HSV-1 RE, was diluted in serum-free RPMI 1640 medium. The virus preparation was then exposed to UV irradiation for 10 minutes. This reduced infectivity from 106 to less than 102 PFU/10 µl. To test for DTH responsiveness, 10 µl of UV-irradiated virus antigen was inoculated into the dorsal side of the mouse’s right ear 7 days after infection using a 50-µl syringe (Hamilton, Reno, NV) and a 30-gauge needle. The left ear (control) received 10 µl RPMI 1640 with 1% newborn calf serum. Ear swelling was measured 24 hours later in a blind fashion using a micrometer (model 7326; Mitutoyo, Paramus, NJ). The results are expressed as ear swelling of the right (antigen-treated) ear minus ear swelling of the left (control-treated) ear in units of 10-4 in.

Measurement of Anti-HSV-1 Antibody Production
To test the effect of local L-Cl2MDP pretreatment on the production of anti-HSV-1 antibodies, an enzyme-linked immunosorbent assay (ELISA) was used. The assay was performed as previously described27 28 with minor modifications. Briefly, HSV-1–infected cell lysate (Cat. No. 10-515-001, Advanced Biotechnologies, Columbia, MD) was diluted to 5 µg/ml in PBS and used as the coating antigen (100 µl/well) on black, high-binding ELISA plates (CoStar 3925; Corning, Corning, NY). HSV-1–specific immunoglobulins were detected with alkaline phosphatase–conjugated, isotype-specific goat anti-mouse immunoglobulins (Southern Biotechnology, Birmingham, AL). ELISA plates were developed with a fluorescent substrate (AttoPhos; Boehringer Mannheim, Indianapolis, IN) and read with a microplate reader (Fluorocount; Packard Instrument, Meriden, CT). A sample dilution was considered positive when the fluorescence for the sample was three times higher than a comparable diluted naive sample.

Assay of Tissues for Infectious HSV-1
To test the effect of L-Cl2MDP treatment on HSV-1 replication in Balb/c mice, individual whole eyes and subconjunctival tissues were excised. The collection of subconjunctival tissue was guided by the following procedure. After enucleation, the eyeball was cut sagittally and was then spread flat in a petri dish. The cornea and retina were detached from the eye under a dissecting microscope. The remaining part was the subconjunctiva. The individually collected tissues were placed in 0.6 ml of 2% fetal bovine serum in RPMI 1640 medium with 1% antibiotics. Preparations were frozen to -70°C and then thawed and homogenized (Ten Broeck homogenizer; Bellco, Vineland, NJ). The homogenates were frozen and thawed again, followed by sonication for 30 seconds using a dismembrator (Sonic 300; Artek Systems, Farmingdale, NY). The clarified supernatants were then titrated for infectious virus content on Vero cell monolayers.

Macrophage Depletion
Cl2MDP was a gift from Boehringer Mannheim (Mannheim, Germany). Multilamellar liposomes containing Cl2MDP or PBS were prepared as described elsewhere.23 To deplete macrophages in different tissues, Balb/c mice were given L-Cl2MDP subconjunctivally (SCJ; 10 µl), intravenously (IV; 0.2 ml), or the combination of both at different times relative to ocular virus infection. Control animals received liposomes containing PBS (L-PBS). For SCJ injections, animals were anesthetized with phenobarbital sodium. A 32-gauge stainless steel needle attached to a dispenser (Hamilton) was used to penetrate the subconjunctiva under a dissecting biomicroscope, and 10 µl of the desired reagent was injected into the bulbar conjunctiva just behind the limbus. SCJ injections resulted in a bleb surrounding the injection site. To obtain an equal distribution of reagents throughout the limbus, three injections were made at different sites, resulting in a circular subconjunctival bleb.

Acid Phosphatase Staining
To evaluate the effectiveness of macrophage depletion in designated tissues, the macrophage-endogenous acid phosphatase staining procedure described elsewhere29 was used. Briefly, mice were killed by cervical dislocation. The eyes, submandibular draining lymph nodes, and spleens were collected and embedded in optimal cutting temperature compound (OCT; Tissue-Tek; Miles, Napierville, IL) and snap frozen in an isopentane dry-ice bath, and 6-µm sections were made at -20°C and mounted on poly-L-lysine coated glass slides. The sections were allowed to air dry for 10 minutes, fixed in cold acetone for 10 minutes, and air dried for another 10 minutes. The slides were then incubated with naphthol NS-BI phosphate and pararosaniline (Sigma, St. Louis, MO) at pH 4.7 to 5.0 for 35 minutes at 37°C. After they were washed with tap water for 10 minutes, all slides were counterstained slightly with hematoxylin before mounting (Permount; Fisher Scientific, Fair Lawn, NJ). For quantitative assay, acid phosphatase–positive cells were counted under a conventional light microscope in a blind fashion.

Immunohistologic Staining
To detect dendritic cells, lymph nodes were enucleated and embedded in OCT and snap-frozen in an isopentane dry ice bath, and 6-µm sections were cut at -20°C with a microtome cryostat (Carl Zeiss, Thornwood, NY). The sections were placed on poly-L-lysine precoated slides (Polysciences, Warrington, PA) and fixed in cold acetone for 10 minutes. After blocking with normal goat serum, slides were then exposed overnight at 4°C to 100 µl of appropriately diluted rat monoclonal antibody specific for murine dendritic cells30 (clone NLDC-145; Serotec, Washington, DC). The sections were then incubated for 30 minutes with secondary biotinylated goat anti-rat IgG (H + L; Jackson Laboratory, Bar Harbor, ME) at a 1:100 dilution that had been absorbed with mouse serum protein and diluted in a mouse skin extract. After two washes with PBS, the sections were exposed to 3% H2O2 in methanol and washed two times for 10 minutes to block endogenous peroxidase activity. The sections were then incubated with avidin-biotin-enzyme complex (Vectastain ABC kit; Vector, Burlingame, CA) for 30 minutes. After two washes with PBS, the sections were incubated in 3,3-diaminobenzamine (DAB substrate kit; Vector) for 8 minutes. The slides were then washed in distilled water and counterstained with Mayer’s hematoxylin for 2 minutes. The microscopic slides containing NLDC-145–positive staining were counted in a coded fashion, with the reader unaware of the treatment given.

Statistical Analysis
The Mann–Whitney test and Student’s t-test were used to determine significant differences between treated and control groups. The level of confidence at which the results were judged significant was P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
L-Cl2MDP’s Effect on Virus Growth in the Eye
Previous investigators have reported that when L-Cl2MDP is administered in SCJ injection, macrophages in the bulbar conjunctiva are depleted,29 whereas IV administration eliminates macrophages mainly in the liver and spleen.31 32 We initially investigated whether one or both routes of administration would result in impaired host resistance to HSV-1 corneal infection. L-Cl2MDP was given 4 and 2 days before virus infection. Eyes were removed 8 days after infection and assayed for infectious virus content. Figure 1A shows that L-Cl2MDP pretreatment by SCJ injection led to an approximately 105-fold increase in virus titer in the eyes, whereas when L-Cl2MDP was given IV before infection, the ocular virus titers were comparable with the L-PBS controls (Fig. 1B) . Additionally, we found that when L-Cl2MDP treatment (SCJ plus IV) was delayed until 2 and 4 days after HSV-1, corneal infection host resistance was not impaired (Fig. 1C) . These data indicate that enhanced ocular virus growth was associated with local macrophage depletion and further that macrophage activity during the first 2 days of infection was critical for controlling ocular HSV growth.



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  		Figure 1. Effect of L-Cl2MDP given before or after infection with ocular HSV-1 growth. Balb/c mice were given L-PBS ({triangleup}) or L-Cl2MDP ({circ}) by the route(s) indicated 4 and 2 days before (A, B, respectively) or after (C) being infected on the scarified cornea with HSV-1 strain RE (4 x 104 PFU). Eyes were excised on day 8 after infection and individually titrated for infectious virus content. *Significantly different (P < 0.01) from the controls.

 
Effect of L-Cl2MDP on Local Macrophage Content
It was important to determine where local SCJ administration of L-Cl2MDP depleted macrophages. Accordingly, tissues excised from the subconjunctival region of pretreated eyes was collected 1 and 4 days after infection and examined for the presence of macrophages by acid phosphatase staining. Figure 2A shows that very few acid phosphatase–positive cells were seen in uninfected subconjunctival tissues. In L-PBS–pretreated hosts, acid phosphatase–expressing cells were evident 1 and 4 days after infection (Figs. 2B 2D , respectively). In contrast, few, if any, acid phosphatase–positive cells were observed at either time point examined in the tissue specimens obtained from L-Cl2MDP pretreated hosts (Fig. 2C 2E) . In agreement with the report of van Klink et al.29 local L-Cl2MDP pretreatment was not found to reduce the number of acid phosphatase–positive cells in infected corneas (data not shown).



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  		Figure 2. L-Cl2MDP administration before infection depleted macrophages in the subconjunctiva. Mice were given 10 µl L-Cl2MDP or L-PBS by SCJ injection 4 and 2 days before infection. Animals were then infected with HSV-1 on day 0. On the days indicated, corneas of two mice in each group were scarified, and subconjunctival tissue excised from each donor was stained for the presence of acid phosphatase–positive cells. (A) Subconjunctival tissue from a normal mouse. (B, D) Subconjunctival tissue samples collected from L-PBS–treated mice on the indicated days after infection. (C, E) Subconjunctival tissues obtained from L-Cl2MDP–treated mice. Arrows: positively stained cells. Magnification, x20.

 
Ocular lymphatic fluid drains into the submandibular lymph nodes.33 Thus, it was also possible that L-Cl2MDP given by SCJ injection eliminated macrophages in this lymphoid tissue. To investigate this hypothesis, sections from right submandibular lymph nodes were stained for acid phosphatase, and positive cells were enumerated. L-Cl2MDP treatment caused a 54% to 82% reduction in lymph node macrophages when compared with the controls (Fig. 3A ). Thus, SCJ administration of L-Cl2MDP profoundly suppressed macrophage content at the site of inoculation and draining lymph nodes for at least the first several days after infection.



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  		Figure 3. Effect of L-Cl2MDP pretreatment on the numbers of macrophages and dendritic cells in submandibular lymph nodes. Balb/c mice were given 10 µl L-Cl2MDP (open bar) or L-PBS (solid bar) by SCJ injection 4 and 2 days before infection. On the days indicated after infection, two mice from each group were killed, right submandibular lymph nodes were collected, and individual frozen sections were prepared. Macrophages were identified by acid phosphatase staining (A), and dendritic cells were determined by reactivity to the monoclonal antibody NLDC-145 (B). The designated cells were counted in six fields (magnification, x40) in two sections from each donor. *Significantly different (P < 0.01) compared with the L-PBS–treated control animals.

 
Effect of L-Cl2MDP on Ocular Langerhans’ Cells and Submandibular Lymph Node Dendritic Cells
Similar to macrophages, Langerhans’ and dendritic cells also originate in the bone marrow and can serve as antigen-presenting cells.34 35 36 37 We determined whether L-Cl2MDP pretreatment by SCJ injection would reduce the numbers of these cell types. An examination of the peripheral, paracentral, and central regions of epithelial sheets revealed that the numbers of Langerhans’ cells from L-Cl2MDP–treated animals was comparable with that seen in the control on both days 2 (Fig. 4A ) and 4 (Fig. 4B) . In addition, L-Cl2MDP local pretreatment did not reduce the number of dendritic cells in the draining lymph nodes (Fig. 3B) . Collectively, these results indicated that L-Cl2MDP pretreatment did not produce a detectable reduction in Langerhans’ and dendritic cells in corneal epithelial sheets or submandibular lymph nodes during the first several days of infection.



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  		Figure 4. Effect of local L-Cl2MDP pretreatment on the numbers of Langerhans’ cells in corneal epithelial sheets. Balb/c mice were given 10 µl L-Cl2MDP or L-PBS by SCJ injection 4 and 2 days before HSV-1 infection. On days 2 (A) and 4 (B) after infection two to three mice from each group were killed. Individual right eyeballs were collected, and epithelial sheets were isolated as described.48 Langerhans’ cells were identified by reactivity with mouse anti-IAd monoclonal antibody (clone AMS-32.1; Pharmingen, San Diego, CA) in immunofluorescence tests. Each epithelial sheet was evenly divided into peripheral (white bar), paracentral (gray bar), and central (black bar) regions. The designated cells were counted in four fields (magnification, x40) in each region of each epithelial sheet.

 
Effect of L-Cl2MDP on Acquired Immunity
Because macrophages are capable of presenting antigens to T cells18 38 39 we postulated that their depletion in the regional draining lymph node would lead to suppression of cell-mediated immunity. To test this hypothesis DTH responsiveness to HSV-1 antigens was analyzed. Figure 5A , representative of three independent experiments, shows that the mean ear swelling in treated animals was substantially lower (70% reduction) than that in the L-PBS–treated controls. When the animals were pretreated IV with L-Cl2MDP (Fig. 5B) or when L-Cl2MDP was given after virus infection (Fig. 5C) , the DTH response was not significantly reduced (P > 0.05).



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  		Figure 5. Effect of local L-Cl2MDP pretreatment on the DTH response to HSV-1 antigens. Mice received L-Cl2MDP (open bar) or L-PBS (solid bar) by the routes indicated 4 and 2 days before (A, B, respectively) or after (C) infection of the scarified cornea with HSV-1. Seven days after ocular infection all the mice were challenged with UV-inactivated HSV-1 antigen, and ear swelling was measured 24 hours later. There were five mice per group. *DTH significantly (P < 0.05) suppressed relative to the control.

 
We also determined whether local L-Cl2MDP pretreatment could influence the development of the specific humoral immune response to herpes virus antigens. Sera from L-Cl2MDP–treated animals were collected 8 days after infection and tested for the presence of HSV antibodies by ELISA. Table 1 shows that local L-Cl2MDP pretreatment resulted in a significant increase in IgM and IgG2a titers (43- and 2.8-fold, respectively). The titers of the anti-HSV IgG1, IgG2b, and IgG3 antibody isotypes were comparable with that seen in the control animals. Thus, L-Cl2MDP pretreatment was associated with enhanced production of selected immunoglobulin isotypes.


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  		Table 1. Effect of Local L-Cl2MDP Pretreatment on the Production of Anti-HSV-1 Antibodies

 
Effect of L-Cl2MDP on Innate Immunity
Macrophages have been shown to phagocytose virus particles and kill virus-infected cells and thus inhibit virus growth in vitro.12 It is therefore possible that macrophages contribute to innate immunity to HSV-1 in the eye. This hypothesis was tested by determining whether local L-Cl2MDP pretreatment was associated with elevated ocular HSV-1 growth shortly after infection. Figure 6 shows that virus titers in the eyes of L-Cl2MDP–treated hosts were not significantly different from those found in L-PBS–treated hosts 2 and 4 days after infection. Analogous results were also seen in subconjunctival tissues (Fig. 6) in which macrophage depletion after L-Cl2MDP treatment was specifically demonstrated (Fig. 4) . IV pretreatment with L-Cl2MDP also was without effect (data not shown). Taken together, these data do not support the hypothesis that in ocular tissue macrophages act to limit initial virus replication.



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  		Figure 6. Effect of L-Cl2MDP pretreatment on early ocular HSV-1 growth. Mice were pretreated with a 10-µl SCJ injection of L-Cl2MDP ({circ}) or L-PBS ({triangleup}) 4 and 2 days before HSV-1 corneal infection. On the days indicated after infection, whole eyes or subconjunctival tissues were prepared and individually titrated for infectious virus content.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study we investigated what effect depletion of macrophages would have on ocular growth after HSV-1 corneal infection. L-Cl2MDP given in SCJ injection before infection was shown to deplete macrophages at the inoculation site and in the regional lymph node. This treatment resulted in virus titers in the eye 8 days after infection that were strikingly enhanced (>5 log) compared with L-PBS–treated controls. In marked contrast, virus replication was not enhanced when L-Cl2MDP was administered solely by the IV route before infection or when given by SCJ injection plus IV 2 and 4 days after infection. Collectively, these results suggest that macrophage participation is critical during the first 2 days after infection, and that macrophages residing in the bulbar conjunctiva and/or draining lymph nodes are the functionally active cells.

The requirement for local macrophages that are active during the first 48 hours after infection suggests that they may serve as accessory cells for generating an acquired immune response. Alternatively, they may contribute to innate resistance. Studies have shown that HSV-1 infection in the naive mouse is largely controlled by cell-mediated immunity. Thus, if macrophages are involved in antigen presentation, then it could be predicted that the cell-mediated immune response would be depressed in hosts depleted of these cells. Indeed, we found that DTH response was substantially reduced in animals given SCJ injection of L-Cl2MDP before infection. However, no significant suppression was seen when the reagent was given IV before or by SCJ injection plus IV after infection. Our results are reminiscent of those reported by Karupiah et al.40 They found that elimination of macrophages through IV plus subcutaneous L-Cl2MDP treatment 4 and 2 days before subcutaneously introduced infection impaired T-cell–mediated immunity and enhanced ectromelia virus growth.

Depletion of macrophages before HSV-1 ocular infection also altered the nature of the humoral immune response. Specifically, the antiviral IgM titer was substantially increased, whereas the IgG2a titer was more modestly elevated. One possible explanation for the elevated IgM titer in our model is that in the absence of macrophages, virus particles efficiently interact with B-cell immunoglobulin receptors, promoting cross-linking and directly facilitating B-cell activation.41 Alternatively, or in addition, the humoral immune response may be heightened because of the increased viral antigen load seen in macrophage-depleted mice. Wijburg et al.42 depleted macrophages by IV administration of L-Cl2MDP before hepatitis virus infection. They found that this treatment was also associated with heightened virus titers and increased production of mainly IgG2a anti-hepatitis virus antibody. In their study IgM antibody levels were not examined. The cytotoxic T-lymphocyte response was not enhanced.

Although the IgM and IgG2a antibody isotypes were elevated, HSV-1 replication was not inhibited in the macrophage-depleted mice. One explanation for this is that the large IgM antibody molecules may diffuse very poorly from vascular into peripheral tissues. In addition, we have observed that passive transfer of high-titer IgG2a neutralizing antibody 24 hours after HSV-1 corneal infection does not accelerate virus clearance from the eye.8 Presumably, this is due at least in part to the ability of HSV-1 to spread from cell to contiguous cell without becoming exposed to extracellular antibody.43 Thus, it is not surprising that the enhanced production of anti-HSV-1 IgM and IgG2a antibodies in the L-Cl2MDP–treated hosts was not associated with suppression of virus replication.

Zisman et al.11 observed that animals depleted of macrophages and then infected intraperitoneally with HSV-1 had an increased viral load early after virus infection. Similar observations have been made in macrophage-depleted mice infected with hepatitis virus,42 yellow fever virus,44 or West Nile virus.45 Thus, we anticipated that macrophages contribute to innate immunity and that L-Cl2MDP given by SCJ injection would result in elevated ocular HSV-1 titers at early time points after infection. However, the virus titers at days 2 and 4 after infection were similar to those seen in the L-PBS control animals. Certain cytokines such as TNF-{alpha} have been shown to play a critical role in promoting macrophage nonspecific anti-herpes activity.46 47 We, therefore, measured the levels of TNF-{alpha} in the ocular tissue of mice infected for 2 and 4 days. Only small amounts of TNF-{alpha} (approximately 40 pg/eye) were detected by ELISA, and local L-Cl2MDP pretreatment did not reduce the level of TNF-{alpha} (data not shown). Thus, we could find no evidence for macrophage-mediated nonspecific inhibition of virus replication. Previous studies in our laboratory have shown that neutrophil depletion was correlated with enhanced virus growth on day 3 after infection.1 Taken together, our studies suggest that in the anterior portion of the eye, neutrophils rather that macrophages may be the dominant mediators of innate resistance.

Langerhans’ cells are normally found in the subconjunctival limbal region of the cornea.48 49 Studies in skin, where they are abundant, have shown that these cells can capture antigen and then migrate to the local lymph node where they can evolve into mature dendritic cells with highly efficient antigen-presenting capability.34 35 36 37 We found that local L-Cl2MDP pretreatment did not deplete ocular Langerhans’ cells. Additionally, the reagent did not deplete dendritic cells, which is in accord with the reports of others.21 50 Because Langerhans’ and dendritic cells can serve as antigen-presenting cells in the generation of cell-mediated immunity, it is surprising that they did not substitute for macrophages. There are at least two possible explanations for these results. First, it may be that macrophages are needed to transfer processed antigen to Langerhans’ and dendritic cells.50 Alternatively, macrophages may secrete cytokines that facilitate dendritic cell maturation into efficient antigen-presenting cells.36 51

In summary, our studies demonstrate that macrophages play a critical role in host resistance to HSV-1 ocular infection. Their function appears to be one of involvement in antigen processing and presentation. The failure to demonstrate a role for these cells in innate immunity to HSV-1 infection may reflect their relatively low numbers in the anterior segment of the eye.


   Footnotes
 
Supported by Grant EY1143 from the National Institutes of Health, and the Lions/USA Eye Research Foundation.

Submitted for publication June 29, 1999; revised October 22, 1999; accepted November 30, 1999.

Commercial relationships policy: N.

Corresponding author: Robert N. Lausch, Department of Microbiology and Immunology, University of South Alabama, MSB 2096, Mobile, AL 36688. lausch{at}sungcg.usouthal.edu


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Tumpey, TM, Chen, S-H, Oakes, JE, Lausch, RN (1996) Neutrophil-mediated suppression of virus replication after herpes simplex virus type 1 infection of the murine cornea J Virol 70,898-904[Abstract]
  2. Su, Y-H, Oakes, JE, Lausch, RN (1990) Ocular avirulence of a herpes simplex virus type 1 strain is associated with heightened sensitivity of {alpha}/ß interferon J Virol 64,2187-2192[Abstract/Free Full Text]
  3. Hendricks, RL, Weber, PC, Taylor, JL, Koumbis, A, Tumpey, TM, Glorioso, JC (1991) Endogenously produced interferon {alpha} protects mice from herpes simplex virus type 1 corneal disease J Gen Virol 72,1601-1610[Abstract/Free Full Text]
  4. Leib, DA, Harrison, TE, Laslo, KM, Machalek, MA, Moorman, NJ, Virgin, HW (1999) Interferons regulate the phenotype of wild-type and mutant herpes simplex viruses in vivo J Exp Med 189,663-672[Abstract/Free Full Text]
  5. Rand, KH, Rasmussen, LE, Pollard, R. B, Arvin, A, Merigan, TC (1977) Cellular immunity and herpesvirus infections in cardiac-transplant patients N Engl J Med 296,1372-1377[Abstract]
  6. Oakes, JE, Rector, JT, Lausch, RN (1984) Lyt-1+ T cells participate in recovery from ocular herpes simplex virus type 1 infection Invest Ophthalmol Vis Sci 25,188-194[Abstract/Free Full Text]
  7. Lausch, RN, Monterio, C, Kleinschrodt, WR, Oakes, JE (1987) Superiority of antibody versus delayed hypersensitivity in clearance of HSV-2 from eye Invest Ophthalmol Vis Sci 28,565-570[Abstract/Free Full Text]
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