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Costimulatory interactions can be critical in developing immune responses to infectious agents. We recently reported that herpes simplex type 1 (HSV-1) infections of the cornea require a functional CD28-CD80/86 interaction to not only reduce the likelihood of encephalitis, but also to mediate herpetic stromal keratitis (HSK) following viral reactivation. In this same spirit we decided to determine the role that CD137 costimulation plays during HSK. Using both B6-CD137L-/- mice, as well as antagonistic and agonistic antibodies to CD137 we characterize the immune response and to what extent CD137 plays an important role during this disease. Immune responses were measured in both the cornea and in the trigeminal ganglia where the virus forms a latent infection. We demonstrate that CD137 costimulation leads to reduced corneal disease. Interestingly, we observed that lack of CD137 costimulation resulted in significantly reduced CD8+ T expansion and function in the trigeminal ganglia. Finally, we showed that viruses that have been genetically altered to express CD137 display significantly reduced corneal disease, though they did present similar levels of trigeminal infection and peripheral virus production following reactivation of a latent infection. CD137 interactions lead to reduced HSK and are necessary to develop robust trigeminal CD8+ T cell responses.

Natural killer T cells (NKT cells) are a unique subset of the immune system that possess characteristics of both an innate and adaptive immune response. This study reviews the reported roles of NKT cells in different solid transplantations such as cardiac, skin, liver, and corneal grafts as well as investigates a novel role of NKT cells in steroid-resistant corneal rejections. It is unknown why there is late corneal graft rejection despite being treated with immunosuppression. Our experimental data suggests NKT cells are playing a crucial part in steroid-resistant late graft rejections. While the pathophysiology of acute rejection is better understood, the process of chronic graft rejection is much less clear. Our data suggests NKT cells as a potential therapeutic target to prevent chronic transplant rejection which needs further investigation.

Herpes stromal keratitis (HSK) represents a spectrum of pathologies which is caused by herpes simplex virus type 1 (HSV-1) infection and is considered a leading cause of infectious blindness. HSV-1 infects corneal sensory nerves and establishes latency in the trigeminal ganglion (TG). Recently, retraction of sensory nerves and replacement with "unsensing" sympathetic nerves was identified as a critical contributor of HSK in a mouse model where corneal pathology is caused by primary infection. This resulted in the loss of blink reflex, corneal desiccation, and exacerbation of inflammation leading to corneal opacity. Despite this, it was unclear whether inflammation associated with viral reactivation was sufficient to initiate this cascade of events.We examined viral reactivation and corneal pathology in a mouse model with recurrent HSK by infecting the cornea with HSV-1 (McKrae) and transferring (intravenous [IV]) human sera to establish primary infection without discernible disease and then exposed the cornea to UV-B light to induce viral reactivation.UV-B light induced viral reactivation from latency in 100% of mice as measured by HSV-1 antigen deposition in the cornea. Further, unlike conventional HSK models, viral reactivation resulted in focal retraction of sensory nerves and corneal opacity. Dependent on CD4+ T cells, inflammation foci were innervated by sympathetic nerves.Collectively, our data reveal that sectoral corneal sensory nerve retraction and replacement of sympathetic nerves were involved in the progressive pathology that is dependent on CD4+ T cells after viral reactivation from HSV-1 latency in the UV-B induced recurrent HSK mouse model.

Herpes simplex virus is responsible for numerous ocular diseases, the most common of which is herpetic stromal keratitis. This is a recurrent infection of the cornea that typically begins with a subclinical infection of the cornea that establishes a latent infection of sensory ganglia, most often the trigeminal ganglia. Recurring infections occur when the virus is reactivated from latency and travels back to the cornea, where it restimulates an inflammatory response. This inflammatory response can lead to decreased corneal sensation, scarring, and blindness. The diagnosis of these lesions as the result of a recurrent herpes simplex virus infection can at times be problematic. Currently, herpetic stromal keratitis is diagnosed by its clinical presentation on the slit-lamp examination, but the literature does not always support the accuracy of these clinical findings. Other diagnostic tests such as polymerase chain reaction assay, enzyme-linked immunosorbent assay, immunofluorescent antibody, and viral cultures have provided more definitive diagnosis, but also have some limitations. That said, accurate diagnosis is necessary for proper treatment, in order to prevent serious consequences. Current treatment reduces the severity of lesions and controls further viral spread, but does not provide a cure.

HSV-1 is a significant human pathogen that can result in the loss of sight as a result of episodic reactivation of latent virus from sensory ganglion neurons. In this study the potential efficacy of anti-viral cytokine expression in preventing latent virus reactivation was investigated. Both type I (IFN-beta) and type II (IFN-gamma) IFN transgene expression following transduction of trigeminal ganglion explant cultures significantly reduced the incident of HSV-1 reactivation that in the case of IFN-beta was dependent on the presence of double stranded RNA-dependent protein kinase and RNase L. In vivo, expression of the IFN-gamma but not IFN-beta transgene significantly delayed and reduced the frequency of reactivation of latent mice exposed to UV light without discernable inflammation. This result is the first report that demonstrates the ability to block reactivation using an ectopic cytokine expression system and warrants further exploration as a means to prevent HSV-1 reactivation.

To evaluate the utility of endothelial/Descemet membrane complex (En/DM) characteristics in diagnosing corneal graft rejection.Diagnostic reliability study.One hundred thirty-nine eyes (96 corneal grafts post penetrating keratoplasty or Descemet stripping automated endothelial keratoplasty: 40 clear, 23 actively rejecting, 24 rejected, and 9 nonimmunologic failed grafts; along with 43 age-matched control eyes) were imaged using high-definition optical coherence tomography. Images were used to describe En/DM and measure central corneal thickness (CCT) and central En/DM thickness (DMT). En/DM rejection index (DRI) was computed to detect the relative En/DM thickening to the entire cornea.In actively rejecting grafts, DMT and DRI were significantly greater than controls and clear grafts (28, 17, and 17 μm and 1.5, 1 and 1, respectively; P .001). Rejected grafts had the highest DMT and DRI compared to all groups (59 μm and 2.1; P .001). DMT and DRI showed excellent accuracy, significantly better than that of CCT, in differentiating actively rejecting from clear grafts (100% and 96% sensitivity; 92.5% and 92.5% specificity), actively rejecting from rejected grafts (88% and 83% sensitivity; 91% and 83% specificity), and nonimmunologic failed from rejected grafts (100% and 100% sensitivity; 88% and 100% specificity). DMT correlated significantly with rejection severity (P.001).In corneal grafts, in vivo relative thickening of the En/DM is diagnostic of graft rejection as measured by DMT and DRI. These indices have excellent accuracy, sensitivity, and specificity in detecting graft immunologic status, superior to CCT. DMT is a quantitative index that correlates accurately with the severity of rejection.

Corneal allograft rejection has been described as a Th1-mediated process involving IFN-γ production. However, recent evidence also implicated IL-17 as being involved in acute corneal allograft responses. Our data support that IL-17 is involved in early acute corneal allograft acceptance. However, we decided to extend these studies to include a later phase of rejection in which there is a peak of IL-17 production that is >15-fold higher than that seen during acute rejection and occurs >45 d postengraftment at the onset of late-term rejection. We demonstrate that neutralizing IL-17A at this time significantly reduced corneal graft rejection. Surprisingly, when corneal grafts that are undergoing this later phase of rejection are treated with anti–IL-17A, there is a reversal of both opacity and neovascularization. Compared with the early phase of rejection, the cellular infiltrate is significantly less, with a greatly reduced presence of Gr-1+ neutrophils and a relative increase in CD4+ T cells and macrophages. We went on to identify that the cells expressing IL-17 were CD4+ IL-17+ T cells and, somewhat surprisingly, IL-17+ F4/80+ macrophages within the rejecting corneal allografts. Taken together, these findings describe a distinct late phase of corneal allograft rejection that is likely mediated by Th17 cells; therapeutic neutralization of IL-17A reverses this rejection. This further suggests that IL-17 might serve as an excellent therapeutic target to reduce this form of corneal allograft rejection.

Interleukin 10 (IL-10), a moderator of Delayed Type Hypersensitivity (DTH) responses, has been demonstrated to be present late in acute HSV corneal infection and may help limit blinding inflammatory lesions there. In contrast, IL-10 is present early in the development of recurrent herpetic stromal keratitis (HSK) lesions in mice. To determine the role of IL-10 and DTH responses in recurrent HSK, we examined DTH responses and disease parameters in latently infected IL-10 knock out (KO) mice, and latently infected normal mice that were untreated or received anti-IL-10 antibodies or recombinant IL-10 following ultraviolet-B stimulated ocular HSV recurrence. Low DTH responses were associated with less severe corneal disease while high DTH responses were associated with greater corneal disease. In IL-10 KO mice, and in normal mice given anti-IL-10 antibodies, corneal opacification was increased and DTH responses were significantly prolonged. Normal mice receiving rIL-10 by ocular and intra-peritoneal routes had less severe corneal lesions. Our results indicate that IL-10 and DTH responses play an important role in the pathogenesis of recurrent HSK in mice.

Summary: Antigen-specific unresponsiveness (or tolerance) has always been an important area of research. Interest in the fate of apoptotic cells and their ability to tolerize has revived interest in some of the older models involving hapten-modified self. Recently, we have examined the mechanisms by which intravenous injection of trinitrophenol-coupled spleen cells leads to systemic tolerance. These studies have revealed an important role for Fas/Fas ligand interactions, caspases, CD40/CD40L, and regulatory CD4+ and CD8+ T cells. Extension of these studies to peripheral deletion of T-cell antigen receptor transgenic T cells has shown that deletion and active regulation of immune responses may be important mechanisms for the control of potentially damaging autoimmune responses.

Virion host shutoff (vhs)-deficient herpes simplex virus (HSV) was tested as a therapeutic vaccine in a mouse model of UV light-induced recurrent herpetic stromal keratitis. Four weeks after primary corneal infection, mice were vaccinated intraperitoneally with vhs− vaccine or control. Four weeks after vaccination, the eyes of latently infected mice were UV-B irradiated to induce recurrent virus shedding and disease. Post-irradiation corneal opacity in latently infected, vhs−-vaccinated mice was significantly reduced compared to control-vaccinated mice (P=0·007 to 0·035). The incidence and duration of recurrent virus shedding were the same in both groups. Antibody titres were increased (P=0·05) and delayed type hypersensitive responses were unaffected by vhs− vaccination. Combined with studies using different vaccination timing and vhs− genotypes, these data suggest that deletion of vhs is a useful strategy in the development of a therapeutic HSV vaccine, and that temporal and genetic factors influence vaccination outcome.

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each year. While several animal models exist for this procedure and mice are the species that is most commonly used. The reasons for using mice are the relative cost of using this species, the existence of many genetically defined strains that allow for the study of immune responses, and the existence of an extensive array of reagents that can be used to further define responses in this species. This model has been used to define factors in the cornea that are responsible for the relative immune privilege status of this tissue that enables corneal allografts to survive acute rejection in the absence of immunosuppressive therapy. It has also been used to define those factors that are most important in rejection of such allografts. Consequently, much of what we know concerning mechanisms of both corneal allograft acceptance and rejection are due to studies using a murine model of corneal transplantation. In addition to describing a model for acute corneal allograft rejection, we also present for the first time a model of late-term corneal allograft rejection.

Recurrent herpetic stromal keratitis (HSK) is a potentially blinding, immune-mediated disease. To better understand the immunopathology of recurrent HSK, we examined the cytokine profile of mouse corneas with the condition.The eyes of latently infected mice were examined for corneal pathology and cytokine content following UV-B-stimulated herpes simplex virus (HSV) reactivation.Peak HSV-induced corneal disease, manifested by stromal opacification, occurred 7-14 days after viral reactivation in latently infected mice. In qualitative RT-PCR analyses, IFNgamma, IL-10, IL-4, and IL-12 p40 mRNA were simultaneously expressed before and during recurrent HSK. Competitive, semi-quantitative RT-PCR evaluation of cytokine mRNA revealed highest IFNgamma expression before and during clinical disease with a decline thereafter. IL-4 levels peaked and declined before day 14, while IL-10 peaked on days 7 or 14 and paralleled IFNgamma at lower levels. Small amounts of IL-12 p40 mRNA were detected late in the disease course. ELISA evaluation of corneal extracts demonstrated similar results, featuring early expression of Th2 cytokines relative to disease.The presence of Th2 cytokines during early stages of recurrent herpetic corneal lesions indicate the presence of a mixed Th1 and Th2 cell infiltrate, which is likely associated with a memory response to viral antigens. These data suggest that disease resolution in corneas with recurrent HSK may depend upon the balance between destructive and protective cytokines at individual sites of viral recurrence.

Background. The relative contribution of CD4 + or CD8 + T cells in allograft rejection remains to be fully characterized. Some reports indicate that there is an absolute requirement for CD4 + T cells in allogeneic rejection, whereas others report that CD4-depleted mice are capable of rejecting certain types of allografts. Methods. We compared the ability of CD4- knockout (KO), CD8 - KO, and normal CD4 + /CD8 + mice to reject allogeneic corneal or skin grafts. We also examined delayed-type hypersensitivity and CTL responses to donor alloantigens. Results. Engraftment of C57BL/6 corneas to C.B6-(n5-7) CD4-KO mice resulted in significantly higher rates of acceptance (>85%) than either C.B6-(n5-7) CD8 - KO (30%) or normal BALB/c mice (40%). Likewise, mean survival times for B6 skin grafts placed on C.B6-(n5-7) CD4- KO mice (29.2 ± 3.5 days) were significantly increased over those of normal BALB/c mice (13.2 ± 1 days), although most CD4 - KO mice (70%) eventually reject their grafts. C.B6-(n5-7) CD4 - KO mice that reject allogeneic grafts fail to develop a delayed-type hypersensitivity response, but they did demonstrate significantly greater cytotoxic T lymphocyte precursor (CTLp) frequencies than did CD4 - KO mice that accepted such grafts or that were not grafted. Conclusions. This study indicates that mice lacking CD4 + T cells have a significantly impaired ability to reject corneal allografts, but are able, in most cases, to reject allogeneic skin grafts. Thus, in the absence of CD4 + T cells, the likely mechanism for rejection appears to involve the generation of CD8 + CTLs.

· Background: The focus of these studies was to determine whether the Y79 human retinoblastoma cell line could function as a good in vitro model system for studying human cytomegalovirus (HCMV) infection. · Methods: Y79 cells were exposed to an HCMV mutant carrying a LacZ gene, and the resulting β-galactosidase expression in infected cells was assessed by flow cytometry. The extent to which the three classes of viral gene products – immediate early, early, and late proteins – were expressed was analyzed by immunohistochemical staining and Western blotting. Infected Y79 cells were also co-cultivated on human foreskin fibroblast (SF cell) cultures to recover virus. · Results: Infection of Y79 cells with the virus resulted in β-galactosidase expression as detected by flow-cytometric analysis. Immunohistochemical staining revealed that a portion of Y79 cells expressed antigens reactive to monoclonal antibodies against immediate early, early, and late HCMV proteins. The 43-kDa early gene product was also detected by Western blotting. Infected Y79 cells co-cultivated on SF cell cultures yielded infectious foci, which turned blue following X-gal staining, demonstrating productive HCMV infection in the Y79 cells. · Conclusion: These results demonstrate that while HCMV can productively infect Y79 cultures, it does so in a highly inefficient manner, leading these authors to conclude that this cell line does not provide a particularly good model system to study HCMV infection.

Herpetic stromal keratitis (HSK) is characterized by an inflammatory response that includes neutrophils, macrophages, NK cells, and T cells. The factors that are responsible for this inflammation are proinflammatory cytokines and chemokines. Many of these factors have been defined for primary disease, but relatively few have been investigated during recurrent HSK. The present study was designed to determine the role that two of these factors, IL-6 and CXCL1, play during recurrent HSK. Results clearly indicate that unlike primary disease, IL-6 plays no role in recurrent HSK. However, the presence of CXCL1 is required for recurrent HSK as evidenced by the lack of corneal disease in mice treated with anti-CXCL1 Ab. This was confirmed using mice lacking the primary receptor for CXCL1, CXCR2. Corneal disease in this strain was significantly reduced compared with wild-type C57BL/6 controls. Unexpectedly, lack of disease occurs even though CXCL1 knockout mice display increased viral shedding at the cornea. The primary mechanism that CXCL1 plays during disease is its ability to stimulate neutrophils to infiltrate the cornea following reactivation. This paper provides further evidence that primary HSK and recurrent HSK possess overlapping yet distinct disease mechanisms.

The protective efficacy of a glycoprotein D subunit vaccine (gD2 SB AS4) was evaluated in a mouse model of human recurrent herpetic stromal keratitis (HSK). When administered before primary infection, gD2 SB AS4 protected mice against corneal pathology, mortality, and latency resulting from ocular viral challenge with herpes simplex virus type 1 (HSV-1) McKrae strain. In addition, gD2 SB AS4 significantly decreased postreactivation corneal disease. A control vaccine, gD2 alum, protected against acute ocular infection only. When administered after primary infection, gD2 SB AS4 vaccination decreased postreactivation ocular shedding but had no other significant effects. Vaccination with gD2 SB AS4 was associated with high anti-gD antibody responses and low delayed-type hypersensitivity responses. These results have identified a prophylactic vaccine, gD2 SB AS4, with activity against acute and recurrent HSK in mice and emphasize the need for vaccine evaluation in both primary and recurrent ocular herpetic disease models.

Although anatomical barriers and soluble mediators have been implicated in immune privilege, it appears that the apoptotic cell death of Fas+ cells by tissue-associated CD95 ligand (Fas ligand, FasL) is an important component. One clinical example of the function of an immune privileged site is the success of human corneal transplants, where a very high percentage of transplants accept without tissue matching or immunosuppressive therapy. Since the mouse cornea expresses abundant Fas ligand and immune privilege has been implicated in the success of these transplants, we examined the role of FasL in corneal transplantation. Our results show that human corneas express functional FasL capable of killing Fas+ lymphoid cells in an in vitro culture system. Using a mouse model for corneal allograft transplantation, FasL+ orthografts were accepted at a rate of 45%, whereas FasL- grafts, or normal grafts transplanted to Fas- mice, were rejected 100% of the time. Histological analysis found that FasL+ grafts contained apoptotic mononuclear cells indicating the induction of apoptosis by the graft, while rejecting FasL- corneas contained numerous inflammatory cells without associated apoptosis. Taken together our results demonstrate that FasL expression on the cornea is a major factor in corneal allograft survival and, thus, we provide an explanation for one of the most successful tissue transplants performed in humans.

Previous studies have shown that keratoplasty buttons obtained at surgery from patients with herpes simplex virus-1 (HSV-1) keratitis have elevated localized expression of the adhesion molecule ICAM-1, which plays a critical role in the initiation and amplification of an immune response. We performed studies to determine whether changes in expression of ICAM-1 and HLA class I are direct effects of productive infection of human corneal fibroblasts with HSV-1.Immunocytologic and flow cytometric analyses were performed to analyze the ability of HSV-1 to induce ICAM-1 and HLA class I expression in a primary cornea-derived keratocyte cell line, E-2. Positive controls for these experiments were E-2 cells infected with human cytomegalovirus (HCMV), which has been shown to increase ICAM-1 expression in selected cells, and E-2 cells treated with IFN-gamma, which upregulates both ICAM-1 and HLA class I expression in most cell types.Kinetic cytometric analysis indicated decreased ICAM-1 expression 3 hours following HSV-1 infection of E-2 cells. In contrast, HCMV led to detectable increases in ICAM-1 expression starting 6 hours after infection. Infections with either HSV-1 or HCMV resulted in reduced HLA class I expression on E-2 and SF cells.These studies suggest that increased ICAM-1 expression seen on corneal stromal cells during clinical HSV-1 infection is not a direct result of productive viral infection, but of other mechanisms such as cytokine release by infiltrating mononuclear cells.

Herpes simplex virus 1 (HSV-1) infection of the cornea leads to a potentially blinding disease, termed herpetic stromal keratitis (HSK) that is characterized by lesions of an immunoinflammatory nature. In spite of the fact that HSK typically presents as a recurrent disease due to reactivation of virus which latently infects the trigeminal ganglia, most murine studies of HSK have employed a primary and not recurrent model of the disease. This report documents the several recurrent models of HSK that have been developed and how data generated from these models differs in some important aspects from data generated following primary infection of the cornea. Chief among these differences is the fact that recurrent HSK takes place in the context of an animal that has a preexisting anti-HSV immune response, while primary HSK occurs in an animal that is developing such a response. We will document both differences and similarities that derive from this fundamental difference in these models with an eye towards possible vaccines and therapies that demonstrate promise in treating HSK.

HSV-1 infection of the cornea leads to a potentially blinding immunoinflammatory lesion of the cornea, termed herpetic stromal keratitis. It has also been shown that one of the factors limiting inflammation of the cornea is the presence of Fas ligand (FasL) on corneal epithelium and endothelium. In this study, the role played by FasL expression in the cornea following acute infection with HSV-1 was determined. Both BALB/c and C57BL/6 (B6) mice with HSV-1 infection were compared with their lpr and gld counterparts. Results indicated that mice bearing mutations in the Fas Ag (lpr) displayed the most severe disease, whereas the FasL-defective gld mouse displayed an intermediate phenotype. It was further demonstrated that increased disease was due to lack of Fas expression on bone marrow-derived cells. Of interest, although virus persisted slightly longer in the corneas of mice bearing lpr and gld mutations, the persistence of infectious virus in the trigeminal ganglia was the same for all strains infected. Further, B6 mice bearing lpr and gld mutations were also more resistant to virus-induced mortality than were wild-type B6 mice. Thus, neither disease nor mortality correlated with viral replication in these mice. Collectively, the findings indicate that the presence of FasL on the cornea restricts the entry of Fas+ bone marrow-derived inflammatory cells and thus reduces the severity of HSK.

The major histocompatibility complex (MHC) of the mouse, which is called the H2 complex, is located on chromosome 17. It contains genes critical to the functioning of the immune system, the products of which are intimately involved in the initiation of immune responses. The proteins made by these genes display a significant amount of polymorphism, or sequence variability. Few genes possess this degree of polymorphism, but it is critical to the immune response and to the survival of a species. The chief characteristic of MHC molecules is their ability to bind small peptides. These bound peptides along with specific areas of the MHC molecule are the structures that interact with the T-cell receptor (TCR) leading to activation of a T-cell immune response, which in turn can active other cells of immune system. Thus, they determine which TCRs are functional which in turn indicates which peptides a T cell will respond to. Key Concepts The historical background and the genetics of MHC development are described. The expression and function of MHC class I genes is described. MHC class I molecules are expressed on virtually all nucleated cells and function to present foreign and self-antigens to the immune system. MHC class I expression is dependent on peptide binding in the peptide binding groove of the molecule. MHC class I polymorphism is important for species resistance to infectious disease. The expression and function of MHC class II genes is described. MHC class II expression is required for generation and function of CD4 T cells. Nonclassical class I molecules bind a variety of peptide and nonpeptide antigens that are commonly expressed by pathogenic organisms. . The function of other genes found within the H2 complex is described. Keywords: H2 complex; MHC; MHC restriction; class I; class II; MHC peptide association; T-cell activation

The role that interferon-gamma (IFNgamma) plays during herpetic stromal keratitis (HSK) has not been definitively determined. In primary HSK most reports suggest that IFNgamma may help control viral replication and contribute to corneal pathology. However, its role in recurrent HSK has not been directly addressed. The present study addresses its role in recurrent HSK by comparing HSK in latently infected normal and IFNgamma gene knockout (GKO) on the C57BL/6 background. We initially evaluated HSK following primary infection and observed that GKO mice had higher tear film virus titers, but virtually identical ocular disease as normal mice. In contrast, following reactivation of latent virus, GKO mice had a greater incidence and severity of opacity, neovascularization, and blepharitis. Interestingly, the incidence of reactivation after UV-B exposure was equivalent in GKO and normal mice, but virus shedding was increased in the GKO groups. We also observed diminished delayed-type hypersensitivity responses in GKO mice, as expected. These data indicate that IFNgamma is important for the control of virus replication in both primary and recurrent ocular HSV infection in C57BL/6 mice. The enhanced recurrent disease seen in GKO mice may be the result of increased viral titers and persistence in these mice which act to prolong the stimulation of an inflammatory response.

Herpetic stromal keratitis (HSK) is a potentially blinding corneal affliction that can accompany herpes simplex virus type 1 (HSV-1) infections. The disease course in HSK begins with a primary infection by HSV-1, typically mucosal surfaces of the mouth or eye. The virus replicates at the site of infection and proceeds via retrograde axonal transport in sensory neurons to sensory (usually trigeminal) and autonomic ganglia.1 Once inside ganglia, the virus establishes latency, in which the pathogenic virus remains dormant yet the genome persists.2 If these latently infected individuals become immunocompromised (as occurs in elderly or malnourished individuals, or with transplant or HIV-infected patients) or the neurons are exposed to a “triggering” immunosuppressive event, such as stress, trauma, sunlight (UV) irradiation, or fever, the virus is reactivated and rapidly replicates. The virus travels via anterograde axonal transport and returns to the original epithelial surface, where another round of replication occurs.1,2 A multitude of studies have shown that the resulting clinical disease is not associated with this viral replication in the cornea, but rather is due largely to the host immune response being restimulated by the presence of the virus.1–5 As a consequence of these events, active viral replication triggers inflammation and neovascularization of the cornea. Once this inflammation is initiated, the disease continues to progress even though infectious virus can no longer be detected. This inflammation consists of different leukocyte lineages that are recruited to the cornea of patients with HSK, including polymorphonuclear leukocytes (PMNs; neutrophils), macrophages, and T cells, CD4+ and CD8+.1–5 Animal studies have shown that the predominant cell type in diseased corneas is neutrophils.3 Facing a conceivably blinding inflammatory attack, the cornea possesses several means to reduce inflammation and neovascularization. These include the presence of immunosuppressive factors, such as TGF-β,6 IL-1 receptor antagonists, and antiangiogenic factors, such as thrombospondin and pigment epithelium-derived factor (PEDF).7,8 Additionally, the presence of Fas ligand (FasL), which is the focus of this study, often is a critical factor in controlling inflammation and neovascularization, thus reducing disease.9–17 Fas is a member of the tumor necrosis factor-R family, which is a group of type I transmembrane proteins. FasL is a ligand of Fas and is synthesized as a transmembrane molecule that undergoes cleavage and processing through a metalloprotease to become soluble FasL (sFasL). This sFasL contains a “death domain,” which is essential for the induction of apoptosis.18 Studies from our laboratory as well as the laboratories of others have demonstrated that the presence of FasL in the eye is an important barrier to inflammatory cells9–11 and new blood vessels.12–14 In fact, we know that control of inflammation is required for the immune privilege of the eye.9,10 FasL expressed on ocular tissues induces apoptosis in Fas+ lymphoid cells that invade the eye in response to viral infection8 or corneal grafting.11,12 FasL expressed in the retina and the cornea also control new vessel growth beneath the retina and in the cornea by inducing apoptosis of Fas+ vascular endothelial cells.13–15 These studies indicate that the presence of FasL in ocular tissues restricts inflammatory responses. Recently we published that the interaction of Fas with FasL is an important factor in controlling HSK during acute infection of the cornea.17 We demonstrated that mice expressing mutations in Fas (lpr) or FasL (gld) experience significantly worse ocular disease than do wild-type mice, regardless of mouse or viral strain.17 However, as mentioned, acute infection rarely leads to clinical disease in humans. Instead, the reactivated virus is responsible primarily for the symptoms that define corneal keratitis.1–5 Thus, we thought it very important to address the role that Fas and FasL have during recurrent disease when the virus is reactivated from latency. To address the role of Fas-FasL interactions during recurrent HSK, we evaluated this interaction in a mouse model of induced recurrent HSK. We used soluble TNF-related apoptosis inducing ligand (sTRAIL) as our control and standard for comparison, since TRAIL and FasL are members of the TNF family and are known to induce apoptosis in lymphocyte lineages. In this study, we demonstrated that mice treated with sFasL experience a decrease in acute and recurrent models of HSK.

Herpes stromal keratitis (HSK) is a vision-threatening, recurrent disease of the cornea that is the leading infectious cause of corneal blindness in the United States. Most of our current knowledge of the immunologic processes that underlie HSK has arisen from studies in mice. Seminal studies by Metcalf et al.1 demonstrated that HSK did not develop in congenitally athymic (and thus T-cell-deficient) nude mice. Subsequent studies confirmed the involvement of T cells by demonstrating that nude mice can be rendered susceptible to HSK by adoptive transfer of T cells before HSV-1 corneal infection.2 It was then established independently in two laboratories that HSK induced by the RE strain of HSV-1 in either A/J or BALB/c mice is regulated primarily by CD4+ T cells3–5 and the Th1 cytokines IFN-γ and IL-2.6–9 Although the capacity of CD4+ T cells to mediate HSK is well documented, the involvement of CD8+ T cells is less clear. One study showed that after corneal infection of A/J mice with the KOS strain of HSV-1, CD8+ T cells were the predominant infiltrating T cell population, and their depletion significantly reduced the incidence and severity of HSK.4 In contrast, studies using the RE and McKrae strains of HSV-1 to infect the corneas of BALB/c, and C57BL/6 mice, respectively, suggested a regulatory role in which CD8+ T cells reduce the incidence and severity of HSK.3,10 Of note, a recent study indicated that this regulatory role of CD8+ T cells may be expressed outside the cornea, perhaps involving control of HSV-1 infection of the trigeminal ganglion.11 Several recent studies have added to the complexities surrounding the involvement of T-cell subpopulations in HSK. In one set of studies, a congenic BALB/c mouse strain developed HSK due to an autoimmune T-cell attack on the cornea induced by a molecular mimicry mechanism involving a homologous epitope on viral and corneal proteins.12–14 In a second study,15 the RE strain of HSV-1 induced HSK in T-cell- and B-cell-deficient SCID mice by a mechanism apparently involving only innate immunity. In a third study,16 HSK was induced in DO11.10 SCID mice, which are incapable of producing HSV-1-specific CD4+ T cells, through apparent cytokine-mediated bystander activation of Ova-specific CD4+ T cells in the infected cornea. The conclusion of these studies is that, depending on the strain of virus and the strain and immune competence of the mice used, HSK can result from an autoaggressive response of CD4+ T cells, an uncharacterized innate immune response, or bystander activation of CD4+ T cells of irrelevant specificity. Although the latter two studies15,16 incorporated a rather elegant design, there is an important caveat to accepting the authors’ conclusions. Both studies used BALB/c SCID mice, in which HSV-1 replication in the cornea never comes under control, and which typically succumb to a lethal encephalitis by 12 days after infection. One cannot assume that the involvement of innate immunity and bystander activation of CD4+ T cells in corneas of BALB/c SCID mice that never control HSV-1 replication will also contribute to HSK in WT mice that control HSV-1 replication in the cornea within 7 days and uniformly survive the infection. Indeed, it was subsequently demonstrated that when HSV-1 replication is controlled in the corneas of SCID mice, bystander activation alone is not sufficient to cause HSK.17 In the current study, viral dose dictated whether HSK development in BALB/c mice occurred in a CD4-dependent or-independent manner. Differences in the HSK is regulated by CD4+ and CD8+ T cells were determined.

Previous studies have shown that the expression of Fas ligand (FasL; CD95L) by donor corneas is critical to their survival when placed on allogeneic recipients. Since there have been reports that the cornea expresses Fas, we tested the idea that FasL on lymphoid cells could be an effector molecule during rejection episodes. When FasL defective BALB/c-gld mice were engrafted with allogeneic corneas, significantly more of these corneas were accepted than by normal BALB/c mice. However, this was not due to impaired FasL-mediated effector function in these mice as the allogeneic corneas did not express detectable Fas by Western blot or RT-PCR analysis. Furthermore, donor corneas without Fas were given no survival advantage, but were rejected similar to wild-type donor allogeneic corneas. Examination of the T cell compartment in gld mice revealed that these cells express higher levels of Fas and are more susceptible to Fas-mediated death than wild-type cells. These results indicate that FasL is not an effector molecule in corneal graft rejection and that gld mice show reduced graft rejection due to greater susceptibility of their T cells to Fas-mediated apoptosis.

Peripheral deletion is one mechanism by which potentially self-reactive clones are removed whether they escape thymic deletion. We have examined the consequences of deleting Ag-specific T cells by i.v. injection of soluble Ag. Deletion of DO11.10 T cells by peptide was mediated predominately via a Fas/FasL mechanism. Animals that underwent deletion were tolerant to subsequent immunization with Ag, even when tolerant mice were given fresh Ag-specific DO11.10 T cells before immunization. Tolerance was mediated by CD8+ T cells that killed the DO11.10-transgenic T cells in vivo. These data demonstrate that the programmed cell death of large numbers of T cells leads to peripheral tolerance mediated by CD8+ CTLs.

In primary ocular herpes simplex virus (HSV) infection, nitric oxide may function to control viral replication and herpetic stromal keratitis (HSK) lesions. Recurrent HSK, manifested as corneal opacity and neovascularization, is the potentially blinding sequel to primary infection. Here, we assess the effects of nitric oxide synthase inhibition on a mouse model of recurrent HSK. In preliminary primary infection experiments, NIH inbred mice treated with aminoguanidine, an inhibitor of inducible nitric oxide synthase (iNOS), experienced no changes in post-infection tear, brain, or ganglia virus titers, but encephalitis-related mortality was elevated. After UV-B stimulated viral reactivation, iNOS inhibition did not affect virus shedding or clinical disease. In contrast to primary HSK, there was no exacerbation of mortality in recurrent disease. Our findings indicate that nitric oxide can be neuroprotective without antiviral effects in primary HSK, and does not play a significant role in the pathogenesis of recurrent HSK. Compared with data from other mouse strains, this work suggests that there may be a genetic component to the importance of NO in controlling ocular HSV infection.

The role that T cell subsets play in herpetic stromal keratitis (HSK) has been the subject of intense research efforts. While most studies implicate CD4+T cells as the principal cell type mediating primary corneal disease, recent reports using knockout mice have suggested that both CD4+and CD8+T cell subsets may play integral roles in modulating the disease. Furthermore, recent studies suggest that CD8+T cells are directly involved in maintaining virus latency in infected trigeminal ganglia. This work has addressed these discrepancies by infecting the corneas of mice lacking CD4+and CD8+T cells with herpes simplex virus type 1 (HSV-1) and monitoring both corneal disease and latent infection of trigeminal ganglia. Results indicated that mice lacking CD8+T cells had more severe corneal disease than either BALB/c or B6 parental strains. In contrast, mice lacking CD4+T cells had a milder disease than parental strains. When mice were evaluated for persistence of infectious virus, only transient differences were observed in periocular tissue and corneas. No significant differences were found in persistence of virus in trigeminal ganglia or virus reactivation from explanted ganglia. These data support the following conclusions. CD4+T cells are not required for resistance to infection with HSV-1 and probably mediate HSK. Mice lacking CD8+T cells do not display differences in viral loads or reactivation and thus CD8+T cells are not absolutely required to maintain latency. Finally, CD8+T cells probably play a protective role by regulating the immunopathological response that mediates HSK.

Ocular herpes simplex virus (HSV) infection results in an immune-mediated inflammation of the corneal stroma known as herpetic stromal keratitis (HSK). Recurrent HSK is a common cause of virus-induced corneal blindness in humans. The role of CD4(+) and CD8(+) T cell subsets in the disease pathogenesis is ill defined and varies with the virus strain and host genetic background. To examine the contribution of T cell subsets to corneal disease, we studied the development of recurrent HSK in CD4 or CD8 gene knockout (KO) mice ocularly infected with HSV-1 McKrae strain. Following UV-B induced viral reactivation, corneal opacity in latently infected BALB/c (HSV sensitive) CD4 and CD8 KO mice was reduced compared to infected BALB/c mice with normal genotype. In contrast, opacity in C57BL/6 (HSV resistant) CD4 and CD8 KO latent mice did not differ from genetically normal latent mice. Virus-induced corneal opacity was not demonstrable in C57BL/6 CD4/CD8 double KO mice. Increased viral shedding, measured by reactivation rate, days shedding or viral titers, occurred in CD4 KO mice of both strains. Our findings indicate that both CD4(+) and CD8(+) cells play a role in the immunopathogenesis of recurrent HSK, and their role is dependent upon the host genetic profile.

Immunization of mice with herpes simplex virus type 1 (HSV-1) mutant viruses containing deletions in the gene for virion host shutoff (vhs) protein diminishes primary and recurrent corneal infection with wild-type HSV-1. vhs mutant viruses are severely attenuated in vivo but establish latent infections in sensory neurons. A safer HSV-1 mutant vaccine strain, Δ41Δ29, has combined vhs and replication (ICP8−) deficits and protects BALB/c mice against primary corneal infection equivalent to a vhs−strain (BGS41). Here, we tested the hypothesis that Δ41Δ29 can protect as well as BGS41 in a therapeutic setting. Because immune response induction varies with the mouse and virus strains studied, we first determined the effect of prophylactic Δ41Δ29 vaccination on primary ocular infection of NIH inbred mice with HSV-1 McKrae, a model system used to evaluate therapeutic vaccines. In a dose-dependent fashion, prophylactic Δ41Δ29 vaccination decreased postchallenge tear film virus titers and ocular disease incidence and severity while eliciting high levels of HSV-specific antibodies. Adoptive transfer studies demonstrated a dominant role for immune serum and a lesser role for immune cells in mediating prophylactic protection. Therapeutically, vaccination with Δ41Δ29 effectively reduced the incidence of UV-B-induced recurrent virus shedding in latently infected mice. Therapeutic Δ41Δ29 and BGS41 vaccination decreased corneal opacity and delayed-type hypersensitivity responses while elevating antibody titers, compared to controls. These data indicate that replication is not a prerequisite for generation of therapeutic immunity by live HSV mutant virus vaccines and raise the possibility that genetically tailored replication-defective viruses may make effective and safe therapeutic vaccines.

CCL2 and CCL3 are proinflammatory chemokines that are produced during the early stages of inflammation and are known to stimulate the migration of mononuclear cells to the site of inflammation,. Previous studies addressing the role of these chemokines during primary herpetic stromal keratitis (HSK), have suggested that CCL2 is involved in reducing corneal disease and that CCL3 is involved in promoting this disease. We addressed the role that these chemokines play in a recurrent model of HSK. Results from these studies did not demonstrate a significant role for CCL2 except for very early time points following reactivation of virus. Surprisingly, mice deficient in CCL3 did not have significantly reduced recurrent disease, , but in fact showed significantly enhanced disease. This argues that CCL3 might play an ameliorative role during recurrent HSK. In addition, we observed that these same CCL3 deficient mice showed increased resistance to viral-induced mortality following infection with HSV-1. Taken together, these results suggest that CCL3 plays a significant protective role during recurrent HSK and is involved in enhancing lethality.

We have studied the ability of three different Mycoplasma species to induce proliferation of bone marrow-derived macrophages (BMM). We observed a significant mitogenic effect when BMM cells from BALB/c, DBA/2J, SJL, and C57BL/6 mice were incubated with membranes derived from Mycoplasma arginini or M. arthritidis but not when they were incubated with an equivalent amount of M. pulmonis membrane. We also determined that pretreatment of mycoplasma membrane preparations with papain eliminated the ability of these preparations to induce BMM proliferation. To determine whether these membrane fractions acted indirectly by stimulating the production of soluble factors known to stimulate proliferation of BMM cells, we performed blocking studies with antibodies directed against colony-stimulating factor 1 (CSF-1), interleukin-3 (IL-3), and granulocyte-macrophage colony-stimulating factor. Our results indicate that antibodies directed against either CSF-1 or IL-3 failed to block mycoplasma-initiated proliferation of BMM cells. However, when anti-GM-CSF was added to proliferative cultures at the time of initiation, we saw a dose-dependent reduction of mycoplasma-initiated proliferation. We conclude that the ability of mycoplasma membranes to initiate the proliferation of BMM is not shared by all species of mycoplasma and that it involves the production of GM-CSF by an as yet undetermined cell.

PURPOSE. The expression of Fas ligand (FasL) in the cornea is essential for corneal allograft acceptance in mice. Because the expression of FasL on the surface of cells is sensitive to cleavage with matrix metalloproteases (MMPs), this study examined whether inhibitors of MMPs would lead to increased FasL expression and improved corneal allograft survival. METHODS. Corneal endothelia derived from mice and humans were treated with MMP inhibitors, and FasL expression was examined. BALB/c mice were engrafted with C57BL/6 or C57BL/6-gld corneas and treated with an ointment containing the MMP inhibitor, doxycycline. Corneal allograft survival was monitored for 50 days. RESULTS. Corneal endothelial cells from both mice and humans displayed increased surface expression of FasL after treatment with MMP inhibitors. The increase in surface expression was further evidenced by the ability of these cells to kill Fasexpressing target cells. Mice treated with doxycycline after corneal allograft transplantation showed significantly prolonged allograft survival and an increase in the overall acceptance rate. CONCLUSIONS. MMP inhibitor treatment of cornea-derived endothelial cells results in increased FasL expression and function. MMP inhibitor treatment prolongs corneal allograft survival and results in a modest increase in corneal allograft acceptance. (Invest Ophthalmol Vis Sci. 2004;45:1169 ‐1173) DOI:10.1167/iovs.03-0932

The recent paper by Ishida et al.1 examines the role of leukocytes, specifically T lymphocytes, in vascular remodeling and vaso-obliteration in the rat eye. The authors conclude that T lymphocytes bind to the vasculature and model it by using Fas ligand (FasL) to prune the developing vessels. The authors also conclude from their examination of oxygen-induced vaso-obliteration that FasL on T lymphocytes induces apoptosis of hyperoxygenated endothelial cells. For these observations to have biological relevance, they should transcend species specificity. Because similar models of retinal angiogenesis exist in the mouse, one should be able to use the mouse to test predictions arising from these important observations. First, if Fas-FasL interactions are involved in retinal vessel development, Fas-deficient (lpr) and FasL-deficient (gld) mice should show vascular defects. Second, vessels in mice without these proteins should not undergo vaso-obliteration to the same degree as do wild-type animals. Third, rodents without T lymphocytes (Rag-/- or severe combined immunodeficient mice and T-cell-deficient rats) and/or cytotoxic lymphocytes (MHC class I− or CD8-deficient) should show aberrant retinal vessel development. Fourth, if the retinal vessels in these mice do not develop normally, this should severely impair normal development of the retina. Fifth, examination of these strains for vaso-obliteration should reveal that this process is severely impaired compared with wild-type animals.

PURPOSE. Neovascularization of the avascular cornea is a significant problem associated with many corneal diseases. Because Fas ligand (FasL) is highly expressed in the cornea, the role of this molecule in controlling corneal neovascularization was examined in this study. METHODS. C57BL/6(B6), FasL (CD95L)– deficient B6-gld, and Fas (CD95)– deficient B6-lpr mice were subjected to the suture model of neovascularization. Corneas were evaluated for neovascularization and representative samples subjected to immunohistochemical analysis for expression of Fas antigen and CD31 (platelet– endothelial cell adhesion molecule [PECAM-1]) on vessels that were present in the tissue. Corneas were also explanted and placed in collagen gel cultures to test the ability of anti-Fas antibody to prevent vessel extension from explanted corneas. RESULTS. Immunohistochemical data demonstrated that quiescent vessels express CD31 alone, whereas vessels that penetrate the cornea coexpressed both the Fas antigen and CD31. A significant increase was observed in neovascularization in FasLdeficient B6-gld corneas compared with B6 corneas, and new vessel growth in both B6 and B6-gld was inhibited by anti-Fas antibody. Whereas Fas-deficient B6-lpr corneas displayed significantly less neovascularization than normal B6, B6-lpr mice express Fas on growing vessels. In corneal explant cultures, vessel growth from B6 and lpr mice corneas was inhibited by anti-Fas antibody, confirming functional Fas expression in B6lpr mice. CONCLUSIONS. These data indicate that FasL is an important factor in controlling corneal neovascularization. (Invest Ophthalmol Vis Sci. 2003;44:93–98) DOI:10.1167/iovs.02-0299

Herpes simplex virus (HSV) infection is one of the leading causes of corneal blindness. This study compared the clinical, virologic, and immunopathologic features of primary and recurrent murine models of herpes simplex keratitis (HSK) in the National Institutes of Health (NIH) inbred mouse strain. Primary infection resulted in multiple epithelial dendrites, followed by diffuse stromal opacification, symptoms that do not mimic what is seen in human HSK. In contrast, recurrent infection presented clinical features that included microdendrites, focal stromal opacities, disciform endotheliitis, and corneal neovascularization, which were similar to those observed in human disease. Immunohistochemical characterizations indicated that the number and duration of T cells and macrophages in recurrent HSK resemble those observed in primary disease. Results also indicated that the amount of infectious virus detected in the cornea during primary and recurrent disease was similar. However, when corneas were stained for HSV-1 antigens, mice with primary HSK displayed diffuse HSV antigen expression throughout the cornea, whereas HSV antigens were more focally distributed in recurrent disease. These data suggest that the clinical differences between the recurrent and primary herpetic keratitis may, in part, reflect the different distribution of HSV-1 antigens within the cornea.

Mouse class II major histocompatibility complex genes have been shown to be regulated at the level of transcription for both tissue-specific and inducible expression. In particular, IFN-gamma induction of the class II genes has been shown to occur at the transcriptional level, although the role that additional post-transcriptional mechanisms of regulation may play in this induction is not known. To evaluate IFN-gamma effects on transcriptional and post-transcriptional events of class II gene expression, we examined the rate of decline of class II transcription, steady-state mRNA, and cell surface protein following the removal of IFN-gamma from maximally stimulated WEHI-3 cells (an IFN-gamma inducible, myelomonocytic cell line). We determined that transcription of class II genes almost completely returned to baseline levels eight hours after removal of IFN-gamma. However, the steady-state level of class II mRNA's required 4 days, and membrane Ia expression required 5 days to return to baseline levels. This decay was linear and allowed us to determine a half-life value of 16-20 h for class II transcripts. These data demonstrate that, following removal of IFN-gamma from fully stimulated cells, transcription of the class II genes declined rapidly, but mRNA was quite long-lived. We also assessed the class II mRNA stability in unstimulated WEHI-3 cells and the B-cell lymphoma. A20/2J, by actinomycin D treatment and northern blot analysis. In agreement with the IFN-gamma washout experiments, transcripts from all four class II genes were quite long-lived in these cell types, with a half-life greater than ten hours. These data support the concept that IFN-gamma acts primarily at the level of class II transcription and argues against IFN-gamma playing a major role in post-transcriptional modulation of class II expression.

Fas (CD95) and Fas ligand (FasL) play major roles in staphylococcal enterotoxin B (SEB)–induced peripheral deletion of Vβ8 + T cells. We found that peripheral deletion was defective in radiation chimeras with nonfunctional tissue FasL, regardless of the FasL status of the bone marrow–derived cells. SEB induced a dramatic upregulation of FasL expression and function in nonlymphoid cells of liver and small intestine. This effect was resistant to inhibition by cyclosporin A, which also failed to inhibit peripheral deletion. In SCID animals nonlymphoid tissues did not express FasL in response to SEB unless transplanted lymphocytes were present. Thus, some immune responses induce FasL in nonlymphoid tissues, which in turn kills activated lymphocytes, leading to peripheral T cell deletion.

We have evaluated the relative contribution of bone-marrow-derived cells to skin allograft immunogenicity in mice differing only at class II major histocompatibility genes by using bone marrow radiation chimeras as donors. The mouse strains used were C57BL/6Kh (B6) and B6.C-H-2bm12 (bm12), which differ only at at A beta gene of the I region of the mouse H-2 complex. Our results demonstrated that skin from (B6----bm12) chimeras was accepted by bm12 recipients and rejected by B6 mice in a manner indistinguishable from that of normal bm12 skin. Likewise, naive bm12 mice rejected (bm12----B6) chimeric skin and normal B6 skin equally well, and B6 animals accepted both types of skin grafts. Our data argues that the donor cell-type leading to graft rejection across limited I region differences is not of bone marrow origin, and that these cells must--at least under certain circumstances--express class II antigens.

Tumorigenicity of 2 sublines, TU1 and TM5, of the C.B6-H-2dm4 (dm4) mouse tumor, TDM4, was tested in dm4 and F1 hybrid mice (from crosses of dm4 with a variety of other strains) to determine the genetics of hybrid resistance to these 2 tumor sublines. Resistance to TU1 was associated with at least 3 genes and required heterozygosity in the H-2D region, with secondary effects attributable to H-2K genes and non-H-2 genes. Resistance to TM5, however, did not require H-2D region heterozygosity, whereas H-2K and non-H-2 genes appeared to have significant effects. For evaluation of possible mechanisms for hybrid resistance, 4 assay systems were used. Comparison of natural killer cell activity in dm4 and various hybrid mice revealed no consistent activity against either TU1 or TM5. Little correlation was seen between resistance and generation of strong T-lymphocyte proliferative responses to the sublines. Likewise, cytotoxic T-lymphocyte (CTL) activity, particularly against TU1, was not associated with increased resistance. In contrast, delayed-type hypersensitivity (DTH) was consistently greater in hybrids with increased resistance to either subline. These results suggest a strong association between DTH responsiveness and hybrid resistance. Furthermore, the association, in some hybrids, of CTL activity with resistance to the TM5 subline, but not to TU1, may mean that different sublines of the same tumor can induce different sets of immune responses.