**3.4. Pro-inflammatory cytokines**

Pro-inflammatory cytokines are usually secreted by inflammatory cells in response to hypoxia or hyperglycemia [64]. Well-known pro-inflammatory cytokines, such as tumor necrosis factor, interleukin, interferon, and receptor tyrosine kinase are found to be elevated in the vitreous of diabetic retinopathy patients, suggesting their important role in the pathogenesis of this disease [5, 64, 65]. Cytokines can induce the progression of diabetic retinopathy directly and indirectly. Direct mechanisms include the direct engagement with target cells to induce neovascularization [64]. While indirect mechanisms induce leukocytes and endothelial cells to produce pro-angiogenic mediators, which in turn induce neovascularization [64, 65]. Therapy targeting these cytokines may be beneficial, but we need better understanding about the cytokine roles to do so [5].

#### *3.4.1. Tumor necrosis factor-α*

Tumor necrosis factors-α (TNF-α), a pro-inflammatory cytokine, is primarily synthesized by macrophages and T cells. Its expression is regulated by NF-κβ and it has been associated with the pathogenesis of several chronic inflammatory diseases including type 2 diabetes. Its function is primarily as an immune-modulator and it also plays a role in neovascularization and fibroplasia [3]. Costagliola et al. suggest that TNF-α is a potent mediator of leukostasis and contributes to blood-retinal barrier breakdown [3, 66]. TNF-α concentration is found elevated in the vitreous of PDR patients and the vitreous/serum ratio of TNF-α is also found higher compared to non-diabetic patients. Costagliola et al. found that TNF-α levels were lower in controls (1.9 pg/mL) than the PDR group (13.5 pg/mL) and increased with the severity of the disease [3, 66]. TNF-α has a short half-life (∼4 min), making its analyzation prone to producing false negative results. Soluble TNF-α receptors (sTNF-α-Rs) have a longer half-life, making it a more reliable marker of the activation of TNF-α system [29, 31, 67–71].

## *3.4.2. Interleukin*

Several studies have shown that there is involvement of interleukins in the development of PDR. The most common interleukins found in DR patients are IL-6 and IL-8, where their concentrations were found increased in the vitreous of patients with PDR and prolonged hyperglycemia [3, 42, 47, 72–82]. Their role in the pathogenesis of PDR is still under investigation but evidence suggests the possibility of a rather direct contribution. IL-6 controls immune cells responses by shifting T-helper cell populations, inhibiting the production of Th1 cells, promoting the differentiation of Th2 and Th17 cells, and infiltration of monocytes and T cells [9, 10, 83]. *In vitro* study of IL-6 reports its ability to increase endothelial cell and vascular cell permeability by rearranging actin filaments and by changing the shape of endothelial cells [3, 65]. Several studies state that IL-6 also plays an important role in angiogenesis by activating VEGF, and regulating expression of metalloproteinases [3, 64]. IL-8 is known to be a potent angiogenic factor and also a potent chemoattractant and activator of neutrophils and T lymphocytes [64, 84, 85]. Increase of IL-8 concentrations in PDR patients, suggest that they are upregulated in response to oxygen stress and contribute to triggering inflammatory reactions. Study by Takahashi et al. shows that there is a significant increase in IL-6 and IL-8 values in PDR patients (918.0 and 2168.0 ng/mL) compared to control patient (517.0 and 343.0 ng/mL) [85]. Elner et al. also found increased levels of IL-8 in active PDR patients (24.7 ± 4.5 ng/mL) compared to control patients (7.5 ± 2.3 ng/mL), however inactive PDR patients (11.6 ± 5.2 ng/mL) did not differ significantly from controls [79]. It is most likely that VEGF expression causes an increase of IL-8 [86]. On the other hand, IL-10 concentration is not increased in the vitreous of patients with PDR. IL-10 is another important immunoregulatory cytokine that is induced by cell hypoxia. IL-10 activates nitric oxide and increases vascular permeability during the development of PDR [3, 65, 84, 85].
