Inflammatory Cytokines Induce Expression of Chemokines by Human Retinal Cells: Role in Chemokine Receptor Mediated Age-related Macular Degeneration

: Chemokine reeptor-3 (CCR-3) was shown to be associated with choroidal neovascularization (CNV) in age-related macular degeneration (AMD). AMD is a vision threatening retinal disease that affects the aging population world-wide. Retinal pigment epithelium and choroid in the posterior part of the retina are the key tissues targeted in the pathogenesis of CNV in AMD. We used human retinal pigment epithelial (HRPE) and choroidal fibroblast (HCHF) cells, prepared from aged adult human donor eyes, to evaluate the expression of major CCR-3 ligands, CCL-5, CCL -7, CCL-11,CCL-24 and CCL-26. Microarray analysis of gene expression in HRPE cells treated with inflammatory cytokine mix (ICM= IFN-  +TNF-  +IL-1  ) revealed 75 and 23-fold increase in CCL-5 and CCL-7 respectively, but not CCL-11, CCL-24 and CCL-26. Chemokine secretion studies of the production of CCL5 and CCL7 by HRPE corroborated with the gene expression analysis data. When the HRPE cells were treated with either individual cytokines or the ICM, both CCL-5 and CCL-7 were produced in a dose dependent manner. Similar to the gene expression data, the ICM did not enhance HRPE production of CCL-11, CCL-24 and CCL-26. CCL-11 and CCL-26 were increased with IL-4 treatment and this HRPE production was augmented in the presence of TNF-  and IL1  . When HCHF cells were treated with either individual cytokines or the ICM, both CCL-5 and CCL-7 were produced in a dose dependent fashion. IL-4 induced low levels of CCL-11 and CCL-26 in HCHF and this production was significantly enhanced by TNF-  . Under these conditions, neither HRPE nor HCHF were demonstrated to produce CCL-24. These data demonstrate that chronic inflammation triggers CCL-5 and CCL-7 release by HRPE and HCHF and the subsequent interactions with CCR3 may

Aging and Disease • Volume 6, Number 6, December 2015 445 AMD [11][12][13][14][15][16]. In addition, the involvement of inflammatory molecules produced by infiltrating leukocytes has been shown to play a key role in the pathophysiological processes of CNV in AMD [9][10][11][12]. RPE and choroidal tissues, located behind the neuroretina, are the primary targets associated with CNV and AMD [4,8,10,12,17]. The RPE with its highly organized basement membrane, known as Bruch's membrane, is essential for normal functioning of the retina [18,19]. The RPE acts as a blood retinal barrier and regulates, a) transport of fluids, nutrients and wastes from the choroid to the retina and vice versa b) engulfment and digestion digest photoreceptor outer segments c) secretion of extracellular matrix materials for retinal adhesion and maintenance of the integrity of Bruch's membrane [18,19]. RPE secretes a number of growth factors, cytokines and chemokines and participates in the regulation of physiology and patho-biology of neuroretina and choroid [20][21][22][23][24][25][26][27][28].
The presence of lymphocytes, macrophages, complement factors C3a and C5, matrix-metalloproteases and other inflammatory molecules in CNV membranes suggests a strong association between inflammatory reactions and AMD [8][9][10][11][12]. An association between genetic polymorphisms in complement factor H and AMD patients also underscore a role of immune system in AMD pathogenesis [7,8]. The trafficking of inflammatory cells to the RPE and choroidal compartments may produce a number of cytokines and chemokines that would further activate resident RPE and choroidal cells to initiate a cascade of events that trigger the pathogenic processes. Recently, we reported that inflammatory cytokines, IFN-, TNF- and IL-1 enhance the secretion of vascular endothelial growth factors (VEGF-A and VEGF-C) by human RPE and choroidal fibroblast cells [21,27,28]. These results suggest a direct link among inflammation, VEGF secretion and neovascularization.
In this study we used human retinal pigment epithelial and choroidal fibroblast cell cultures prepared from adult donor eyes to evaluate the expression and production of CCR-3 ligands CCL-5, -7, -11, -24, and 26. Here, we report that inflammatory cytokines IFN-, TNF-, IL-1 and IL-4 induce CC ligands, predominantly CCL-5 and CCL-7.

Cell Cultures
Aging and Disease • Volume 6, Number 6, December 2015 446 Adult human donor eyes (70-85y old), obtained from Eye Banks, were used for the preparation of human retinal pigment epithelial (HRPE) and choroidal fibroblast (HCHF) cells [20,23,27]. HRPE and HCHF cells were grown in minimum essential medium supplemented with 10% fetal bovine serum, non-essential amino acids, penicillin (100 u/ml), streptomycin (100 ug/ml) and amphotericin B (25 ng/ml). All of HRPE cells reacted positively to immune-staining with cytokeratin, cytoskeletal protein marker for epithelial cells. HRPE cell cultures at passages 7 to 10 and HCHF cell cultures at passages 4 to 6 were used in these studies.

Microarray analysis of gene expression in HRPE cells treated with cytokines
Confluent cultures of HRPE cells grown in 100 mm dishes (Falcon, Primaria) were treated with inflammatory cytokine mix (IFN- (100 u/ml), TNF- (10 ng/ml) and IL-1 (10 ng/ml) for 8 h in serum free medium. Purity and integrity of total RNA prepared was verified by absorption spectra and in Agilant 2100 Bioanalyzer. Affymetrix GeneChip arrays (HG U133 plus 2.0) were used for global gene expression profiling. All procedures were performed according to the detailed protocol provided by Affymetrix (Santa Clara, CA) and as described earlier for HRPE [26]. Affymetrix GeneChip Operating Software (GCOS) and GeneSpring software (Silicon Genetics /Agilant, CA) were used for normalizing the gene expression analysis and for comparison between control and treated samples.

Effect of inflammatory cytokines on CCL-5, CCL-7, CCL-11, CCL-24 and CCL-26 secretion by HRPE and HCHF cells
HRPE and HCHF cultures were grown to confluence in 24 well culture plates in 10% FBS containing media. To avoid the effects and interactions of various growth factors, cytokines and other proteins present in the serum, all treatments were performed in serum free medium. The cultures were washed once with serum-free medium (SFM) and left in SFM for 5-6 h. Then media were removed and SFM containing various concentrations and combinations of inflammatory cytokines IFN-, TNF- and IL-1 or inflammatory cytokine mix (ICM) were added to the wells (1 ml/well). Cultures were incubated for 24h and culture supernatant fluids collected were frozen at -70 0 C.

Effect of other cytokines and growth factors on CCL-11, -24 and -26 secretion by HRPE and HCHF cells
Since the individual inflammatory cytokines or ICM did not induce the production of eotaxins (CCL-11, -24 and -26) by HRPE and HCHF cells, we examined the effects of a number of other cytokines and growth factors. HRPE and HCHF cells grown to confluence in 24 well plates were treated with a number of cytokines such as interleukin (IL) -2, -4, -5, -6, -8, -10, -11, -12, -13, and -18, and growth factors TGF-, bFGF, PDGF, EGF-1 and TGF-. Since IL-4 induced the secretion of CCL-11 and CCL-26, we studied the effect of ICM on IL-4 induced CCL-11 and CCL-26 secretion. CCL-24 secretion was not observed under any of these conditions.

Effect of IL-4 on inflammatory cytokine induced CCL-5, CCL-7, CCL-11and CCL-26 secretion by HRPE and HCHF cells
Next, we sought to examine the effect of ICM in the presence of IL-4. Cultures were incubated with 10 ng/ml of IL-4 and IFN- (100u/ml), TNF- (10 ng/ml) or IL-1 (10 ng/ml) for 24h in serum free medium. Culture supernatants collected were used for the analysis of CCL-5 and CCL-7 by Elisa.

Statistical analysis
Comparisons were made among the various treatment groups belonging to the same batch of cells grown under similar conditions. Statistical analysis of secreted chemokines by HRPE and HCHF cells treated with various agents were performed by Students "T" test. "P" values less than 0.05 were considered as significant.

Microarray analysis of gene expression by HRPE treated with inflammatory cytokines
We used the Affymetrix GeneChip human genome U133 plus 2.0 to evaluate the global gene expression profiles in HRPE cells that were treated with inflammatory cytokine mix (ICM) containing IFN-, TNF- and IL-1. About 1400 genes were up or down regulated by more than 2 fold (data not shown). The gene expression profile of chemokines presented as fold changes are shown in Table.1. CCR -3 ligands CCL-5 and CCL-7 were upregulated by 75 and 23 fold respectively. However, the expression of other CCR-3 ligands CCL-8, -13, -15 and eotaxins (CCL-11, -24 and -26) was not observed under these conditions. To validate these results, we conducted detailed experiments to study secretion of CCR-3 chemokine ligand proteins. Aging and Disease • Volume 6, Number 6, December 2015 449

Inflammatory cytokines induce CCL-5 and CCL-7 secretion by HRPE cells
All experiments were performed in serum free medium to avoid the interference of cytokines, growth factors and other constituents present in fetal bovine serum. HRPE cells did not secrete CCL-5 (RANTES) and CCL-7 (MCP-3) constitutively. IFN- did not influence CCL-5 secretion, but significantly enhanced TNF- and IL-1 induced CCL-5 secretion (Fig.1A). On the other hand, IL-1 is the most potent inducer of CCL-7 secretion. TNF- is the second most potent inducer and IFN- is the least potent inducer of CCL-7 (Fig.1B). The addition of TNF- or IFN- to IL-1 had no significant effect on CCL-7 secretion. In these studies, we used IFN- (100 u/ml), TNF- (10 ng/ml) and IL-1 (10 ng/ml) concentrations (ICM 10x). Then, we sought to evaluate the effects of ICM at lower concentrations (ICM, 1x; ICM, 0.1x and ICM, 0.01x) to simulate the conditions under various pathological conditions. At ICM 0.1x, there was a significant secretion of CCL-5 and CCL-7 by HRPE cells compared to untreated cells (Fig.1C, D). These data demonstrate that low concentrations of inflammatory cytokines can stimulate HRPE cells to produce CCL-5 and CCL-7.

Inflammatory cytokines induce CCL-5 and CCL-7 secretion by HCHF cells
Choroidal tissue is the primary site to initiate neovascularization from choriocapillaris in CNV in AMD [4,8,17,33]. CCL-5 and CCL-7 were not secreted constitutively by HCHF. TNF- or IL-1 induced the secretion of CCL-5 and this was substantially enhanced when TNF- and IFN- were present together (Fig. 4A). CCL-7 secretion was induced by IL-1 and this was substantially enhanced in the presence of IFN- (Fig. 4B).
We also evaluated the dose responses to ICM at lower concentrations (1x, 0.1x and 0.01x). Even at low concentrations of ICM (0.01x), there was a significant secretion of CCL-5 and CCL-7 (Fig. 4C, D). These results show that HCHF cells are more sensitive to stimulation with ICM than HRPE cells.

IL-4 induces secretion of CCL-11 and CCL-26 but not CCL-24 by HCHF cells
When exposed to 10 ng/ml of IL-4, HCHF induced both CCL-11 and CCL-26 (Fig. 5A, B). These chemokines were not produced at a concentration of 1 ng/ml of IL-4(data not shown). HCHF produced higher levels of CCL-11 and CCL-26 compared to HRPE. The synergistic effects of TNF- and IL-1 were observed on IL-4 induced secretion of CCL-11 by HCHF cells (Fig.5A). In the case of CCL-26, TNF- and IL-1 slightly enhanced IL-4 induced secretion that was not statistically significant (Fig.5B). IFN- had no effect on CCL-11 and CCL-26 secretion by HCHF cells (data not shown). The secretion of CCL-24 was not detected under any of these conditions by HCHF cells. CCR3 expressed on eosinophils, basophils, mast cells and lymphocytes plays a critical role in the allergic reactions in skin, lungs and other tissues [36][37][38][39]42]. CCR3 is also expressed on microglial, dendritic and airway epithelial and endothelial cells [31,32,43]. CCR3 is known to be highly promiscuous and can bind to a number of CC ligands with varying affinities [31,32,38,44]. CCL-11, 24 and 26 are the ligands with specific and potent binding affinities only for CCR3 while CCL-5 and CCL-7 can also bind to CCR1, CCR2, and CCR5 in addition to CCR3 [31,32,38,44]. CCR3 with its ligands CCL11 and CCL24 has been reported to play an essential role in eosinophil recruitment to the lungs and gut mucosa [32,40,42].
The role of CCR3 in developmental or pathological angiogenesis has received very little attention [33,35,45,46]. Expression of CCR3 in retinal and choroidal tissues has not been reported in normal healthy conditions. In our previous study, we demonstrated that HRPE cells expressed CCR1 but not CCR3 or other CC receptors. [22]. Using immune-histochemical staining, Takeda et al [33] demonstrated the presence of CCR3 proteins on endothelial cells of choroidal blood vessels obtained from AMD patients but not in age-matched control specimens. In these AMD patients, the presence of CCR3 ligands, CCL-11, CCL-24 and CCL-26, was also demonstrated by immunostaining in choroidal tissue stroma primarily consisting of fibroblasts [33]. Expression of CCL-11, CCL-24 and CCL-26 mRNA in human RPE cells was shown by RT-PCR, but the secretion of these eotaxins by HRPE was not demonstrated [33]. Microarray analysis of global gene expression profile of HRPE cells treated with inflammatory cytokine mix did not detect mRNA expression of any of the eotaxins (Table 1). We performed additional secretion studies to validate these results. The secretion of eotaxins by HRPE and HCHF cells was not detected constitutively or in the presence of inflammatory cytokines (Fig. 2, 5). IL-4, only at high concentrations (10 ng/ml) but not at low concentrations (1 ng/ml), induced secretion of CCL-11 and CCL-26.
The promiscuity of CCR3 reactivity to various ligands suggests potential involvement of ligands other than eotaxins in CNV in AMD [32,44]. The expression of CCL-5 and CCL-7, major ligands with binding affinities for CCR3 have not been examined in CNV and retinal pigment epithelium tissues in AMD patient specimens [33,35,46]. Microarray studies with HRPE cells showed several fold increases in CCL-5 and CCL-7 mRNA upon treatment with inflammatory cytokine mix ( Table 1). Both HRPE and HCHF cells produced significant quantities of CCL-5 and CCL-7 proteins in the presence of low concentrations of inflammatory cytokines (Fig 1 & 4). The secretion of CCL-5 and CCL-7 was also observed in RPE cells prepared from human fetal and adult donor eyes [22,25,47]. Therefore, it is likely that CCL-5 and CCL-7 produced under inflammatory conditions could act as ligands for CCR3 in pathological angiogenesis in CNV. The binding affinities of CCL-5 and CCL-7 to CCR3 are comparable to eotaxins in endothelial cells present in some tissues [43,48]. The affinities of choroidal endothelial cell CCR3 to its various ligands are not known. Since the secretion of CCL-5 and CCL-7 by HRPE and HCHF cells is greater than a hundred fold under inflammatory conditions, CCL-5 and CCL-7 may be ligands associated with CCR3 mediated CNV in AMD [33,35,46]. Recent study has shown increased expression of CCR3 in retina and RPE/choroid tissues of aged (91 years) human donor eyes compared to young (25 years) human donor eyes [49]. Thus enhanced CCR3 expression in retinal and choroid tissue in aged human eyes may be accessible to interaction with CCL5 and CCL7 chemokines produced during inflammatory conditions. CCL-5 and CCL-7 ligands can interact effectively with other chemokine receptors such as CCR1, CCR2 and CCR5 [31,32]. CCL-5 and CCL-7 may play role in pathophysiological angiogenic processes by enhancing the homing of endothelial progenitor cells and by influencing expression of angiogenesis associated genes [50][51][52][53][54][55]. CCR-1 and CCR-5 are expressed both on several hematopoietic and non-hematopoietic cells [31,32]. In addition, CCR-1 and CCR-5, reported to be expressed on endothelial, epithelial and smooth muscle cells, play roles in neovascularization and wound healing [32,48,51,53]. Inflammatory cytokines TNF- and IFN- are known to upregulate CCR2 and CCR5 on endothelial cells and macrophages [43,48]. However, the expression of CCR1, CCR2 and CCR5 on choroidal endothelial cells in normal or in AMD patients is unknown [33,49]. It is possible that CCL-5 and CCL-7 ligands may be associated with Aging and Disease • Volume 6, Number 6, December 2015 453 CCR3 and other CCR receptors in CNV processes in AMD. Inflammatory reactions are widely thought to be one of the initiating and /or promoting factors in CNV in AMD [56][57][58][59]. Recent studies on the role of chemokines and their receptors in CNV in AMD [60][61][62] indicate potential new avenues for exploration of chemokine targeting in search of AMD therapies. We demonstrate in this report that cytokines associated with chronic inflammation trigger CCL-5 and CCL-7 release by HRPE and HCHF. The interactions of these ligands with CCR3 may participate in the pathologic processes in AMD.