In the current study, we first performed a broad cytokine array test containing 48 different cytokines, and some highlighted cytokines levels (IL-8, GROα, and ENA-78) were validated by ELISA analysis to predict Omalizumab response in CSU patients compared with the control. Additionally, we compared the changes in these protein levels for age classifications. According to the findings, different cytokines regulated Omalizumab response in CSU patients. However, age could affect the levels after Omalizumab treatment among CSU patients. We first determined that some cytokines significantly increased following Omalizumab in CSU patients compared with the control. Therefore, we mainly focused on increased protein levels in this study to assess the predictor role of cytokines in response to omalizumab. Furthermore, we conducted an ELISA analysis to verify the array results. Our results indicated that IL-8, GROα, and ENA-78 serum levels significantly decreased over 45 years of age in CSU patients following Omalizumab treatment (p < 0.05). However, these protein levels were not reduced among younger CSU patients, and we observed an increase in the T1 group.
High-affinity IgE receptor (FcƐRI) or functional antibodies against IgE activate basophils in the vessel and mast cells of the tissue, causing the release of many inflammatory mediators, chemokines, and cytokines. At the same time, the activation of the complement cascade (for example, through the production of anaphylatoxin C5a and eosinophils initiates the coagulation pathway via tissue factor [10]. Mast cells communicate with other inflammatory cells via interleukins, chemokines, and growth factors and provide the connection between innate and acquired immunity [11, 12]. Therefore, these parameters could have a potential role in the pathogenesis of CSU patients.
IL-8, as a proinflammatory cytokine, is a small (8kDa) soluble protein belonging to the CXC chemokine subfamily and is mainly secreted by monocytes, macrophages, fibroblasts, keratinocytes, and endothelial cells under inflammatory stimulation [13–15]. Additionally, IL-8 level is also secreted by lymphocytes, epithelial cells, and tumor cells [14, 15]. Recruitment of inflammatory neutrophils and basophils via IL-8 leads to the stimulation of histamine release, histamine receptors increasing the production of proinflammatory cytokines and chemotactic factors, and cell-receptor interaction [11, 14, 16]. For its biological activity, IL-8 binds to two cell surface G protein-coupled receptors, CXCR2 [13]. Platelet-derived GROα and ENA-78 play a role in ensuring the selectivity of IL-8 in chemotaxis via the CXCR2 receptor [14]. While these receptors exert the most robust response on neutrophils, they are also effective in monocytes, basophils, and eosinophils [8]. GROα is an approximately eight kDa proinflammatory chemokine key in neutrophil migration and activation. GROα interacts with CXCR2 on endothelial cells in the circulatory system to promote lymphatic tube formation and angiogenesis [17, 18]. ENA-78 is an eight kDa proinflammatory member of the CXC subfamily of chemokines [19]. GROα shares some sequence identity with IL-8. Inflammatory stimulation increases ENA-78 production in multiple hematopoietic cell types, fibroblasts, endothelial cells, and vascular smooth muscle cells. In vivo, ENA-78 is elevated at inflammation and pulmonary fibrosis sites, where it promotes neutrophil infiltration, activation, and angiogenesis [20–22]. Following this information, a significant association among these protein levels could be a significant predictor marker in CSU patients to assess omalizumab efficacy.
Indeed, it is hypothesized that platelets may play a role in the inflammatory response associated with CSU, representing the link between inflammation, coagulation, and histamine release [16, 23]. This activation of platelets is thought to be related to the chemokines IL-8, GROα, and ENA-78 [14]. A few studies have reported that platelet indices such as D-Dimer show significant changes in CSU patients in correlation with clinical severity, autoreactivity, and/or inflammatory status [24]. In our data, a decrease in IL-8, GROα, and ENA-78 in correlation with treatment and their correlation with D-dimer value suggest that platelet activation could be more involved in the CSU pathogenesis in cases over 45 years of age.
In a study conducted with asthmatic patients, mast cell chymase changes the expression of adhesion molecules and induces the expression of the chemokine IL-8, GROα, CXCL5, CXCL6, and CCL2 [11]. In our study, IL-8, GROα, and ENA-78 in CSU patients significantly decreased in correlation with omalizumab treatment in the patient group over 45 years of age. In comparison, no significant change was observed in the patient group under 45 years. On the other hand, the recovery status of these two groups of patients (under and over the age of 45) was similar, as shown by the UAS and UCT scores. The data obtained could be potentially useful for both the pathogenesis of CSU and the importance of changes in these cytokine levels depending on the age of patients. Therefore, although the clinical outcome does not change between young and middle-aged patient groups, the processes involved in pathogenesis may work differently. The factor that negatively affects the understanding of the pathogenesis of CSU and, indirectly, the number of patients could affect the obtained results. Therefore, further studies should be required in a large number of CSU patients compared to the control group to identify the role of age in CSU pathogenesis and the association between Omalizumab response and cytokines level.
Furthermore, plasma levels of vascular endothelial growth factor (VEGF), secreted by eosinophils, mast cells, and basophils, are increased in patients with CSU and associated with disease severity. It is also mentioned that VEGF induces mast cell production. Recently, it has also been shown that IL-8 plays an essential role in angiogenesis by activating the VEGF pathway [25]. VEGF is the most potent direct-acting regulator of angiogenesis, inducing proliferation, migration, and tube formation of endothelial cells [26]. In this pathway, IL-8 induces VEGF expression in endothelial cells, and VEGF acts as an autocrine and paracrine factor on vascular endothelium. Activation of endothelial cells is significant in the pathogenesis of CSU due to increased permeability, cellular infiltration, and vascular remodeling. CSU is also associated with elevated plasma levels of markers of thrombin generation [27]. These findings shed light on histamine-independent mechanisms that play a role in vascular leakage in CSU [27]. We understand that processes other than histamine released from the mast cell may be involved in the pathogenesis of complex CSU disease, which is the prototype of mast cell-mediated diseases. Lattanzio and colleagues hypothesize that this interaction of IL-8 and VEGF synergizes to induce and maintain angiogenesis [27]. Transcriptional analysis by Buhl et al. indicates that the upregulation of IL-8 and other chemokines, including CXCL1 and CXCL5 in rosacea, play a role in the inflammation step via angiogenic properties [28, 29]. The results of our study support the literature in terms of the elevation of IL-8 and platelet-derived GRO-α and ENA-78 in the CSU patients over 45 years of age.