John J. Hooks

Immune Interferon in the Circulation of Patients with Autoimmune Disease

The observation that type II, or immune, interferon could be produced by peripheral-blood leukocytes in vitro on an immune-specific basis suggested that it also might be produced in vivo in various autoimmune disorders. We found immune interferon in the serums of patients with systemic lupus erythematosus, rheumatoid arthritis, scleroderma and Sjögrens syndrome. Among 28 patients with systemic lupus erythematosus, 71 per cent of those with active and 21 per cent of those with inactive disease showed interferon in their serums. Serial serum samples showed a good correlation between interferon titers and disease activity. Moreover, interferon titers correlated positively with antibodies to DNA and negatively with serum levels of the third component of complement. It is possible that the production of interferon may contribute to immunologic aberrations in auto-immune diseases and also protect the already compromised host from viral infections.

Transforming growth factor‐β induces expression of vascular endothelial growth factor in human retinal pigment epithelial cells: Involvement of mitogen‐activated protein kinases

Vascular endothelial growth factor (VEGF) is a major agent in choroidal and retinal neovascularization, events associated with age‐related macular degeneration (AMD) and diabetic retinopathy. Retinal pigment epithelium (RPE), strategically located between retina and choroid, plays a critical role in retinal disorders. We have examined the effects of various growth factors on the expression and secretion of VEGF by human retinal pigment epithelial cell cultures (HRPE). RT‐PCR analyses revealed the presence of three isoforms of mRNA corresponding to VEGF 121, 165, and 189 that were up regulated by TGF‐β1. TGF‐β1, β2, and β3 were the potent inducers of VEGF secretion by HRPE cells whereas bFGF, PDGF, TGF‐α, and GM‐CSF had no effects. TGF‐β receptor type II antibody significantly reversed induction of VEGF secretion by TGF‐β. In contrast activin, inhibin and BMP, members of TGF‐β super family, had no effects on VEGF expression in HRPE. VEGF mRNA levels and protein secretion induced by TGF‐β were significantly inhibited by SB203580 and U0126, inhibitors of MAP kinases, but not by staurosporine and PDTC, protein kinase C and NF‐κB pathway inhibitors, respectively. TGF‐β also induced VEGF expression by fibroblasts derived from human choroid of eye. TGF‐β induction of VEGF secretion by RPE and choroid cells may play a significant role in choroidal neovascularization (CNV) in AMD. Since the secretion of VEGF by HRPE is regulated by MAP kinase pathways, MAP kinase inhibitors may have potential use as therapeutic agents for CNV in AMD. Published 2003 Wiley‐Liss, Inc. J. Cell. Physiol. 197: 453–462, 2003© 2003 Wiley‐Liss, Inc.

Class II antigen expression and gamma interferon modulation of monocytes and retinal pigment epithelial cells from patients with retinitis pigmentosa

Monocytes from retinitis pigmentosa patients express diminished amounts of class II (HLA-DR) antigens in comparison to normal individuals, normal siblings of retinitis pigmentosa patients, glaucoma patients, and macular degeneration patients. This observation is correlated with a subnormal production of IFN-gamma, a potent regulator of class II antigen expression. When monocytes from retinitis pigmentosa patients are treated with IFN-gamma, the decreased expression of HLA-DR on the cell surface is restored to levels found on monocytes from normal individuals. HLA-DR antigens were detected on the surfaces of human retinal pigment epithelial cells grown in vitro and the expression of these antigens can be enhanced following IFN-gamma treatment. These data demonstrate an altered expression and regulation of class II, HLA-DR, antigens in a human eye disease. Since class II antigens and IFN-gamma are currently known to be necessary regulating proteins for efficient immune reactivity, these findings may indicate an immune disturbance in retinitis pigmentosa patients. Alternatively, this alteration may have nonimmune implications. For example, these studies may demonstrate an imbalance in systemic regulatory control systems and hence raise the possibility that these or related regulatory proteins and cell surface receptors may be instrumental in maintaining retinal integrity.

Inflammatory Cytokines Regulate MicroRNA-155 Expression in Human Retinal Pigment Epithelial Cells by Activating JAK/STAT Pathway

Inflammatory response of the retinal pigment epithelium plays a critical role in the pathogenesis of retinal degenerative diseases such as age-related macular degeneration. Our previous studies have shown that human retinal pigment epithelial (HRPE) cells, established from adult donor eyes, respond to inflammatory cytokines by enhancing the expression of a number of cytokines and chemokines. To investigate the role of microRNA (miRNA) in regulating this response, we performed microarray analysis of miRNA expression in HRPE cells exposed to inflammatory cytokine mix (IFN-γ+TNF-α+IL-1β). Microarray analysis revealed ∼11-fold increase in miR-155 expression, which was validated by real-time PCR analysis. The miR-155 expression was enhanced when the cells were treated individually with IFN-γ, TNF-α or IL-1β, but combinations of the cytokines exaggerated the effect. The increase in miR-155 expression by the inflammatory cytokines was associated with an increase in STAT1 activation as well as an increase in protein binding to putative STAT1 binding elements present in the MIR155 gene promoter region. All these activities were effectively blocked by JAK inhibitor 1. Our results show that the inflammatory cytokines increase miR-155 expression in human retinal pigment epithelial cells by activating the JAK/STAT signaling pathway.

Expression of Ia antigen on retinal pigment epithelium in experimental autoimmune uveoretinitis

Recently, human retinal pigment epithelial (RPE) cells have been demonstrated to express class II, HLA-DR, antigens both in vivo and in vitro. HLA-DR antigens were detected on RPE cells from patients with uveitis and retinitis pigmentosa. In addition, in vitro studies revealed that not only does this cell express HLA-DR antigen but also that this antigen can be modulated by the lymphokine, interferon (IFN)-gamma. In this study we evaluated the development of the murine class II, Ia, antigens on RPE cells in experimental autoimmune uveoretinitis (EAU). Ia antigen was evaluated with the avidin-biotin-peroxidase technique. Ia antigen was not detected on RPE cells from normal rats. However, Ia antigen was detected on the surface of RPE cells from EAU rats four days prior to the development of clinical and histopathological EAU. Moreover, the expression of Ia antigen on RPE cells from EAU rats continued to persist until one and one-half months after immunization. This study demonstrates that during the course of EAU the RPE cell is activated to express Ia antigens. This antigen expression may be important in the initiation and/or perpetuation of immune reactivity in the eye.

Expression of HLA-DR Antigen on Retinal Pigment Epithelial Cells in Retinitis Pigmentosa

Class II (HLA-DR) antigens are cell surface molecules that play a major role in the initiation and perpetuation of immune responses. Although most cells do not constitutively express class II antigens, selected cells can be stimulated to do so in some immunologically mediated disorders. When retinal pigment epithelial cells were evaluated by either immunoperoxidase or immunofluorescent staining of frozen eye sections from normal individuals, HLA-DR antigens were not detected. In contrast, retinal pigment epithelial cells from two patients with retinitis pigmentosa did express HLA-DR antigens. These findings demonstrated that at some time during the course of retinitis pigmentosa, the retinal pigment epithelial cell is activated to express HLA-DR.

Toxoplasma gondii Infection Induces Gene Expression and Secretion of Interleukin 1 (IL-1), IL-6, Granulocyte-Macrophage Colony-Stimulating Factor, and Intercellular Adhesion Molecule 1 by Human Retinal Pigment Epithelial Cells

ABSTRACT We have used human retinal pigment epithelial (HRPE) cultures to investigate the primary cellular responses of retinal resident cells to intracellular Toxoplasma gondii replication. At 4 days postinoculation, when all of the cells were infected, the secretion of interleukin 1β (IL-1β), IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and intercellular adhesion molecule 1 (ICAM-1) was augmented by 23-, 10-, 8-, and 5-fold, respectively, over the control. Northern and reverse transcriptase PCR analyses showed significant upregulation of steady-state levels of mRNA for IL-1β, IL-6, GM-CSF, and ICAM-1. The secretion of these molecules by HRPE cells may play a critical immunoregulatory role in the pathophysiological processes associated with T. gondii-induced retinochoroiditis.

Regulation of VEGF expression in human retinal cells by cytokines: Implications for the role of inflammation in age‐related macular degeneration

Chronic inflammation is implicated in the pathogenesis of age‐related macular degeneration (AMD). Choroidal neovascularization (CNV) observed in exudative form of AMD results in vision loss. Human retinal pigment epithelial cell (HRPE) layer and choroidal tissue are the primary pathological sites in AMD. Pathological and therapeutic evidences have strongly indicated the vascular endothelial growth factor (VEGF) molecules as critical components in CNV pathogenesis. In these studies, we used human primary HRPE and choroidal fibroblast cells (HCHF) prepared from adult donor eyes. The effects of inflammatory cytokine (IFN‐γ + TNF‐α + IL‐1β) mix (ICM) on global gene expression profiles in HRPE cells, revealed 10‐ and 9‐fold increase in VEGF‐A and VEGF‐C expression, respectively. The microarray results were validated by quantitative RT‐PCR and secretion of VEGFs proteins. IL‐1β is the most potent in inducing VEGFs secretion followed by IFN‐γ and TNF‐α, and the secretion was more effective in the presence of 2 and 3 cytokines. NF‐κB and JAK‐STAT pathway, but not HIF‐1α, Sp‐1, Sp‐3, and STAT‐3, transcription factors were upregulated and translocated to nucleus by ICM treatment. The mRNA levels of VEGF‐A and VEGF‐C and secretion of these proteins were also significantly enhanced by ICM in HCHF cells. The secretion of other angiogenic molecules, PEDF, SDF‐1α, endostatin, and angiopoietins was not affected by ICM. Our results show that the inflammatory cytokines enhance secretion of VEGF‐A and VEGF‐C by HRPE and HCHF cells. These studies indicate that VEGFs secreted by these cells initiate and promote pathological choroidal and retinal noevascularization processes in AMD. J. Cell. Physiol. 227: 116–126, 2012.

Herpes simplex virus DNA identification from aqueous fluid in Fuchs heterochromic iridocyclitis

PURPOSE To report the presence of herpes simplex virus DNA in the aqueous humor of an eye with Fuchs heterochromic iridocyclitis. METHODS In an eye with a clinical diagnosis of Fuchs heterochromic iridocyclitis, samples of aqueous humor and anterior capsule of the lens were obtained during cataract surgery. Polymerase chain reaction was performed on the samples to detect the presence of viral DNA including herpes simplex virus, varicella-zoster virus, and cytomegalovirus. Serologic analysis was also performed for antiviral immunoglobulins. RESULTS Herpes simplex virus DNA was identified in the aqueous humor but not in the anterior capsule. Serum immunoglobulin G was positive for herpes simplex virus, varicella-zoster virus, and cytomegalovirus. CONCLUSIONS The presence of herpes simplex virus DNA in the aqueous humor of an eye with Fuchs heterochromic iridocyclitis suggests that herpes simplex virus infection may play a role in the pathogenesis of Fuchs heterochromic iridocyclitis.

Immune regulation in the retina.

Immune reactivity in the retina can be critically important in inflammation and infections, but regulation of this response is essential. The retinal pigment epithelial (RPE), a unique retinal cell, displays a number of essential functions to support the health of the retina. In this review, we highlight how the RPE cell plays a pivotal role in immune defense. The RPE cell orchestrates both innate and adaptive immunity since it expresses TLRs, complement components, MHC class I and II molecules, and serves as an antigen presenting cell. Moreover, both of these immune responses result in the production of a plethora of cytokines, mainly proinflammatory. In order to counteract these inflammatory factors and silence unwanted immune reactivity, the RPE cell also generates suppressive molecules. Recently, chronic immune reactivity has been implicated in a number of retinal diseases, such as age-related macular degeneration (AMD). Current evidence suggests that the generation of excessive retinal inflammation may be the consequence of a loss of RPE immunosuppressive factors. Herein, we summarize the varied interactions of the RPE cell with the immune response and highlight how the RPE cell survives and participates in this dynamic environment.