Cytokine Analysis: A Fresh Perspective

Introduction

Cytokines are small proteins secreted by multiple cells in the body, may act by autocrine or paracrine or endocrine action, and do not have a single target organ unlike hormones. It is recognized that cytokines display the following properties: pleiotropism, redundancy, synergism, and antagonism. They have role in regulation of immunity, inflammation, hematopoiesis, tumor regulation, regulation of blood vessel formation, and so on; some cytokines playing multiple roles (pleiotropism) and some cytokines sharing the same action (redundancy). It is also recognized that a balance of different cytokines (synergistic versus antagonistic cytokines) determines the outcome in regulation, either stimulation or inhibition.

Need for Fresh Perspective on Cytokine Analysis

Cytokines are secreted in response to a stimulus leading to their effect on target cell. Some cytokines promote an action, whereas others oppose the action. In a normal individual, a few cytokines carry out certain actions at very low concentrations, whereas there are other cytokines maintaining an equilibrium between opposing actions (Srinivasan and Kilpatrick 2017). Loss of balance can lead to disease and development of disease can alter the balance between cytokines. With such a complex relationship of cytokines, comparing only the absolute cytokine levels is not advisable. It is also not appropriate for the following reasons. First, variable secretion of cytokines: Not all cytokines are present in systemic circulation. Some are detectable in circulation only when they are amplified by 10- to 1,000-fold due to underlying disease. Others, although detectable, have a highly variable secretion between individuals. Second, a single cytokine is secreted by multiple cells. Third, a single cytokine plays multiple roles depending on the circumstance and is influenced by various other secretions by cells. Fourth, cytokines having opposing actions maintain a balance and genetic predisposition determines the tilt of this balance leading to disorders (Srinivasan and Kilpatrick 2017). Therefore, it would be appropriate to consider the balance or relation of cytokines having opposing actions and consider cytokine ratios instead of only absolute values of cytokines (Biswas and others 2010; de Brito and others 2016; Miranda and others 2019).

This article proposes to present a brief review of multiple actions of cytokines and possible ratios that ought to be compared to deduce the true level of balance with regard to regulation of various actions.

Proinflammatory Versus Anti-Inflammatory Cytokines

The following cytokines have been found to have proinflammatory role: interleukin (IL)-1, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-23, granulocute monocyte-colony stimulating factor (GM-CSF), monocyte chemoattractant protein (MCP)-1, macrophage inflammatory proteins (MIP)-1α, MIP-1β, regulated on activation, normal T cell expressed and secreted (RANTES), CXC chemokine ligands (CXCL)-1 (growth-related oncogene alpha [GROα]), CXCL-5 (epithelial neutrophil activating peptide [ENA]-78), CXCL-8 (IL-8), CXCL-9 (monokine induced by gamma interferon [MIG]), CXCL-10 (interferon gamma-induced protein 10 [IP-10]), CXCL-11 (I-TAC), CC chemokine ligands (CCL)-2 (MCP-1), CCL-3 (MIP-1α), CCL-4 (MIP-1β), CCL-5 (RANTES), CCL-11 (eotaxin), CCL-17 (thymus and activation-regulated chemokine [TARC]), CCL-20 (MIP-3α), tumor necrosis factor (TNF)-α, interferon (IFN)-γ, leptin, adiponectin, B cell-activating factor (BAFF), a proliferation-inducing ligand (APRIL) (Wilson and others 2010; Su and others 2012; Palomino and Marti 2015; Tsalamandris and others 2019).

The following cytokines have been found to have anti-inflammatory role: IL-1ra, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, G-CSF, eotaxin, transforming growth factor (TGF)-β (Marie and others 1996; Wilson and others 2010; Scheller and others 2011; Su and others 2012).

Some cytokines seem to play both roles, but the most distinct among them known to have both pro- and anti-inflammatory roles are IL-6 (Scheller and others 2011). Other cytokines may perform opposing roles within the framework of the broader function such as inflammatory cytokine IL-2 that contributes to both induction and termination of inflammation (Hoyer and others 2008).

Inflammation is described by some as a homeostatic model wherein proinflammatory cytokines mount an initial effective immune response and anti-inflammatory cytokines counter the same to avoid excessive damage to healthy tissues (Guest and others 2008). The earlier listed cytokines must, therefore, be not only measured and compared individually but also ratios of each proinflammatory cytokine (numerator) to each anti-inflammatory cytokine (denominator) will give a better understanding of the equilibrium between these within the individual (Srinivasan and Kilpatrick 2017). IL-6 may be included in numerator and in denominator to assess possible role of IL-6 where further mechanistic study is not pursued.

Exploring inflammation is about identifying pro- and anti-inflammatory cytokines, cytokines that influence polarization of macrophages, cytokines produced by classically or alternately activated macrophages, cytokines influencing or produced by white blood cells and cytokines inducing different T cell types. Within the list of cytokines and a few others included, it may be beneficial to identify subgroups that highlight other equilibriums that are related.

Cytokines Related to M1 Versus Cytokines Related to M2

Cytokines involved in induction/activation of M1/M2 macrophages: cytokines known to activate M1 macrophages are IFN-γ, LPS, and TNF-α (Owen and Mohamadzadeh 2013; Orecchioni and others 2019).

Cytokines known to activate M2 or promote polarization of M2 macrophages are IL-4, IL-10, IL-13, CCL2/MCP-1, CCL17/TARC, and CCL22 (Owen and Mohamadzadeh 2013; Van Dyken and Locksley 2013; Makita and others 2015).

Cytokines produced by M1/M2 macrophages: cytokines produced by M1 macrophages (biomarkers of M1) are IL-1, IL-6, IL-8, IL-12, type I IFN, TNF-α, MCP-1, MIP-1α, CCL-5 (RANTES), CXCL-1 (GROα), CXCL-2 (MIP-2α), CXCL–3, CXCL-5 (ENA-78), CXCL-8 (IL-8), CXCL-9 (MIG), CXCL-10 (IP-10), and CXCL-11 (I-TAC) (Mantovani and others 2004; Owen and Mohamadzadeh 2013; Melton and others 2015; Carson and others 2017; Atri and others 2018; Saqib and others 2018). M1 macrophages secrete mostly proinflammatory cytokines.

M2 macrophages produce (biomarkers of M2): IL-4, IL-10, CCL-17 (TARC), CCL-22, CCL-24/eotaxin-2 (Owen and Mohamadzadeh 2013; Sheng and others 2018; Celik and others 2020). Cytokines produced by M2 macrophages are mostly anti-inflammatory cytokines; but it also produces very low levels of proinflammatory cytokines such as IL-12 among many (Saqib and others 2018).

The dysregulation of the immune pathway is contributed to by an imbalance between M1 and M2 macrophages, hence the need for ratios of individual cytokines instead of only comparing absolute values of single cytokines (Saqib and others 2018). Comparing ratios of individual cytokines activating or promoting polarization of M1 to those promoting M2 polarization gives a better perspective of the equilibrium. Similarly, ratios of individual cytokines produced by M1 macrophages to those produced by M2 macrophages would give further insight.

Cytokines Related to Th1 Versus Cytokines Related to Th2

Cytokines inducing Th1 versus cytokines inducing Th2 cells: cytokines inducing Th1 differentiation from naive Th0 cells are IL-12 and IFN-γ (Hamza and others 2010).

Cytokines inducing Th2 from naive Th0 cells are IL-4 and IL-6 (Diehl and Rincón 2002; Doran and others 2017). The following cytokines are known to inhibit Th1 induction: IL-6 and IL-10 (Diehl and others 2000; Diehl and Rincón 2002; Schülke 2018).

Cytokines produced by Th1 versus cytokines produced by Th2 cells: cytokines produced by Th1 cells are IL-2, IL-12, IFN-γ, and TNF-β, (Rincón and others 1997; Soboslay and others 1999; Davoine and Lacy 2014).

Cytokines produced by Th2 cells are IL-4, IL-5, IL-6, IL-10, and IL-13, (Rincón and others 1997; Soboslay and others 1999).

Ratios of cytokines inducing Th1 to those inducing Th2 and ratios of cytokines produced by Th1 to cytokines produced by Th2 would lend more insight to the cytokine milieu in the body.

Angiogenic Versus Angiostatic Cytokines

The cytokines that are noted to have proangiogenic role are IL-2, IL-6, IL-8, MCP-1, GROα, MIP-1α, TNF-α, ENA-78, leptin, RANTES (CCL-5), and eotaxin (CCL-11) (2018; Salcedo and others 2001; Claffey 2002; Mehrad and others 2007; Suffee and others 2012; Middleton and others 2014; Amjadi and others 2016).

The following cytokines are noted to have angiostatic roles: IP-10, MIG, I-TAC, IFN-γ, IL-4, and IL-13 (Mehrad and others 2007; Zídek and others 2009; Owen and Mohamadzadeh 2013).

Even as inflammation is molding the environment, the balance of cytokines listed earlier determine whether it is going to lead to angiogenesis or not. And this cannot be determined by just individual cytokines but by calculating the ratios of cytokines that cause angiogenesis to cytokines that are angiostatic.

Conclusion

It is obvious from the aforementioned discussion that a single cytokine can be produced by multiple cells, it can have multiple effects and multiple cytokines can produce the same action. It augurs well to conclude that studying the absolute concentration of a few cytokines or studying their expression in just a cell does not relay much about the homeostasis in the body. Therefore, it would be beneficial if future studies select cytokines in a way that can be used to calculate various ratios and decipher more about homeostasis in the body. This would be a step toward studying ratios of 1 group of cytokines and another group of cytokines, wherein the group involves cytokines having synergistic or mechanistic relation.