Carol Wallace

CXCR4 receptor expression on human retinal pigment epithelial cells from the blood-retina barrier leads to chemokine secretion and migration in response to stromal cell-derived factor 1 alpha

Retinal pigment epithelial (RPE) cells form part of the blood-retina barrier and have recently been shown to produce various chemokines in response to proinflammatory cytokines. As the scope of chemokine action has been shown to extend beyond the regulation of leukocyte migration, we have investigated the expression of chemokine receptors on RPE cells to determine whether they could be a target for chemokine signaling. RT-PCR analysis indicated that the predominant receptor expressed on RPE cells was CXCR4. The level of CXCR4 mRNA expression, but not cell surface expression, increased on stimulation with IL-1β or TNF-α. CXCR4 protein could be detected on the surface of 16% of the RPE cells using flow cytometry. Calcium mobilization in response to the CXCR4 ligand stromal cell-derived factor 1α (SDF-1α) indicated that the CXCR4 receptors were functional. Incubation with SDF-1α resulted in secretion of monocyte chemoattractant protein-1, IL-8, and growth-related oncogene α. RPE cells also migrated in response to SDF-1α. As SDF-1α expression by RPE cells was detected constitutively, we postulate that SDF-1–CXCR4 interactions may modulate the affects of chronic inflammation and subretinal neovascularization at the RPE site of the blood-retina barrier.

Control of chemokine production at the blood–retina barrier

Chemokine production at the blood–retina barrier probably plays a critical role in determining the influx of tissue‐damaging cells from the circulation into the retina during inflammation. The blood–retina barrier comprises the retinal microvascular endothelium and the retinal pigment epithelium. Chemokine expression and production by human retinal microvascular endothelial cells (REC) have never been reported previously, so we examined the in vitro expression and production of monocyte chemoattractant protein‐1 (MCP‐1), regulated on activation of normal T‐cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)‐1α, MIP‐1β, interleukin (IL)‐8, epithelial cell‐derived neutrophil activating protein‐78 (ENA‐78) and growth related oncogene α (GROα) in these cells, both unstimulated and stimulated by cytokines likely to be present during the evolution of an inflammatory response. We compared this to expression and production of these chemokines in vitro in human retinal pigment epithelial cells (RPE). MCP‐1 was expressed and produced constitutively by REC but all the chemokines were produced in greater amounts upon stimulation with the proinflammatory cytokines IL‐1β and tumour necrosis factor‐α (TNF‐α). MCP‐1 and IL‐8 were produced at much higher levels than the other chemokines tested. MIP‐1α and MIP‐1β were present only at low levels, even after stimulation with IL‐1β and TNF‐α. Cytokines with greater anti‐inflammatory activity, such as IL‐4, IL‐10, IL‐13, transforming growth factor‐β (TGF‐β) and IL‐6, had little effect on chemokine production either by REC alone or after stimulation with IL‐1β and TNF‐α. RPE, although a very different cell type, showed a similar pattern of expression and production of chemokines, indicating the site‐specific nature of chemokine production. Chemokine production by REC and RPE is probably significant in selective leucocyte recruitment during the development of inflammation in the retina.

Involvement of CCR5 in the passage of Th1-type cells across the blood-retina barrier in experimental autoimmune uveitis

Although the recruitment of T helper cell type 1 (Th1)/Th2 cells into peripheral tissues is essential for inflammation and the host response to infection, the traffic signals that enable the distinct positioning of Th1/Th2 cells are unclear. We have determined the role of CC chemokine receptor 5 (CCR5) in this using experimental autoimmune uveitis (EAU) as a model system. In EAU, Th1‐like cells are preferentially recruited into the retina across the blood‐retina barrier, partly as a result of expression of the adhesion molecules P‐selectin glycoprotein ligand 1 and lymphocyte function‐associated antigen‐1 on these cells. CD3+ T cells, infiltrating the retina, also expressed the chemokine receptor CCR5, and CCR5 ligands, macrophage‐inflammatory protein‐1α (MIP‐1α), MIP‐1β, and regulated on activation, normal T expressed and secreted (RANTES), were strongly expressed in the retina at peak EAU. Th1‐like cells, polarized in vitro, expressed high levels of CCR5. The trafficking of these CCR5+ cells was examined by tracking them after adoptive transfer in real time in vivo at an early disease stage using scanning laser ophthalmoscopy. Treatment of the cells with antibody against CCR5 prior to transfer resulted in a reduction in their infiltration into the retina. However, rolling velocity, rolling efficiency, and adherence of the cells to retinal endothelium were not reduced. CCR5 is clearly important for Th1 cell recruitment, and this study demonstrates for the first time in vivo that CCR5 may act at the level of transendothelial migration rather than at the earlier stage of rolling on the endothelium.

The mitochondria targeted antioxidant MitoQ protects against fluoroquinolone-induced oxidative stress and mitochondrial membrane damage in human Achilles tendon cells

Tendinitis and tendon rupture during treatment with fluoroquinolone antibiotics is thought to be mediated via oxidative stress. This study investigated whether ciprofloxacin and moxifloxacin cause oxidative stress and mitochondrial damage in cultured normal human Achilles’ tendon cells and whether an antioxidant targeted to mitochondria (MitoQ) would protect against such damage better than a non-mitochondria targeted antioxidant. Human tendon cells from normal Achilles’ tendons were exposed to 0–0.3 mm antibiotic for 24 h and 7 days in the presence of 1 µm MitoQ or an untargeted form, idebenone. Both moxifloxacin and ciprofloxacin resulted in up to a 3-fold increase in the rate of oxidation of dichlorodihydrofluorescein, a marker of general oxidative stress in tenocytes (p<0.0001) and loss of mitochondrial membrane permeability (p<0.001). In cells treated with MitoQ the oxidative stress was less and mitochondrial membrane potential was maintained. Mitochondrial damage to tenocytes during fluoroquinolone treatment may be involved in tendinitis and tendon rupture.

Effect of anti-macrophage inflammatory protein-1α on leukocyte trafficking and disease progression in experimental autoimmune uveoretinitis

This study has enabled us to identify the influence of the chemokine, macrophage inflammatory protein‐1α (MIP‐1α), on leukocyte behavior at the blood‐retina barrier in vivo and its link with the inflammatory process and disease pathogenesis. MIP‐1α has not previously been thought to be effective under conditions of physiological shear flow. However, short‐term anti‐MIP‐1α treatment inhibited leukocyte slowing and accumulation and subsequent extravasation of leukocytes at the blood‐retina barrier in animals with experimental autoimmune uveoretinitis. This was effective predominantly in the post‐capillary venules which have been shown to be the main site of passage of leukocytes across the blood‐retina barrier. Long‐term anti‐MIP‐1α treatment also prevented decreased leukocyte velocity and reduced disease severity as measured clinically, histologically and in terms of blood‐retina barrier breakdown.

Cytokine regulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) production by human retinal pigment epithelial cells

GM‐CSF is an important regulator of macrophage, granulocyte and dendritic cell behaviour and function. These cell types have been implicated in the retinal damage characteristic of endogenous posterior uveitis. Dendritic cells in the choroid have access to retinal antigens processed by the retinal pigment epithelial (RPE) cells of the blood–retinal barrier and are thought to be candidates for the presentation of antigen in uveoretinitis. We therefore investigated the production of GM‐CSF and its regulation in human RPE cells. IL‐1β, tumour necrosis factor‐alpha (TNF‐α) and transforming growth factor‐beta (TGF‐β) all stimulated GM‐CSF production by RPE cells and a combination of these cytokines increased GM‐CSF production over five‐fold compared with that with the individual cytokines alone. Interferon‐gamma (IFN‐γ) rapidly down‐regulated these responses. IFN‐γ did not appear to be acting directly on IL‐1β or via the synthesis of another protein. GM‐CSF mRNA expression showed the same pattern of response to these cytokines, indicating transcriptional or pre‐transcriptional regulation, and there was no evidence that IFN‐γ was acting by destabilizing GM‐CSF mRNA. These results are generally important in understanding the ways in which cytokine regulation differs between different cell types and also more specifically for determining ways in which a cytokine with a significant role in the development of autoimmune uveoretinitis may be manipulated.

CX3CR1‐deficiency is associated with increased severity of disease in experimental autoimmune uveitis

The role of CX3CR1 in regulating the function of monocytes and microglia was examined in mice in which CX3CR1 had been replaced by green fluorescent protein (GFP). Induction of experimental autoimmune uveitis (EAU) in these mice resulted in increased disease severity at day 23 postimmunization with uveitogenic peptide when compared with CX3CR1‐positive mice and increased apoptosis of neuronal cells in the inner nuclear layer. Resident microglia within the retina were activated equally as EAU developed in mice with or without CX3CR1, as determined by changes in morphology, suggesting that the microglial cell response did not account for the differences. Although the inflammatory infiltrate had increased in mice without CX3CR1 at day 23 postimmunization, the percentage of natural killer cells in the infiltrate was not changed in these mice. Similarly, increased disease severity at this stage was not associated with an overall increased percentage of macrophages in the retinal inflammatory infiltrate or in increased activation of these cells. The increased recruitment of monocytes to the retina in response to EAU induction in CX3CR1GFP/GFP mice compared with CX3CR1GFP/+ mice was not reflected in increased migration away from vessels, leading to marked clustering of GFP+ cells around veins and venules in these mice. It is possible that this monocyte/macrophage clustering leads to the increased severity of disease seen in the mice by focusing and so intensifying the inflammatory response.

Development of Experimental Autoimmune Uveitis: Efficient Recruitment of Monocytes Is Independent of CCR2

PURPOSE Macrophages are major contributors to the damage occurring in the retina in experimental autoimmune uveitis (EAU). CCR2 may be needed for efficient recruitment of monocytes to an inflammatory site, and the aim of this study was to determine whether this was the case in EAU. METHODS EAU was induced and graded in C57BL/6J and CCR2(-/-) mice. Macrophage infiltration and CCR2 expression were assessed using immunohistochemistry. Retinas were examined for MCP-1 expression using RT-PCR. Rolling and infiltration of labeled bone marrow monocytes at the inflamed retinal vasculature were examined by scanning laser ophthalmoscopy and confocal microscopy, respectively. Effect of CCR2 deletion or blockade by antibody and antagonist was determined. RESULTS Expression of mRNA for MCP-1 increased as EAU developed and was localized to the retina. CCR2 was associated with infiltrating macrophages. However, EAU induced in CCR2(-/-) mice was not reduced in severity, and neither was the percentage of macrophages in the retina. CCR2(-/-) monocytes, 48 hours after adoptive transfer to mice with EAU, showed no significant difference in percentage rolling or infiltration into the retina compared to WT. CCR2-independent rolling of monocytes was confirmed by CCR2 neutralizing antibody and antagonist treatment. CONCLUSIONS CCR2 does not have a primary role in the recruitment of monocytes to the inflammatory site across the blood-retina barrier in well-developed EAU. Therapeutics targeting CCR2 are unlikely to be of value in treating human posterior uveitis.

Recognition of DHN-melanin by a C-type lectin receptor is required for immunity to Aspergillus

Resistance to infection is critically dependent on the ability of pattern recognition receptors to recognize microbial invasion and induce protective immune responses. One such family of receptors are the C-type lectins, which are central to antifungal immunity. These receptors activate key effector mechanisms upon recognition of conserved fungal cell-wall carbohydrates. However, several other immunologically active fungal ligands have been described; these include melanin, for which the mechanism of recognition is hitherto undefined. Here we identify a C-type lectin receptor, melanin-sensing C-type lectin receptor (MelLec), that has an essential role in antifungal immunity through recognition of the naphthalene-diol unit of 1,8-dihydroxynaphthalene (DHN)-melanin. MelLec recognizes melanin in conidial spores of Aspergillus fumigatus as well as in other DHN-melanized fungi. MelLec is ubiquitously expressed by CD31+ endothelial cells in mice, and is also expressed by a sub-population of these cells that co-express epithelial cell adhesion molecule and are detected only in the lung and the liver. In mouse models, MelLec was required for protection against disseminated infection with A. fumigatus. In humans, MelLec is also expressed by myeloid cells, and we identified a single nucleotide polymorphism of this receptor that negatively affected myeloid inflammatory responses and significantly increased the susceptibility of stem-cell transplant recipients to disseminated Aspergillus infections. MelLec therefore recognizes an immunologically active component commonly found on fungi and has an essential role in protective antifungal immunity in both mice and humans.Resistance to infection is critically dependent on the ability of pattern recognition receptors to recognize microbial invasion and induce protective immune responses. One such family of receptors are the C-type lectins, which are central to antifungal immunity. These receptors activate key effector mechanisms upon recognition of conserved fungal cell-wall carbohydrates. However, several other immunologically active fungal ligands have been described; these include melanin, for which the mechanism of recognition is hitherto undefined. Here we identify a C-type lectin receptor, melanin-sensing C-type lectin receptor (MelLec), that has an essential role in antifungal immunity through recognition of the naphthalene-diol unit of 1,8-dihydroxynaphthalene (DHN)-melanin. MelLec recognizes melanin in conidial spores of Aspergillus fumigatus as well as in other DHN-melanized fungi. MelLec is ubiquitously expressed by CD31+ endothelial cells in mice, and is also expressed by a sub-population of these cells that co-express epithelial cell adhesion molecule and are detected only in the lung and the liver. In mouse models, MelLec was required for protection against disseminated infection with A. fumigatus. In humans, MelLec is also expressed by myeloid cells, and we identified a single nucleotide polymorphism of this receptor that negatively affected myeloid inflammatory responses and significantly increased the susceptibility of stem-cell transplant recipients to disseminated Aspergillus infections. MelLec therefore recognizes an immunologically active component commonly found on fungi and has an essential role in protective antifungal immunity in both mice and humans.

Cutting Edge: Failure of Antigen-Specific CD4+ T Cell Recruitment to the Kidney during Systemic Candidiasis

Candida albicans is the leading cause of systemic candidiasis, a fungal disease associated with high mortality and poor treatment options. The kidney is the target organ during infection and whose control is largely dependent on innate immunity, because lymphocytes appear redundant for protection. In this article, we show that this apparent redundancy stems from a failure of Ag-specific CD4+ T cells to migrate into infected kidneys. In contrast, Ag-specific CD8+ T cells are recruited normally. Using Ag-loaded immunoliposomes to artificially reverse this defective migration, we show that recruited Ag-specific CD4+ T cells polarize toward a Th17 phenotype in the kidney and are protective during fungal infection. Therefore, our data explain the redundancy of CD4+ T cells for defense against systemic infection with C. albicans and have important implications for our understanding of antifungal immunity and the control of renal infections.