Robert L. Hendricks

Endogenously produced interferon α protects mice from herpes simplex virus type 1 corneal disease

Intravenous (i.v.) injection of u.v. light-inactivated herpes simplex virus type 1 (UV HSV-1) at the time of HSV-1 corneal infection reduced the cytotoxic T lymphocyte (CTL) response to HSV-1, and significantly reduced the incidence of HSV-1-induced corneal stromal disease in A/J mice. The spread of HSV-1 through the eye after corneal infection, detected using engineered HSV-1 (US3::Tn5-lacZ) with the lacZ gene under the transcriptional control of the viral late gene promoter for glycoprotein C, was also markedly reduced by i.v. UV HSV-1 injection. The restriction of HSV-1 corneal invasiveness in i.v. UV HSV-1-injected mice preceded the onset of a detectable specific cell-mediated or humoral immune response to HSV-1, and was accompanied by an elevated serum titre of interferon (IFN-alpha), reversed by anti-IFN-alpha/beta antibody, and mimicked by systemic IFN-alpha treatment. IFN-alpha-treated mice developed a normal CTL response to HSV-1 after corneal infection, but the corneal invasiveness of the virus was markedly reduced and none of the treated mice developed corneal stromal disease. Together with our previous findings that HSV-1-specific CTLs participate in the pathogenesis of corneal stromal disease, these results indicate that i.v. injection of UV HSV-1 at the time of corneal infection may prevent stromal disease by the combined effects of IFN-mediated reduction of the spread of virus in the cornea and inhibition of the activity of the HSV-specific T lymphocytes that induce tissue destruction in the corneal stroma.

Herpes Simplex Keratitls in Patients with Acquired Immune Deficlency Syndrome

Abstract Acquired immune deficiency syndrome (AIDS) is associated with a wide spectrum of systemic and ocular infectious diseases. Little information is known about herpes simplex virus type 1 (HSV-1) keratoconjunctivitis in association with AIDS. The authors present six cases of recurrent HSV keratitis occurring in AIDS patients. Features of the herpetic keratitis in these patients included unilateral dendritic or geographic epithelial keratopathy; predilection for peripheral versus central corneal involvement; one to three recurrences per patient over a mean observation period of 17 months, with a median dendritefree interval of 7 months; and a moderately prolonged clinical course with a median healing time of 3 weeks using topical antiviral therapy. Only one of six cases had stromal infiltrative involvement. These cases raise the question of whether the immunologic abnormalities associated with AIDS may affect the clinical characteristics and course of HSV keratitis.

Concurrent regeneration of T lymphocytes and susceptibility to HSV-1 corneal stromal disease.

In these studies, mice were simultaneously depleted of CD4 and CD8 T lymphocytes (T cell-depleted) by weekly intraperitoneal injections of rat monoclonal antibodies specific for L3T4 and Lyt-2 respectively, beginning 1 day before topical corneal infection with KOS herpes simplex virus type 1 (HSV-1). Control mice were mock-depleted by weekly injections of saline. All mice developed dendritic epithelial lesions 2-3 days after infection, which healed within 2 days. Fifty percent of the mock-depleted mice developed severe HSV-1 stromal disease, which began 9-14 days after infection. No skin lesions were observed in mock-depleted mice. In contrast, none of the T cell-depleted mice developed HSV-1 corneal stromal disease through an initial 30 day observation period. These mice did develop severe periocular skin lesions. After 30 days, the T cell-depleted mice were subdivided into two groups. In one group, T cell depletion was continued and the mice remained largely free of stromal disease for an additional 30 days (one of 30 mice developed a mild stromal haze). T cell depletion was discontinued in the second group. During the subsequent 30 days the CD4 and CD8 T cells in their lymph nodes and spleens recovered to approximately 50% of normal, and 43% (13 of 30) of the mice developed severe HSV-1 stromal disease. The skin lesions healed in all T cell-depleted mice between days 30 and 60, even when T cell depletion was maintained. Our findings demonstrate that T cells are both protective (preventing the spread of HSV-1 in the skin) and detrimental (inducing the destruction of the corneal stroma).(ABSTRACT TRUNCATED AT 250 WORDS)

Ex vivo model of leukocyte migration into herpes simplex virus-infected mouse corneas

We are investigating neutrophilic infiltration into herpes simplex virus type 1 (HSV‐1) ‐infected mouse corneas. Using a novel ex vivo corneal migration assay, we demonstrated two waves of neutrophil chemotaxis in HSV‐1‐infected corneas. An early chemotactic gradient developed within 48 h in concert with the appearance of HSV‐1 epithelial lesions, peaked by 4 days post‐infection (p.i.), and degraded by 6–8 days p.i. A second chemotactic gradient appeared 10 days after infection, just before the initiation of chronic inflammation. The gradient was maintained in corneas that developed inflammation but rapidly degraded in corneas that failed to develop inflammation. The early chemotactic gradient was established in the absence of T lymphocytes, but the second chemotactic gradient was virtually abrogated by T cell depletion. We conclude that HSV‐1 infection induces two temporally separated neutrophil chemotactic gradients in the mouse cornea: an early, transient, T cell‐independent gradient; and a later, chronic, T cell‐dependent gradient. J. Leukoc. Biol. 60: 167–173; 1996.

Involvement of LFA-1 and ICAM-1 in the herpetic disease resulting from HSV-1 corneal infection

Herpes simplex virus type 1 (HSV-1) corneal infection in immunologically normal mice results in a transient epithelial lesion followed in about 2 weeks by a potentially blinding inflammatory response in the corneal stroma, and a mild blepharitis. Similarly infected T cell-deficient mice do not develop corneal stromal inflammation, but exhibit severe periocular skin disease and succumb to viral encephalitis. The role of certain adhesion molecules in both T cell activation, and in the extravasation of inflammatory cells from the blood into inflammatory sites is now being established. These studies investigated the involvement of the adhesion pair LFA-1/ICAM-1 in the disease that results from HSV-1 corneal infection in mice. Treatment of mice with mAb to LFA-1 beginning 1 day before HSV-1 corneal infection resulted in a delay in the onset of stromal inflammation, but ultimately stromal inflammation developed to a normal extent. This treatment also caused a significant exacerbation of periocular skin disease, but did not render mice susceptible to encephalitis. Treatment with mAb to ICAM-1 beginning 1 day before HSV-1 corneal infection caused an acceleration of both stromal inflammation and periocular skin disease, and rendered mice uniformly susceptible to lethal encephalitis. Treatment with either mAb beginning 6 days after HSV-1 corneal infection did not significantly affect the clinical course of herpetic disease. Our findings suggest that LFA-1 may play a role in the early phase of corneal stromal inflammation following HSV-1 corneal infection. Both LFA-1 and ICAM-1 appear to be important for protection of the skin from HSV-1 infection.(ABSTRACT TRUNCATED AT 250 WORDS)

Melanin can deplete immunosuppressive substances from the aqueous humor

Heavily pigmented eyes tend to experience greater inflammation than lightly pigmented eyes following trauma and surgery. The purpose of these studies was to test the possibilities that: (i) melanin augments T cell-responses by depleting or neutralizing anti-inflammatory substances that are normally present in the aqueous humor, and (ii) melanin augments extraocular T cell-mediated inflammatory responses. Two types of experiments were performed. First, the capacity of melanin-adsorbed and non-adsorbed rabbit aqueous humor to inhibit the proliferative response of the T cell line D10.G4.1 to IL-1 was tested. Non-adsorbed aqueous humor inhibited T cell proliferation to the background level in unstimulated cultures, whereas melanin-adsorbed aqueous humor enhanced the proliferation of stimulated, but not resting T cells. Next, mice were sensitized to the antigen conalbumin, and challenged in the ear pinna with conalbumin alone, conalbumin + melanin, or melanin alone and delayed-type hypersensitivity (DTH) was measured. Challenge with melanin alone caused some ear swelling, and melanin increased the DTH response to conalbumin. Our findings are consistent with the notion that melanin can augment intraocular inflammation by depleting or neutralizing the inhibitory components of normal aqueous humor, possibly exposing stimulatory components that are normally masked.

Presence of an enlarged pool of MOPC-315-specific cytotoxic T lymphocyte precursors in the thymuses of mice that eradicated a large MOPC-315 tumor as a consequence of low-dose melphalan therapy*

SummaryWe have previously shown that, as a consequence of low-dose melphalan (l-phenylalanine mustard) treatment, thymocytes from mice bearing a large, day-10 MOPC-315 tumor, but not thymocytes from normal mice, acquire the ability to generate an enhanced level of antitumor cytotoxicity upon in vitro stimulation with MOPC-315 tumor cells plus low concentrations (9.0–90 IU/ml) of exogenous interleukin-2 (IL-2) [23]. Here we show that the time interval between tumor inoculation and low-dose melphalan therapy as well as the magnitude of tumor burden at the time of the chemotherapy are important for the ability of the drug to render thymocytes more responsive to in vitro stimulation with MOPC-315 tumor cells plus low concentrations of recombinant IL-2 (rIL-2). Specifically, the chemotherapy was found to be effective in enhancing the thymic antitumor reactivity only if the mice bore a large, late-stage tumor. Comparison of thymocytes from untreated mice bearing a large, late-stage tumor to thymocytes from normal mice revealed that tumor-bearer thymocytes contained approximately a three-fold higher frequency of cytotoxic T lymphocyte precursors (CTLp) for MOPC-315-associated antigens. Following curative low-dose melphalan therapy of mice bearing a large, latestage MOPC-315 tumor, the frequency of CTLp for MOPC-315-associated antigens increased further, reaching a level approximately tenfold higher than that found among thymocytes of normal mice. At the same time, the frequency of CTLp for an antigenically unrelated allogeneic tumor (EL4) as well as the overall percentage of mature cells was not increased. The cells responsible for the exertion of the enhanced antitumor cytotoxicity following in vitro stimulation of thymocytes from mice treated with low-dose melphalan when they have a large, latestage MOPC-315 tumor are of the CD8+/CD4− phenotype. Thus, the enhanced level of antitumor cytotoxicity generated by thymocytes from mice that are treated with low-dose melphalan when they have a large, late-stage MOPC-315 tumor is due, at least in part, to the presence of an enlarged pool of CTLp specific for MOPC-315-associated antigens, which mature into CD8+/CD4− effector cells upon stimulation with MOPC-315 tumor cells plus low concentrations of rIL-2.

Murine trabecular meshwork cells in tissue culture

Trabecular meshwork cells from an inbred strain of mice (A/J) were established in tissue culture. Within 1 hour of enucleation, tissue containing the cornea and the chamber angle was excised and placed in tissue culture. Two to five days later, three cell types grew from the explants. Two of these cell types, corneal endothelium and fibroblasts, grew together, with the fibroblasts preferentially spreading on top of the endothelial cells. The trabecular meshwork cells extended from the explant as a distinct morphological type. The corneal endothelium and its associated fibroblasts were then removed from the culture flask with a sterile cotton swab, leaving a monolayer of pure trabecular meshwork cells. These cells required 3-4 weeks to reach confluency and could be passaged five times. They were actively phagocytic in culture and exhibited immunoreactivity to antibodies against two extracellular matrix components, laminin and collagen type IV. Mouse trabecular meshwork cells also expressed receptors for acetylated low-density lipoprotein, a property shared by trabecular meshwork cells derived from other species. The availability of trabecular meshwork cells from an inbred strain of mice will facilitate future in vivo functional studies of these cells in a syngeneic system, as well as investigations of potential immunoregulatory properties of the trabecular meshwork.

Interferon-gamma production and HLA-DR expression in patients with retinitis pigmentosa**

Other investigators have reported deficient production of interferon-gamma (IFN-gamma) and reduced expression of class II major histocompatibility (HLA-DR) antigens on monocytes from patients with retinitis pigmentosa (RP). Our previous investigation did not demonstrate deficient IFN-gamma production by lymphocytes from patients with RP. We have extended our previous study by determining the frequency of HLA-DR-positive monocytes in the peripheral blood of well-defined groups of RP patients and by including a larger sample size of patients and subdividing the autosomal dominant and recessive subpopulations. Our present results confirm and extend our previous finding that lymphocytes from patients with RP are not deficient in the production of IFN-gamma as assessed with a commercially available radioimmunoassay test kit. In addition, using two-color immunofluorescence staining and flow cytometry, we also demonstrated normal expression of HLA-DR antigens on monocytes from these patients. In both this and our previous study, using techniques employed in our laboratory, we have been unable to detect significant cell-mediated immune abnormalities in a large and well-characterized group of RP patients.