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Department of Microbiology, University of Tennessee, Knoxville, TN 37996
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Whether herpetic disease, including HSK, becomes manifest in mice is profoundly influenced by genetic background. For example, C57BL/6 mice are highly resistant, and strains such as BALB/c, CAL-20, and A/J are susceptible (9), but the molecular explanation for differential disease susceptibility remains ill defined. Of notable interest, two congenic mouse strains, which differ only at the Ig heavy chain locus (Igh-1) on chromosome 12, exhibit markedly different susceptibilities to HSK induction. Accordingly, strain C.B-17 (H2d, Igh-1b) is resistant, while strains CAL-20 (H2d, Igh-1d) and BALB/c (H2d, Igh-1a) are susceptible (10, 11). Precisely how the gene products of the Igh locus, or perhaps a closely linked gene cluster, influence HSK expression following HSV infection is unknown. In the susceptible BALB/c mouse, HSK development requires that virus replicates in the cornea and that a CD4+ T cell-mediated immune response occurs (12). Conceivably, resistance might be explained either by the lower level of HSV replication or, perhaps, by the lack of induction of T cell immunity, both of which are needed for HSK expression (12). Alternatively, a concomitant regulatory control response may occur in the resistant mouse that modulates that immunopathologic function of CD4+ T cells. A further explanation is that HSK represents a virus-induced autoimmune disease (13, 14). Recent work indicated that a possible candidate corneal autoantigen might cross-react with the Ighb allotype responsible for resistance in C.B-17 mice (14). Thus, tolerization of susceptible mice to the Ighb allotype made them resistant to the expression of HSK. Furthermore, disease could be induced in HSV-infected athymic mice if they were given adoptive transfers of T cells reactive to an Ighb peptide. Accordingly, these data implied that C.B-17 mice were resistant because they had their T cell repertoire tolerant to peptides of Igh-1b and, hence, tolerant toward cognate corneal Ags.
In the present report we have further compared the HSK susceptibilities of C.B-17 and BALB/c mice and have measured the effectiveness of adoptive transfers of T cells from both strains to confer HSK to HSV-infected C.B-17 SCID mice. Our results show that HSV replicates equally well in C.B-17 and BALB/c mice, and both generate comparable anti-HSV immunity. Interestingly, whereas we confirm the HSK resistance of C.B-17 mice, CD4+ T cells from such mice were as effective as BALB/c T cells at transferring HSK to SCID recipients. Additional experiments failed to incriminate the presence of a regulatory cell control in donor populations, which probably serves to modulate CD4+ T cell function. Our data show that HSK causing pathogenic T cells exist in the resistant C.B-17 mouse, although such cells do not exhibit their pathogenic potential in the intact animals. Thus, the data presented in this report imply that the microenvironment either of the SCID lymphoid tissue or possibly of the HSV-infected eye provides an activating milieu that drives quiescent effector cells, perhaps of multiple specificity, as well as those recognizing cognate Ag. Such activated CD4+ T cells then release numerous chemokines and cytokines, which orchestrates the inflammatory response typical of HSK.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six- to eight-week-old female BALB/c (Harlan Sprague-Dawley, Indianapolis, IN), C.B-17 mice (Igh-1b; The Jackson Laboratory, Bar Harbor, ME), and C.B-17 SCID mice (Taconic Farms, Germantown, NY) were employed in this study. The immunocompetent BALB/c and C.B-17 mice were housed conventionally, while the SCID mice were housed in microisolator cages, with all food, water, bedding, and instruments autoclaved or disinfected. All manipulations involving SCID mice were performed in a laminar flow hood. All experimental procedures were in complete agreement with the ARVO resolution on the use of animals in research.
Virus
HSV-1 RE strain obtained from Dr. Robert L. Hendricks (University of Chicago, Chicago, IL) was propagated and titrated on Vero cells using standard protocols (15). Virus stocks were aliquoted and stored at -80°C.
Mouse immunizations and corneal HSV infections
Corneal infections of all mouse groups were conducted under deep anesthesia induced by the inhalant anesthetic methoxyflurane (Metofane, Pittman Moore, Mondelein, IL). BALB/c and C.B-17 mice were scarified on their corneas with a 27-gauge needle, and a 4-µl drop containing 1 x 106 plaque-forming units (pfu) of HSV-1 RE was applied to the eye and gently massaged with the eyelids. The eyes of these animals were examined on different days postinfection with a slit lamp biomicroscope (Kowa, Nagoya, Japan), and the severity of clinical keratitis of individually marked mice was recorded as described previously (6). The scoring system was as follows: 0, clear cornea; +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. Anesthetized C.B-17 SCID mice were infected with 5 x 105 pfu of HSV-1 RE after scarification of the cornea with a 27-gauge needle.
Adoptive transfer protocol
C.B-17 and BALB/c mice infected through the eye were sacrificed 3 wk later, and their spleens were harvested. Single cell suspension of spleens were made. The splenocyte preparation was treated with Tris ammonium chloride solution to lyse the RBC. The single-cell suspension was then enriched for T cells or CD4+ T cells using affinity columns (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions or were left unfractionated. Naive C.B-17 mice were also used as donors whose splenocytes were enriched for T cells or CD4+ T cells as described above. Twenty-four hours after corneal infection of SCID mice, 8 to 10 x 106 T cells (HSV-immune or naive), 7 to 9 x 106 CD4+ T cells (HSV-immune or naive), or 8.5 x 107 whole unfractionated splenocytes in 500 µl of PBS were slowly injected i.v. into the tail veins of the recipient SCID mice.
Virus recovery
At various time points postinfection, swabs of corneal surface were taken. The swabs were put into sterile tubes containing 500 µl of McCoy’s medium with 100 IU/ml of penicillin and 100 µg/ml of streptomycin (Life Technologies, Grand Island, NY). Such swabs were stored at -80°C. For detection and quantitation of HSV in the swabs, the samples were thawed and vortexed. Swabs from individual mice were titrated on Vero cell cultures according to standard protocols (15).
Histologic and immunohistologic examination
At the termination of the experiments, the animals were sacrificed, and their eyes were enucleated. They were fixed in 10% neutral buffered formalin, and 5-mm paraffin sections were prepared and stained with hematoxylin and eosin, mounted, and coverslipped for microscopic examination. For immunohistochemical staining, eyes were embedded in OCT compound (Miles, Elkhart, IN), and frozen sections of 6- to 8-mm thickness were cut, fixed in cold acetone for 5 min, washed with PBS, and blocked with 5% BSA for 30 min. The sections were then treated with biotinylated Abs to CD4 (PharMingen, San Diego, CA) for 30 min at room temperature. Cells with bound Abs were identified by a peroxidase reagent (ABC kit, Vector Laboratories, Burlingame, CA) for 30 min at room temperature. The sections were then treated with diaminobenzidine substrate (Bio-Genex, San Ramon, CA) for about 5 to 10 min, washed, and mounted in Aquamount (Lerner Laboratories, Pittsburgh, PA) for microscopic examination and photography.
Measurement of immune responses
Cellular immune responses to HSV were determined using the lymphoproliferation assay. Briefly, animals were sacrificed on day 21 postinfection, and single cell suspensions of their splenocytes were incubated in RPMI 1640 (Sigma, St. Louis, MO) containing 5% FCS with either x-irradiated ultra-violet inactivated-HSV-infected or uninfected stimulator cells at various responder/stimulator ratios. On the fourth day the cells were pulsed with [3H]thymidine (ICN Radiochemicals, Irvine, CA) at the rate of 1 µCi/well for the last 18 h. The plates were harvested and read using a beta scintillation counter (Trace 96, Inotech, Wohlen, Switzerland). The results were expressed as the mean counts per minute ± SD.
Ab assays were performed on sera collected from individual mice on day 21 postinfection and checked for HSV-specific total IgG by standard ELISA as described previously (16). Briefly, ELISA plates were coated with 100 µl of a 1/200 dilution of HSV-1-infected cell lysate (Advanced Biotechnologies, Columbia, MD) in carbonate buffer (pH 9.6) overnight. The plates were washed with PBS containing 0.05% Tween-20, serum samples diluted 1/200 were added with twofold serial dilution, and the plates were incubated for 2 h at 37°C. Wells coated with goat anti-mouse IgG (1 µg/ml; Southern Biotechnology Associates, Birmingham, AL; catalogue no. 1030-01) were treated similarly with serially diluted standard mouse IgG (Southern Biotechnology Associates; catalogue no. 107-01). After subsequent washings, a 1/1000 dilution of horseradish peroxidase-conjugated goat anti-mouse IgG (Southern Biotechnology Associates; catalogue no. 1030-05) was added for 30 min. Plates were thoroughly washed. The substrate 2,2-azino-bis3-ethylbenzthiazoline-6-sulfonic acid (Sigma) was added, and the color changes were read at 405 nm. From the graph generated by the standard IgG, concentrations of IgG in the test sera were estimated.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We observed, as reported by others (10, 11), that following ocular
infection with HSV-1 RE, BALB/c mice developed HSK but C.B-17 mice were
resistant. While BALB/c mice showed a high incidence and earlier onset
of HSK, with 9 of 14 eyes showing positive clinical signs by day 9
postinfection, C.B-17 mice, in marked contrast, were almost free from
clinical keratitis, with only 2 of 12 eyes showing mild lesions. By day
21, while peak clinical lesions were observed in BALB/c mice, C.B-17
mice appeared completely resistant (Fig. 1
). Histologically, C.B-17 eyes on day 21
postinfection appeared relatively free from signs of inflammation and
cellular infiltration, while eyes from BALB/c displayed histopathologic
signs typical of HSK (Fig. 2, Aand B).
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Previously, it was shown that HSK induction requires productive
viral replication and the development of a T cell response to virus
(12). Since C.B-17 were highly resistant to HSK, it was necessary to
rule out the possibility that resistance resulted from a failure of
adequate virus replication and a consequent inferior immune response.
Titration of virus concentration in ocular swabs from both C.B-17 and
BALB/c mice yielded similar titers and duration of detection in both
mouse strains (Table I). Thus the
necessary step of virus replication occurred equally well in the
resistant strain. In addition, measurements of T cell proliferative
response levels and humoral Ab responses in both BALB/c and C.B-17 mice
were approximately equal (Table I).
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As reported previously, although HSV-infected SCID mice fail to manifest HSK, they readily do so if reconstituted with CD4+ T cells from BALB/c mice (17). The SCID mouse provided a test system to determine whether CD4+ T cells from resistant C.B-17 mice would also be able to transfer susceptibility to HSK.
According to previous reports, transfer of disease would not be
expected to occur, since C.B-17 T cells should lack responsiveness to
the HSK cognate Ag(s) (14). To evaluate this idea, experiments were
performed in which both naive and HSV-immune CD4+ T cells
from both BALB/c and C.B-17 mice were transferred into SCID mice
infected 24 h previously with HSV. The results are shown in Figure 3. While none of the unreconstituted SCID
mice developed clinical lesions, unexpectedly, transfers of either
naive or HSV-immune CD4+ T cells from C.B-17 mice supported
HSK development just as efficiently as did the equivalent
CD4+ T cell population from BALB/c mice. Thus, lesions
induced by C.B-17 CD4+ T cells were similar in severity and
proceeded with approximately the same development pattern as occurred
with BALB/c CD4+ T cells. While 100% of recipients of
HSV-immune CD4+ T cells derived from C.B-17 and BALB/c mice
developed peak lesions by day 15 postinfection, recipients of naive
CD4+ T cells from both mouse strains showed a delayed
expression of peak lesions, which manifested only by day 18.
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, C and D).
In typical HSK lesions, the characteristic lymphocyte population that
is readily observed is the CD4+ T cell (7). While such
cells were absent in the corneas of HSV-infected unreconstituted SCID
mice (data not shown), they were readily detectable in the corneas of
HSV-infected SCIDs reconstituted with either C.B-17 or BALB/c
CD4+ T cells (Fig. 2, E andF), thus adding further proof that the ocular disease
manifestation was similar in both treatment groups. Failure to demonstrate regulatory cells in the donor C.B-17 population
In some systems, CD4+ T cell-mediated
immunopathology is modulated by regulatory cells, usually of the
CD8+ phenotype (18). Moreover, in the murine model HSK is
usually more severe if CD8+ T cell function is inhibited
(19). To test the possibility that CD4+ T cell transfer of
C.B-17 cells might function only because cells that regulate
CD4+ cells were removed from adoptive transfers,
experiments with unfractionated T cells as well as whole splenocyte
populations were performed. The results are shown in Figure 4, A and B. Once
again, C.B-17 unfractionated T cells or whole spleen cells transferred
HSK just as effectively as the corresponding BALB/c population. The
responses observed with naive C.B-17 T cells and the whole splenocyte
population, which were delayed compared with those of the HSV-immune
C.B-17 population, were nonetheless positive and essentially similar to
that shown with naive BALB/c CD4+ T cells (Fig. 3).
Histologic examinations revealed no appreciable differences between the
eyes of SCID recipients of C.B-17 or BALB/c T cells or those of
recipients of whole splenocytes (not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Support for the idea that host Ags may be involved in driving HSK has come from several directions (reviewed in 13 , but the most provocative evidence was provided by Avery et al. (14), who suggested that the resistance of C.B-17 mice was explained by their expression of the IgG2a allotype from the Igh-1b allele. In support of this idea, they showed that susceptible CAL-20 mice (Ighd) became resistant if tolerized to IgG2ab and also that the disease could be transferred to HSV-infected nude mice with CD4+ T cell clones reactive to a peptide derived from IgG2ab (14). Their data were consistent with the idea that virus infection led to the availability of a corneal Ag cross-reactive with the IgG2ab-derived peptides and that mice tolerant to such peptides would be unable to generate the appropriate pathologic specificity of CD4+ T cells. It was also reported earlier that transfer of T cells from HSV-immune, resistant C.B-17 mice did not cause significant disease when adoptively transferred to HSV-infected syngeneic nude recipients (20).
Our data concur with the observation that C.B-17 mice are resistant to HSK expression, yet such mice do develop virus-specific CD4+ T cells and show replication of virus in the eye comparable to that observed in susceptible BALB/c mice. Consequently, C.B-17 mice have the potential to exhibit the clinical disease. However, our data do not support the idea that HSK fails to occur because C.B-17 CD4+ T cells lack the appropriate reactivity due to tolerance to the putative cognate Ag (14). Accordingly, we found that adoptive transfers of C.B-17 HSV-immune or even naive CD4+ T cells to SCID recipients routinely caused lesions, and these were indistinguishable from those induced by similar transfers of BALB/c CD4+ T cells. In other words, C.B-17 T cells were not irreversibly tolerant to HSK-inducing Ags, but readily caused disease upon transfer to HSV-infected SCID mice. Our findings appear at variance with the aforementioned observations of Avery et al. (14) and Akova et al. (20). We have no explanation for the discrepancy, but the systems used for analysis were different. Thus, we used SCID rather than nude mice as recipients of adoptive transfers; in addition, our studies used a more virulent HSK-inducing virus strain (HSV RE) compared with that (HSV-KOS) used by the Foster group (14, 20).
In our study we observed a discordance in the functional behavior of CD4+ T cells in the ocularly infected intact C.B-17 mouse with their activity when transferred to syngeneic HSV-infected SCID mice. Although we lack a mechanistic explanation for the discordance, possibilities include the operation of some cellular regulatory mechanism or perhaps maintenance of tolerance by the continuous presence of the putative IgG2ab tolerogen in the intact C.B-17 mouse (14). The latter mechanism would seem unlikely since the Ighb tolerized cells used by Avery et al. failed to lose tolerance upon transfer into animals lacking the Ighb tolerogen (14). Attempts to explain the discordance by the presence of regulatory cells in the C.B-17 euthymic host were not supported by experimental data. However, it is difficult to formally reject this hypothesis. Moreover, there is ample precedence for the operation of such regulatory effects in other inflammatory diseases. For instance, in experimental inflammatory bowel disease, evidence for cell populations that down-regulate pathogenic CD4+ T cells has been forthcoming (21). In this model, CD4+ T cells separated on the basis of surface markers such as CD45RB played different roles. While adoptive transfer of CD4/CD45RBhigh cells transferred colitis in SCID mice, CD4/CD45RBlow cells did not. Furthermore, SCID recipients of whole lymph node preparations containing both populations did not manifest the disease. Thus, the CD45RBlow population exerted a negative regulatory effect on pathogenic autoaggressive CD4+ T cells (22). In addition, CD8+ T cells are known to exert regulatory roles in some situations, such as in EAE (18, 23, 24). Additional experiments are under way to exclude the operation of a similar regulatory mechanism in the HSK system.
An alternative explanation for our finding that C.B-17 CD4+ T cells do confer HSK to SCID mice could be that the microenvironment of the SCID animals favors the activation and effector function of adoptive cell transfers. Thus, we routinely observe that naive T cell transfers can induce HSK (17), and lesions may occur before the demonstration of T cell reactivity to HSV in recipient SCIDs (25). It could be that either the microenvironment of the lymphoid system or the HSV-infected eye that becomes rich in several chemokines, proinflammatory cytokines, and particularly IL-12 serves directly or indirectly as a nonspecific activator of CD4+ T cells.4 We showed previously that HSV is a potent stimulus for IL-12 expression (26). Moreover, recently Shevach’s group observed (27) that in vitro culture of MBP-reactive CD4+ T cells from the EAE-resistant strain with IL-12 converted them from quiescent nonlesion-forming cells into an aggressive EAE-causing function. We suggest that quiescent T cells, Ag specific or not, become activated in the HSV-infected SCID microenvironment, and these cells support the subsequent inflammatory response. Additional experiments are underway to further evaluate such ideas.
In conclusion, it is clear that pathogenic CD4+ T cells exist in C.B-17 mice, although such mice resist HSK. Our results show that C.B-17 CD4+ T cells are not irreversibly tolerant to HSK inducing Ags and readily cause disease upon adoptive transfer into HSV infected SCID mice. Although the existence of regulatory effects in C.B-17 mice remains a possible explanation, we could not support this hypothesis with experimental data. Rather, we favor the idea that HSV, which replicates abundantly in the SCID mouse cornea, produces a rich proinflammatory microenvironment (a cytokine storm) which induces any ingressing effector CD4+ T cell to be activated and to contribute to orchestrating an ongoing inflammatory lesion (HSK).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Barry T. Rouse, Department of Microbiology, M 409 Walter’s Life Sciences Building, University of Tennessee, Knoxville, TN 37996-0845.
3 Abbreviations used in this paper: HSV, herpes simplex virus; HSK, herpetic stromal keratitis; HSV RE, RE strain of herpes simplex virus-1; pfu, plaque-forming units; high, high levels; low, low levels; EAE, experimental autoimmune encephalitis.
4 J. Thomas, S. Kanangat, and B. T. Rouse. Submitted for publication.
Received for publication September 24, 1997. Accepted for publication December 22, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-2 are protective in skin but pathologic in the corneas of HSV-1 infected mice. J. Immunol. 149:3023.[Abstract]
knockout mice. Doctoral dissertation, University of
Tennessee, Knoxville, TN.
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