INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Infection of the human cornea with herpes simplex virus (HSV) may lead to a blinding immunoinflammatory disease termed herpetic stromal keratitis (HSK) (33, 34). The lesion can be modeled in a susceptible mouse strain, where it regularly occurs as a consequence of primary infection of the cornea (36). In mice, and likely in humans, the lesion appears to be a T-cell-mediated immunoinflammatory process (10, 33, 34). For example, the lesion fails to occur in nude mice, which lack T cells yet possess functional B cells (34). Additionally, in ocularly HSV-infected SCID mice, which lack their own T or B lymphocytes, adoptive transfer of CD4⁺ T cells can lead to lesion expression (23). Other observations, however, argue that T-cell-mediated immunity may not totally account for the lesions and suggest a role for B-cell-mediated immunity in HSK pathogenesis. For instance, humoral antibody can protect animals from lesions if it is present in sufficient concentrations soon after infection (9, 22, 28, 30). Furthermore, B cells may be important during the clinical phase of the disease, as it has been reported that suppression of B-cell function, as can be done by immunoglobulin M (IgM) treatment, diminishes the severity of HSK (16). HSK thus represents a complex syndrome in which the role of cellular components, such as B cells and their products, remains ill defined. With the availability of a convenient animal model in which B-cell function appears abrogated (18), the pathogenesis of HSK was reassessed. Our results show that in contrast to previous observations, B-cell-deficient (µK/O) mice were more susceptible and developed severe HSK in the absence of B cells. This greater susceptibility appeared to have two explanations. First, the production of antibody limited the duration of virus persistence and dissemination within the cornea. In consequence, in immunocompetent mice, the virus was virtually confined to the corneal epithelium and was absent by day 5 postinfection, well before the time of HSK induction. Surprisingly, in µK/O mice the virus spread to the corneal stroma from the epithelium and persisted for a longer period, extending into the time of lesion expression. At the lesion site, the virus likely acted as an indirect proinflammatory stimulus for invading CD4⁺ T cells, causing their bystander activation and subsequent participation in HSK lesion expression. Second, mice lacking B cells generated minimal Th2 responses compared to wild-type mice during the peak clinical phase of HSK lesions. Since the products of Th2 cells are known to modulate lesion severity (5, 19), it is likely that the diminished Th2 responses in µK/O mice also contribute to the greater severity of the infection.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Mice. BALB/c mice (4 to 6 weeks old) were purchased from Harlan Sprague-Dawley (Indianapolis, Ind.). B-cell-deficient mice (µK/O; H-2^d background), made by targeted disruption of the membrane exon of the Ig µ chain gene, were provided by Werner Muller (Institute for Genetics, University of Cologne, Germany) (18). The µK/O mice were bred in our pathogen-free animal facility. The lack of IgM⁺ B cells in these mice was confirmed by fluorescence-activated cell sorter analysis. All experimental procedures were in complete accordance with the Association for Research in Vision and Opthalmology resolution on the use of animals in research.

Virus. HSV type 1 (HSV-1) RE and HSV-1 KOS strains were propagated and titrated on monolayers of Vero cells (ATCC CCL81) using standard protocols (31). All virus stocks were aliquoted and stored at 80°C.

Corneal HSV infections and clinical observations. Corneal infections of all mouse groups were conducted under deep anesthesia induced by the inhalant anesthetic methoxyfurane (Metofane; Pittman Moore, Mondelein, Ill.). The mice were scarified on their corneas with a 27-gauge needle, and a 4-µl drop containing the required virus dose was applied to the eye and gently massaged with the eyelids. The eyes were examined on different days postinfection with a slit lamp biomicroscope (Kowa Co., Nagoya, Japan), and the clinical severity of keratitis of individually scored mice was recorded. The scoring system was as follows: +1, mild corneal haze; +2, moderate corneal opacity or scarring; +3, severe corneal opacity but iris visible; +4, opaque cornea, iris not visible; and +5, necrotizing stromal keratitis.

Virus recovery and titrations. At various time points postinfection, swabs of the corneal surface were taken. The swabs were put into sterile tubes containing 500 µl of Dulbecco modified Eagle medium with 10 IU of penicillin/ml and 100 µg of streptomycin (Life Technologies, Grand Island, N.Y.)/ml and stored at 80°C. For detection and quantification of HSV in the swabs, the samples were thawed and vortexed. Duplicate 200-µl aliquots of each sample of thawed swab medium were plated on Vero cells grown to confluence in 24-well plates at 37°C in 5% CO₂ for 1 h and 30 min. The medium was aspirated, and 1 ml of 2× Dulbecco modified Eagle medium containing 1% low-melting-point agarose was added to each well. The cultures were observed daily for the development of typical cytopathic effect. The titers were calculated as PFU per milliliter in accordance with standard protocols (31).

Passive antibody transfer. To assess the role of virus replication in lesion development, mice were infected on the cornea and 5 days later were given intravenously 300 µl of anti-HSV serum (36.5 µg/ml; HSV-specific IgG). Sera were collected from HSV-1 RE-immunized BALB/c mice and checked for HSV-specific total IgG by standard enzyme-linked immunosorbent assay (ELISA) as described previously (21). Briefly, the ELISA plates were coated with 100 µl of HSV antigens or anti-mouse IgG (1 µg/ml; PharMingen) as a standard in carbonate buffer (pH 9.8) overnight at 4°C. Serum samples were diluted 1:200 in phosphate-buffered saline (PBS) and run in triplicate with purified mouse IgG as a standard (PharMingen), followed by horseradish peroxidase-conjugated goat anti-mouse IgG (PharMingen). Quantification was performed with Spectramax ELISA reader softmax, version 1.2.

Delayed-type hypersensitivity (DTH). Eight and 18 days after virus infection on the scarified corneas of mice, test antigens in 20 µl of PBS were injected in the ear pinnae of anesthetized mice, and ear thickness was measured 48 h postinjection with a screw gauge meter (Oditest; H. C. Kroeplin GmBH, Schluechtern, Germany) as described elsewhere (17). The test antigens used were UV-inactivated HSV-1 KOS (10⁵ PFU prior to UV inactivation) and Vero cell extract in the right and left ears, respectively. The mean increase between the thicknesses of the left and right ears was calculated and expressed as 10² mm.

HSV-specific lymphoproliferation assay. To test whether HSV-specific T-cell responses of the µK/O and BALB/c mice were comparable, the mice were sacrificed 8 and 18 days after virus infection on scarified corneas. Individual spleens and cervical and mandibular lymph nodes were used as responders for lymphoproliferation assays. This method has been described in detail elsewhere (21). Briefly, these responders were restimulated in vitro with irradiated syngeneic splenocytes infected with UV-inactivated HSV-1 KOS cells (multiplicity of infection, 1.5) or irradiated naïve splenocytes and incubated for 5 days at 37°C. Eighteen hours before the cells were harvested, [³H]thymidine (1.0 µCi/well) was added to all culture wells, and the plates were read with a -scintillation counter (Trace 96; Inotech, Lansing, Mich.). The results were expressed as mean counts per minute ± standard deviation for six replicates per sample.

Quantification of cytokines by ELISA. Mice infected with virus on their scarified corneas were sacrificed 8 and 18 days after virus infection. Single-cell suspensions of splenic cells and cervical and mandibular draining lymph node (DLN) cells (2 × 10⁶/ml) were restimulated in vitro with UV-inactivated HSV-1 RE at an MOI of 1.5 and incubated for 72 h at 37°C. The supernatants were analyzed for interleukin 4 (IL 4), IL 10, and gamma interferon (IFN-) cytokine production by ELISA. Concanavalin A (ConA)-stimulated (5 µg/10⁶ cells/ml) and unstimulated cells were used as positive and negative controls, respectively. Ninety-six-well microtiter plates were coated with 2 µg of rat anti-mouse IL-2, IL-4, IL-10, and IFN- antibody (PharMingen) per ml at 4°C overnight. The plates were then washed three times with PBS containing 0.5% Tween 20 and blocked with 3% nonfat dry milk for 1 h at 37°C. After the plates were washed, serially diluted samples and standards (recombinant IL-4 [rIL-4], rIL-10, and rIFN-) were added and the plates were incubated overnight at 4°C. The plates were washed with PBS, and 1 µg (each) of biotinylated anti-IL-2, anti-IL-4, anti-IL-10, and anti-IFN- antibody (PharMingen) per ml were added to the wells, and the mixtures were incubated at 37°C for 2 h. Peroxidase-conjugated streptavidin (Jackson ImmunoResearch, West Grove, Pa.) was added at 37°C for 1 h. The color was developed by adding the substrate solution (11 mg of 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid in 25 ml of 0.1 M citric acid, 25 ml of 0.1 M sodium phosphate, and 10 µl of hydrogen peroxide). Quantification was performed with Spectramax ELISA reader software version 1.2.

Quantification of cytokine-producing cells by ELISPOT. Mice were sacrificed 8 and 18 days after virus infection, and their splenic and lymph node cells were used as responders. The resulting samples were analyzed for IL-4, IL-10, and IFN- spot-forming cells by enzyme-linked immuno-SPOT (ELISPOT). To generate cytokines, the responders were stimulated with enriched dendritic-cell populations obtained by the method of Nair et al. (25) that had been pulsed with UV-inactivated HSV (MOI, 5.0) for 3 h before being added to the responders. The responders and stimulator dendritic cells (naïve or pulsed) were added at a responder-to-stimulator ratio of 50:1, 25:1, or 12.5:1 in 200 µl of RPMI with 10% fetal bovine serum per well in ELISPOT plates coated with various anti-cytokine antibodies. After 72 h of incubation, the plates were washed and biotinylated anti-cytokine antibodies were added. After 1 h of incubation at 37°C, alkaline phosphatase-conjugated streptavidin in PBS (1 µg/100 µl) was added and the plates were incubated for another hour at 37°C. The spots were developed by using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate as a substrate and counted 24 h later with a dissecting microscope.

Immunohistochemical staining. Eyes were frozen in optimum cutting temperature compound (Miles, Elkart, Ind.) on different days postinfection. Six-micrometer-thick sections were cut, air dried, and fixed in cold acetone for 5 min. The sections were then blocked with heat-inactivated goat serum and stained for the presence of HSV antigens by the use of rabbit anti-HSV anti-serum (Dako Corp., Carpentaria, Calif.), which was followed with biotinylated anti-rabbit Ig (1/20 dilution; Biogenex, San Ramon, Calif.). The sections were then treated with horseradish peroxidase-conjugated streptavidin (1:1,000) and 3,3'-diaminobenzidine (Vector, Burlingame, Calif.) and counterstained with hematoxylin.

Statistical analysis. Wherever specified, the data obtained were analyzed for statistical significance by Student's t test.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Increased susceptibility of µK/O mice to HSK. Infection of the corneas of BALB/c mice with HSV results in an inflammatory reaction in the stroma, which involves the essential participation of CD4⁺ T cells (10, 34). To evaluate a role for B-cell-mediated immunity in HSK pathogenesis, lesion development was compared in B-cell-deficient mice (µK/O) and control immunocompetent BALB/c mice after infection with different doses of HSV-1 RE (5 × 10⁶ to 1 × 10⁴ PFU). Unexpectedly, µK/O mice were more susceptible than BALB/c mice to HSV-1 ocular challenge and succumbed to death from encephalitis at doses of virus (5 × 10⁶ to 1 × 10⁶ PFU) well tolerated by BALB/c mice (Fig. 1). Furthermore, low doses of virus successfully generated HSK in µK/O mice but not in BALB/c mice (Fig. 2). Thus, at an infecting dose of 10⁴ PFU, 100% of µK/O mice developed HSK compared to only 10% of infected BALB/c mice. At higher doses of infectious virus, the severity of lesions in the µK/O animals usually exceeded lesion severity in the BALB/c mice exposed to an identical infectious dose of virus (Fig. 2). Thus, our data demonstrate that the presence of B cells influences resistance to HSV infection.

View larger version (16K): [in this window] [in a new window]   FIG. 1.   Susceptibility of µK/O mice to encephalitis after infection with high doses of HSV-1 RE. Groups of BALB/c and µK/O mice were infected with 5 × 106 PFU (n = 6) and 106 PFU (n = 10) of HSV-1 RE on their scarified corneas and examined for signs of sickness, wasting, paralysis, and encephalitis. The percentage of survival following virus infection is plotted.

View larger version (64K): [in this window] [in a new window]   FIG. 2.   Susceptibility of µK/O mice to HSK following HSV-1 RE infection. Groups of BALB/c and µK/O mice (n = 10 to 15) were infected with 106, 105, and 104 PFU of HSV-1 RE on their scarified corneas. The mice were examined clinically by slit lamp microscope, and the severity of lesions was scored on a 0-to-5 scale as described in Materials and Methods. The percentage of mice showing a mean clinical score of 4.0 at day 21 postinfection is plotted. The mean clinical scores at day 21 are shown above the bars.

How do B cells influence HSK? Virus clearance from the cornea usually occurs prior to HSK lesion manifestation in immunocompetent animals (1). As shown in Table 1, µK/O mice were significantly impaired in their ability to clear virus from the eye following corneal infection. Differences were most evident at low infecting doses of virus. Whereas BALB/c mice cleared the virus by 4 days postinfection, in µK/O mice the virus persisted for at least 8 days. Of most striking interest, the locations of viral antigen in the corneas of infected BALB/c and µK/O mice showed some differences. Thus, the virus was virtually confined to the epithelium until day 4 postinfection in both mouse strains (data not shown). However, after that time the predominant location of virus in the µK/O mice was in stromal tissues. In fact, viral antigen was demonstrable in the stromata of some µK/O animals until at least 10 days postinfection (Fig. 3).

View larger version (80K): [in this window] [in a new window]   FIG. 3.   Immunoperoxidase staining for viral antigen (as indicated by the arrows) in the corneas of µK/O mice on day 2 (A) and day 10 (C) and BALB/c mice on day 2 (B) and day 10 (D) following infection with 5 × 105 PFU of HSV-1 RE on their scarified corneas. Magnification, ×200.

View larger version (23K): [in this window] [in a new window]   FIG. 4.   Kinetics of HSK in HSV-1 RE-infected µK/O mice and BALB/c mice following passive transfer of HSV immune serum at day 5 postinfection. Groups of BALB/c (n = 6) and µK/O (n = 5) mice were infected with 5 × 105 PFU of HSV-1 RE on their scarified corneas. The mice were examined clinically by slit lamp microscope, and the severity of lesions was scored on a 0-to-5 scale as described in Materials and Methods. The mean clinical scores at days 7, 9, 12, 15, and 21 are plotted for all groups. Error bars (standard deviations) for untreated µK/O and immune serum-treated µK/O mice are also shown. Significantly different (P < 0.05) mean scores between untreated and immune serum-treated µK/O mice are indicated by asterisks. The mean scores for untreated and immune serum-treated BALB/c mice are not different (P > 0.05).

T-cell responsiveness in BALB/c and µK/O mice. The greater susceptibility of µK/O mice to express HSK could be associated with an inadequate or unbalanced CD4⁺-T-cell response involved in the pathogenesis of the ocular lesion (5, 8, 35). To test this idea, the extent and nature of the CD4⁺-T-cell immune responses were compared at two different time points postinfection in µK/O mice and BALB/c mice. Several observations supported the idea that CD4⁺-T-cell responses in µK/O mice were both delayed and changed in their cytokine balance compared to those of BALB/c animals. The cervical and mandibular DLN and splenic HSV-specific proliferative responses, as well as the DTH reactions, in the µK/O mice were depressed soon after infection (day 8) compared to those of BALB/c mice. Ultimately, at the time of the peak clinical phase (day 18), however, responses of µK/O mice were comparable to those of BALB/c animals (Fig. 5 and 6). These data indicate that T-cell responsiveness is decreased early in infection in mice lacking B cells. Similarly, a comparison of the cytokine profiles in the DLNs and spleens of the µK/O mice and the BALB/c mice by ELISA and ELISPOT assays revealed a decreased IFN- (Th1 cytokine) production at the early clinical phase (day 8 postinfection) in the µK/O mice (Fig. 7). At the time of peak clinical lesion (day 18 postinfection), comparable IFN- responses were observed for both BALB/c and µK/O mice (Fig. 8). On the other hand, with regard to Th2 cytokines (IL-4 and IL-10), whereas BALB/c mice generated weak yet positive responses, such responses were absent or marginal in B-cell-deficient mice (Fig. 8). Therefore, in the absence of B cells, mice may lack negative regulators of Th1 immune response and may, in consequence, suffer more severe HSK lesions.

View larger version (50K): [in this window] [in a new window]   FIG. 5.   Delayed kinetics of DTH reactions in µK/O mice. Mice infected with 105 PFU of HSV-1 RE on their scarified corneas were challenged for DTH reactions at days 8 and 18 postinfection (p.i.). UV-inactivated HSV-1 KOS strain or Vero cell extract was injected in 20-µl volume into the right or left pinnae, respectively, of both groups of mice (n = 10 to 12). The increase in ear thickness was measured 48 h later by a blinded individual. The mean difference in the right and left ear pinna thickness is plotted with error bars (± standard deviations). A significant difference is observed between µK/O and BALB/c mice at day 8, indicated by an asterisk (P < 0.005), and between µK/O mice at days 8 and 18, indicated by a double asterisk (P < 0.05).

View larger version (53K): [in this window] [in a new window]   FIG. 6.   Delayed HSV-specific proliferation of spleen cells and cervical and mandibular DLN cells in µK/O mice. BALB/c mice and µK/O mice were infected on their scarified corneas with 105 PFU of HSV-1 RE and sacrificed at days 8 and 18 postinfection. Spleen cells and cervical and mandibular DLN cells were used as responders for proliferation. The responders were mixed with either irradiated syngeneic splenocytes exposed to UV-inactivated HSV-1 KOS strain (MOI, 1.5) or irradiated syngeneic naïve splenocytes and incubated for 5 days as described in Materials and Methods. ConA (2 µg/ml) was added to the responder culture as a polyclonal simulator positive control, and the mixture was incubated for 3 days. Differences in ConA stimulation for days 8 and 18 postinfection are likely due to differences in radiolabel incorporation or general health of the cultures. T-cell proliferation for DLNs and spleen at days 8 and 18 postinfection are shown. The means of counts for responders and irradiated syngeneic splenocytes are plotted with error bars (± standard deviations). The data represent mean proliferation of triplicates from two experiments with four individual mice in each group. Significant difference in the values for HSV-specific proliferation between µK/O and BALB/c mice are indicated by asterisks (P < 0.05).

View larger version (33K): [in this window] [in a new window]   FIG. 7.   Cervical and mandibular DLN and splenic cells (2 × 106/ml) were obtained on day 8 postinfection from mice infected with 105 PFU of HSV-1 RE on their scarified corneas. (Top row) Cells were restimulated in vitro with UV-inactivated HSV-1 RE at an MOI of 1.5 and incubated for 72 h at 37°C. The supernatant from each of the groups was collected and analyzed by ELISA for cytokine production as described in Materials and Methods. ConA-stimulated (5 µg/106 cells/ml) and unstimulated cells were used as positive and negative controls, respectively. The results are means of triplicates from three separate experiments. DLN cells from the mice (n = 3) were pooled, while spleen cells from individual mice were used in each experiment. Values are expressed in picograms per milliliter. SFC, spot-forming cells; UD, undetectable; *, P < 0.05. (Bottom row) Cells (2 × 105) in a 100-µl volume per well were restimulated in vitro for 4 days with either irradiated enriched dendritic cells that were pulsed with UV-inactivated HSV-1 RE or irradiated naïve enriched dendritic cells at stimulator-to-responder ratios of 1:50, 1:25, and 1:12.5. Frequencies of cytokine SFC were measured by ELISPOT assay as described in Materials and Methods. The number of SFC after naïve-dendritic-cell stimulation was substracted from the values of stimulation by UV-inactivated-HSV-pulsed dendritic cells. The results, expressed as SFC/106 cells, are means of four replicates from three separate experiments. DLN cells from the mice (n = 3) were pooled, while spleen cells from individual mice were used in each experiment. *, P < 0.05 (two-tailed t test).

View larger version (35K): [in this window] [in a new window]   FIG. 8.   Cervical and mandibular DLN and splenic cells were obtained from mice infected with 105 PFU of HSV-RE on their scarified corneas at day 18 postinfection. (Top row) Cells were restimulated in vitro with UV-inactivated HSV-1 RE at an MOI of 1.5 and incubated for 72 h at 37°C. The supernatant from each of the groups was collected and analyzed by ELISA for cytokine production as described in Materials and Methods. ConA-stimulated (5 µg/106 cells/ml) and unstimulated cells were used as positive and negative controls, respectively. The results are means of triplicates from three separate experiments. DLN cells from the mice (n = 3) were pooled, while spleen cells from individual mice were used in each experiment. The values are expressed in picograms per milliliter. SFC, spot-forming cells; UD, undetectable; *, P < 0.05. (Bottom row) Cells (2 × 105) in 100 µl per well were restimulated in vitro for 4 days with either irradiated enriched dendritic cells that were pulsed with UV-inactivated HSV-1 RE or irradiated naïve enriched dendritic cells at stimulator-to-responder ratios of 1:50, 1:25, and 1:12.5. Frequencies of cytokine SFC were measured by ELISPOT assay as described in Materials and Methods. The number of SFC after naïve-dendritic-cell stimulation were substracted from the values of stimulation by UV-inactivated HSV-pulsed dendritic cells. The results, expressed as SFC/106 cells, are means of four replicates from three separate experiments. DLN cells from the mice (n = 3) were pooled, while spleen cells from individual mice were used in each experiment. *, P < 0.05 (two-tailed t test).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

HSV infection of the eye may result in a blinding immunoinflammatory lesion which is orchestrated by CD4⁺ T lymphocytes (10, 33, 34). In this report, we demonstrate that mice lacking B cells due to deletion of the Ig µ chain (µK/O) are more susceptible to HSV infection and develop lesions of HSK at infecting doses of virus well below those necessary to induce HSK in immunocompetent mice. At infecting doses required to induce HSK in immunocompetent mice, most µK/O mice succumbed to herpes encephalitis. The reason for the greater susceptibility of µK/O mice was assumed to be at least twofold. First, in the absence of antibody production, virus persisted for longer periods in the eye and disseminated to tissues such as the corneal stroma, the site of inflammation. Second, the T-cell response necessary for both protection and the mediation of lesions was delayed and changed in cytokine balance in µK/O mice compared to immunocompetent animals. The compromised T-cell response combined with the absence of antibody might explain the spread of virus to the brain and death from encephalitis. The change in the Th1-Th2 subset balance could account for the lesion severity in µK/O mice. Thus, such animals developed diminished Th2 responses known to downregulate Th1-mediated anti-HSV immunoinflammatory reactions (5, 8, 35). Consequently, our studies indicate a role for B-cell-mediated functions following ocular infection by HSV.

The protective role for antibody in HSV infections may relate to its function of containing or neutralizing virus and preventing its spread to the central nervous system (9, 22, 28). Consequently, early (day 0 to day 3) administration of immune serum following ocular infection of immunocompetent mice leads to virus neutralization and complete amelioration of HSK (30). Here, we report that antibody is also involved in limiting the replication of virus and preventing its dissemination to surrounding sites, such as the corneal stroma. Thus, in immunocompetent individuals, the severity of lesions may be minimized as a consequence of limiting virus replication to the corneal epithelium. Strikingly, in the µK/O mice the virus persisted in the cornea for prolonged periods and spread from the epithelium to the stroma, the site of the inflammatory reaction. Of interest, if the µK/O mice were given antibody 5 days postinfection, such spread to the stroma failed to occur and the treated mice expressed only mild lesions. Accordingly, in the absence of early antibody or perhaps natural antibody production in µK/O mice, a mechanism of immunopathology may come into play that represents only a minor component of HSK in immunocompetent mice. This additional mechanism is termed bystander activation and was observed to account for HSK lesions in T-cell receptor transgenic mice on a SCID or RAG background (12, 13). Such mice possessed CD4⁺ T cells, but the cells were unable to recognize HSV antigens. Bystander activation as a mechanism of tissue pathology has also been observed in some other model systems (3, 15, 27). We contend that the severe lesions evident in µK/O mice may largely represent bystander activation reactions. They occur because the persistence of virus in the stroma drives proinflammatory cytokines and chemokines at the site. Such mediators in turn drive arriving CD4⁺ T cells (likely largely non-HSV specific) to orchestrate the HSK lesions. Accordingly, whereas it may appear that HSK lesions in immunocompetent and µK/O mice are identical, the pathological mechanisms at play for their expression may be different. Further experiments are ongoing to verify these ideas.

An additional process to account for the heightened susceptibility to HSV infection in the µK/O mice must also be considered. Thus, B cells could play a role in anti-HSV immune defense other than by functioning as antibody producers (2). Such cells may act as antigen-presenting cells for the induction of T-cell-mediated immunity, although whether this occurs during primary T-cell induction remains in dispute (6, 7, 11, 24, 26). Some reports show that B cells play a significant role in inducing CD4⁺ T cells of the Th2 cytokine-producing profile against protein antigen (4, 7, 20, 29, 32). In consequence, the absence of B cells may result in impaired Th2 induction. This may result in a failure to modulate Th1 T-cell-mediated immunoinflammatory lesions, which in consequence become more severe and prolonged. Such a situation was described previously for experimental allergic encephalomyelitis (29, 37) and in addition may at least partially explain our findings. Thus, we observed that the balance of T-cell responsiveness to HSV was changed in µK/O mice compared to immunocompetent controls. In fact, the µK/O mice made an almost exclusively Th1 CD4⁺-T-cell response. Such cells were shown by our group as well as others to act as the main orchestrators of HSK lesions (14, 34). However, it is well known that the potency of Th1 cells in mediating pathology or immunity can be modulated by cytokines generated by Th2 cells. Our previous studies showed modulation effects on HSK and cutaneous inflammatory lesions of transforming growth factor , IL-4, and IL-10, all products of Th2 T cells or B cells (5, 19).

In conclusion, B cells influence the pathogenesis of HSV infection. It appears likely that B cells are involved in multiple events, which include antibody production as well as antigen-presenting-cell activity for regulatory subsets of T cells. The antibody likely functions principally to limit the extent of virus spread to critical sites, such as the corneal stroma and the central nervous system. The former, if it occurs, may result in the immunoinflammatory lesions of HSK, and the latter may cause a lethal viral encephalitis.