Cytokine Storm Overview

Cytokine release syndrome (CRS) is a systemic inflammatory response that can be triggered by a variety of factors, including infectious and noninfectious diseases, as well as certain immunotherapeutic agents [1,2,3]. While the general concept of an excessive or uncontrolled release of pro-inflammatory cytokines is well known, a definition of what constitutes a cytokine storm is lacking. Current areas of research include investigation of the molecular events that precipitate a cytokine storm, the contribution that a cytokine storm makes to pathogenesis, and the therapeutic interventions that might be used to prevent the storm or quell it once it has started [1].

The acronym CRS was introduced in the early 1990s, when the anti–T-cell antibody muromonab-CD3 (OKT3) was being studied as an immunosuppressive treatment for solid organ transplantation [4,5]. The term “cytokine storm” was first used in 1993 to describe the events modulating the onset of the graft-versus-host disease, a condition characterized by a powerful activation of the immune system [3]. CRS represents one of the most frequent adverse effects of T cell–engaging immunotherapeutic agents and is often observed following administration of nonprotein-based cancer drugs [6,7].
 

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Cytokines and chemokines play a significant role in immunity and immunopathology during viral infections (Table 1). As infected cells die by apoptosis or necrosis, the release of inflammation mediators initiates a variety of responses. Inflammation associated with a cytokine storm starts locally and spreads throughout the body via circulation. Expression of inflammatory, antiviral, and apoptotic genes increases, accompanied by abundant immune cell infiltration and tissue damage [1]. Reparative processes follow clearance of infection and tissue damage, leading to a resolution and homeostasis of the tissue [8]. When severe inflammation damages local tissue structures, healing occurs with fibrosis and can result in persistent organ dysfunction.

A rapid and well-coordinated innate immune response is the first line of defense against viral infections, but dysregulated and excessive immune responses can cause immunopathology [9,10,11]. Acute lung injury (ALI) occurs as a consequence of a cytokine storm in the lung alveolar environment and circulatory system. It is most commonly associated with suspected or proven infections in the lungs or other organs [12]. Pathogen-induced lung injury can progress into a more severe form of ALI, known as acute respiratory distress syndrome (ARDS). The cytokine storm and consequent ARDS result from the effects of a combination of many immune-active molecules [12].

Increased circulating levels of pro-inflammatory cytokines (IFNα, IFNγ, IL-1B, IL-6, IL-12, IL-18, IL-33, TNFα, TGFβ), immunosuppression cytokines (IL-4 and IL-10), and chemokines (CXCL10, CCL2, CXCL8, CXCL9, CCL3, CCL5) intensify pulmonary inflammation and extensive lung damage in SARS and COVID-19 patients, and MERS-CoV infection [13,14,15,16]. Cytokine storm involves the mTOR and JAK pathways and activation of Th1 and Th17 cells. It can also result in Th2 cell immune-oriented immunosuppression [17,18,19,20,21,22].

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By its biological properties, C-reactive protein is classified as an acute inflammatory protein [6,23,24]. The acute expression increases up to 1,000-fold at sites of an inflammation or infection. Initiation signals from the immune system lead to synthesis and secretion of CRP, primarily from the liver hepatocyte cells [6,23]. CRP can also be synthesized by macrophages, endothelial cells, and adipocytes. CRP is a homopentameric protein and belongs to the pentraxin protein family [6,23,24]. As part of the soluble innate immune system, CRP acts as a pattern recognition molecule. In the presence of calcium, it can bind to microbial polysaccharides like phosphocholine (PCh). CRP then binds to C1q protein complex, triggering the complement pathways of the innate immune system [6,23,24]. CRP also binds to other ligands, such as chromatin, histones, and small nuclear ribonucleoprotein particles. After binding to these ligands, it binds to the Fc-gamma (Fcy) receptors on phagocytic cells, initiating the ligand elimination.

CRP levels increase dramatically in response to inflammation, injury, and infection [23,25,26]. Following an inflammatory event, cytokines such as IL-1B, IL-6, and IL-17 signal the transcription of the gene that encodes CRP in hepatic cells. Increasing evidence points toward CRP having a functional role in the inflammatory process [6,25,26]. Increased CRP concentration is used as a diagnostic marker for inflammatory events. Even though CRP aids in this inflammation, sustained high-CRP levels can be a sign of high-level inflammation or cytokine storm [23,24, 27].

Melody et al. evaluated CRP and ferritin levels in B-cell lymphoma patients treated with chimeric antigen receptor (CAR) T-cell therapy [28]. They analyzed patients who required admission to the intensive care unit (ICU) as a result of CAR-T associated toxicity versus non-ICU patients. They found that all 13 patients admitted to the ICU developed CRS symptoms and had increased CRP levels (> 20 mg/L) [28], which is considered a significant increase as compared with CRP serum levels of ~ 8 mg/L in healthy individuals [6,24,25,28]. Wu et al. investigated the cytokine profile of 57 patients with H7N9 avian influenza [26]. They found that patients with high CRP levels had poor outcomes. They also observed that cytokine levels of IL-6, IL17A, and monocyte chemoattractant protein-1 (MCP-1) positively correlated with increased CRP levels [26]. These and other recent studies suggest that increased CRP levels can be a reliable marker of CRS.
 

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The innate immune system acts as the first line of defense against pathogens. This system can include a variety of cell types, including neutrophils, macrophages, and monocytes [1,29]. These cells act to recognize the pathogens, to release cytokines, and to eradicate pathogens through phagocytosis. Other innate immune system cells include dendritic cells, natural killer cells, and the gamma-delta subset of T cells. These cells respond to pathogens by recognizing the foreign antigens and secreting cytokines that initiate the adaptive immune system [29].

Even though both adaptive and innate immune system can be involved in a cytokine storm, innate cells are most often correlated with CRS pathogenesis [1,29]. Innate cells, including neutrophils, macrophages, and natural killer (NK) cells, play an important in generating a cytokine storm [1,30]. Neutrophils are responsible for generating a fibrous network that amplifies cytokine production and thrombi formation [1,29,30]. During a cytokine storm, macrophages can be activated to oversecrete inflammatory cytokines, leading to severe tissue damage [1,30]. Excess IL-6 from a cytokine storm can mediate the cytolytic function of NK cells, leading to prolonged inflammation and lower perforin/granzyme production [1,31]. In a cytokine storm, Th1 helper T cells of the adaptive arm can induce an increased inflammatory response [1,32]. This inflammatory response includes overproduction of interferon-gamma and increased activation of macrophages [1,29,32]. In certain cases, CAR-T cell therapy has been known to induce cytokine storm in cancer patients [28], suggesting the ability of cytolytic T cells to initiate CRS.

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Cytokines function as indicators of inflammation or disease progression, and their release serves as a means of manipulating cellular responses in vivo and in vitro. Due to their secreted nature, these molecules can be detected in serum, plasma, and cell culture supernatant. The enzyme-linked immunosorbent assay (ELISA) remains the gold standard for cytokine detection and allows quantitation of specific mediators in the cytokine storm. In addition, the key cytokine mediators and other immune regulatory targets can be quantified using an innovative alternative to ELISA: the Invitrogen ProQuantum High-Sensitivity Immunoassays.

Since many different causes and pathologic conditions for the induction of a cytokine storm exist, and not all syndromes involving cytokine release result in the same pathogenic cytokine profile, it is of great interest to analyze the level of a broader panel of immunomodulatory markers to get a more complete picture of a patient’s immune status. For studying a broader set of biomarkers and to acquire a more holistic representation of biomarkers involved in the cytokine storm syndrome, multiplex approaches such as the Invitrogen ProcartaPlex Panels are recommended. For example, the Invitrogen Immune Monitoring 65-Plex Human ProcartaPlex Panel is a preconfigured multiplex immunoassay kit that measure 65 protein targets, including cytokines, chemokines, and growth factors, using Luminex xMAP technology.

Another approach to the study of immune responses is to monitor changes in gene expression profiles associated with pathologic conditions using multiplex analysis of relevant RNA targets. The Invitrogen QuantiGene Plex Gene Expression Assay is a fast, high-throughput solution that allows the simultaneous measurement of up to 80 genes of interest in a single well of a 96- or 384-well plate. With carefully selected content, gene expression analysis using real-time PCR enables a closer look at relevant targets and allows the generation of high-quality data for a broad range of studies.
 

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Upon elimination of infection, the resolution of the inflammatory responses is necessary to return the affected tissue back to homeostasis. This process involves release of pro-resolving mediators that prevent the migration of granulocytes and increase leukocyte apoptosis (Table 2). Macrophages are involved in restorative and resolutive roles through reprogramming by pro-resolving molecules [33]. Whereas anti-inflammatory mediators block leukocyte recruitment, endothelial activation, and vascular permeability, pro-resolving molecules alter the inflammatory process by releasing endogenous mediators that affect signaling cascades, cellular interactions, and inflammatory switches [33]. This process can include termination of effector leukocyte infiltration, regulation of cytokine levels, induction of apoptotic neutrophil efferocytosis by macrophages, reprogramming of macrophages from classically activated to alternately activated, and production of anti-inflammatory mediators [33]. Pro-resolving mediators include bioactive lipids, certain peptides, gaseous mediators, purines, neuromodulators, and reactive oxygen species (ROS) [33]. Furthermore, inflammation promotes tissue repair at the site of tissue damage by recruitment of phagocytes. This repair process can also include alternately activated macrophages and the establishment of new blood supplies that promotes the division of epithelial and fibroblasts [34]. Fibrinogen also leaks out of inflamed vessels to provide a scaffold for these cells [33].
 

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