General Pathology Series: Inflammatory Cells and Mediators
Foreword
Inflammation is a vital reaction mechanism to threats, and it is part of a beneficial defense system that has evolved and been conserved evolutionarily over thousands of years. But why is this process associated with diseases? While inflammation is primarily protective and helps maintain tissue equilibrium, uncontrolled inflammation can become detrimental, leading to the progression of chronic inflammatory diseases. This series aims to provide a comprehensive understanding of the inflammatory process by emphasizing its historical context, intricate mechanisms, and events that will determine its end or the development of diseases. Through six captivating chapters, readers will obtain a holistic perspective on inflammation and its significance in health and disease.
This series is divided into six articles including:
General Pathology Series: Inflammation - Historical Perspectives
General Pathology Series: Inflammatory Cells and Mediators
General Pathology Series: Inflammatory Cells Recruitment and Clearance
General Pathology Series: Inflammatory Cells and Mediators
The body’s defense system (immune system) responds to injury or irritation through a natural response known as inflammation. During this process, several cellular and biochemical events are triggered as a consequence of tissue damage. Central to the formation of inflammation are the inflammatory mediators, which include molecules of different natures such as proteins, peptides, glycoproteins, cytokines, arachidonic acid metabolites (prostaglandins and leukotrienes), nitric oxide, and oxygen free radicals. These compounds are produced by the cells present in the affected area, including epithelial cells (lining cells), endothelial cells (cells that form the blood vessel wall), and infiltrating inflammatory cells (cells that migrate from the blood circulation to the affected tissue). Inflammatory mediators produced at the onset of the inflammatory response induce dilation and increased leakiness of blood vessels, favoring the migration of inflammatory cells (leukocytes) from the bloodstream to the affected tissue. Infiltration of inflammatory cells from the circulation is essential since these cells are primarily responsible for the elimination of the harmful agent, removing bacteria, particles, and cell debris, facilitating tissue repair (Figure 1).
What are the Essential Elements of the Inflammatory Response?
The inflammatory response is a complex and finely regulated process that consists of four basic components: inducers, sensors, mediators, and target tissues (Figure 2). Inducers —for instance, bacteria— trigger the inflammatory response as they are detected by sensors present on the surface of the cells. Sensors are known as receptors, molecules that recognize and bind to an inducer. Sensors, such as Toll-like receptors (TLRs), are expressed on specialized sentinel cells, specific cells in the tissue that monitor the presence of dangerous and injurious substances. These cells produce and release inflammatory mediators after recognizing a harmful agent (inducers). Then, these mediators act on various target tissues and cells, which respond by altering their functional states and by producing further mediators to build an effective inflammatory response against damaging stimuli. Furthermore, these inflammatory mediators are responsible for the appearance of clinical signs of inflammation such as fever, swelling, and pain (Medzhitov, 2010).
Inflammatory Cells: Leukocytes
The inflammatory cells (also known as leukocytes) are important players during an inflammatory reaction. There are several types of leukocytes, including neutrophils, monocytes, eosinophils, lymphocytes, macrophages, mast cells, and basophils (Figure 3). They can be classified into granulocytes (where small granules can be observed inside them by using specific tools, e.g., a microscope) and agranulocytes (where granules are absent or are too small to be identified). Each of these cell types has a unique role to play in the body's defense system. For example, neutrophils and eosinophils play a more active role in the initiation or acute response to an infection or injury. In contrast, lymphocytes and macrophages play an important role in the prolonged or chronic response to infection or injury.
Another classification for leukocytes is based on the shape of their nucleus. Leukocytes can be categorized as mononuclear cells when the cell displays a round nucleus (e.g., lymphocytes, monocytes, macrophages) or polymorphonuclear cells when the cell presents a lobed nucleus (e.g., neutrophils, eosinophils). Leukocytes are also commonly known as white blood cells and they perform a distinct role from red blood cells (other blood cell type in circulation), whose principal function is to carry oxygen to all organs. Both white and red blood cells are derived from stem cells present in the bone marrow, a specialized tissue located in the center of the bones responsible for the production and renewal of blood cells (Ahmad, Awais, Kausar, & Akram, 2023).
Neutrophils
Neutrophils are important players in the body's defense system, being the most prevalent cells found in the bloodstream. These cells are the first leukocytes to migrate to inflammatory sites (Nathan, 2002) and several mediators involved in leukocyte recruitment (migration of inflammatory cells to an inflammatory site) have been extensively studied by researchers (Borregaard, 2010; Zlotnik, Yoshie, & Nomiyama, 2006). Neutrophils are produced in the bone marrow in large numbers, approximately 1011 cells per day, and they typically constitute about 70% of all white blood cells in the body. These cells are constantly renewed. In the blood circulation, neutrophils survive for about 6 to 8 hours, but after migrating to the tissue, their survival can be prolonged from three to five days due to the actions of inflammatory mediators produced locally, ensuring sufficient time for neutrophils to exercise their antimicrobial functions (Summers et al., 2010). Three main antimicrobial functions are recognized for neutrophils: phagocytosis, degranulation, and the release of nuclear material in the form of neutrophil extracellular traps (NETs) (Figure 4). When neutrophils recognize microbial pathogens (harmful microorganisms), these different mechanisms to destroy them can be activated. Phagocytosis involves the ingestion of the microorganism by forming a vacuole: a space within the cell, enclosed by a membrane and containing the ingested microorganism. Inside this structure, the microorganism is destroyed by the action of an acidic environment (low pH) and degrading proteins. Neutrophils also release the contents of their intracellular granules (degrading proteins, bactericidal substances) into the extracellular environment with the purpose of killing pathogens, a process that is known as degranulation. When the microorganism is too large to be ingested, extracellular traps (NETs) formed by DNA fibers and granule proteins can be released by neutrophils to capture, neutralize, and destroy it (Rosales, 2018). NETs are also called NETosis and are a specialized type of cell death of neutrophils.
Although these neutrophil antimicrobial mechanisms are effective in fighting infections, they can also be deleterious. The degrading enzymes released from neutrophils can damage tissues, favoring the additional recruitment of leukocytes to the inflammatory site (Soehnlein & Lindbom, 2010). It is important to control the number and length of stay of neutrophils in the inflammatory sites. This is because the excessive accumulation and permanence of these cells in the tissue can be detrimental, exacerbating the inflammatory process.
Monocytes and Macrophages
Macrophages and monocytes are leukocytes that play an important role during the inflammatory response. Macrophages are monocyte-derived cells that originate in the bone marrow from a common stem cell. In response to a growth factor known as macrophage colony-stimulating factor (M-CSF), bone marrow stem cells divide and differentiate into monocytes, which leave the bone marrow, enter the bloodstream, and migrate to different organs and tissues. Once in the tissue, monocytes can differentiate locally, giving rise to different types of macrophages that can be resident or transient —staying temporarily in the inflamed tissue (Nathan, 2008; Wynn, Chawla & Pollard, 2013). Macrophages are considered “professional phagocytes”, displaying a primary role in phagocytosis, the process of engulfing and eliminating foreign particles and pathogens. Macrophages are also involved in a process called efferocytosis, a mechanism similar to phagocytosis, but associated with the removal of cellular debris derived from dead cells that are generated after tissue damage and during the inflammatory process (Nathan, 2008; Poon, Lucas, Rossi & Ravichandran, 2014).
Different stimuli (for instance, inflammatory mediators) can cause different macrophage responses, leading to the activation of these cells in tissues. Mediators produced locally in the tissue can direct the phenotype of these cells, giving rise to macrophages with distinct characteristics (Mosser & Edwards, 2008). There is a classification widely used by researchers to determine the different phenotypic types of macrophages (Figure 5). These can be termed “M1 macrophages”, pro-inflammatory macrophages associated with host defense; or “M2 macrophages”, anti-inflammatory macrophages responsible for healing and repairing the tissue. M2 macrophages are also classified into other subtypes (M2a, M2b, M2c, and M2d phenotypes), which have been reported to differ greatly in their composition and function. All of these macrophage phenotypes express distinct molecules: inflammatory mediators (cytokines and chemokines) and receptors, which are directly associated with their different functions (Ariel & Serhan, 2012; Wang et al., 2020).
Eosinophils
Eosinophils are specialized white blood cells that are often involved in allergic responses and play an essential role in host defense against parasitic infections. Like neutrophils, eosinophils are a part of the granulocyte family and are characterized by their distinctive granules, which contain various proteins and enzymes that can help kill microorganisms, especially parasites. These cells usually account for less than 5% of the white blood cells in blood circulation (Jackson, Akuthota & Roufosse, 2022).
Basophils and Mast Cells
Basophils and mast cells are essential players in the inflammatory response, particularly in allergic reactions. Basophils circulate in the blood, while mast cells are in tissues, patrolling against pathogens. Mast cells and basophils contain granules and can release several substances (Figure 6), especially histamine, a molecule that increases blood vessel permeability (small molecules or cells flow in and out of the blood vessel), causing tissue edema (swelling), and hyperemia (redness due to increased blood flow) (Varricchi, Raap, Rivellese, Marone & Gibbs, 2018).
Lymphocytes
Lymphocytes are another type of white blood cell and are found primarily in lymphoid tissues: components of the lymphatic system that are complementary to the circulatory system and that work as filters for foreign substances, including lymph nodes, spleen, and tonsils. Lymphocytes can also circulate in the bloodstream in a range of 20-40% of total white blood cells. There are different types of lymphocytes with distinct functions. B lymphocytes (B cells) develop in the bone marrow and are responsible for producing antibodies, which are proteins that specifically bind to foreign substances, including bacteria and viruses, to neutralize and eliminate them. When B cells encounter an antigen (e.g., a specific molecule of a microorganism), they undergo what is called “clonal expansion”, amplifying the number of cells, and differentiating into plasma cells, which are specialized antibody-producing cells (LeBien & Tedder, 2008). T lymphocytes (T cells), on the other hand, mature in the thymus gland, which is why they were named T cells. There are other subtypes of T cells, including killer T cells (cytotoxic T cells) and helper T cells. Killer T cells directly attack and eliminate infected or abnormal cells, such as those infected with viruses or cancer cells. Helper T cells assist B cells in making antibodies. Helper T cells can also be activated by macrophages (Kumar, Connors, & Farber, 2018).
Inflammatory mediators
As previously discussed (Figure 2), the inflammatory response is mediated by specific molecules (inflammatory mediators) that will act on target organs and cells to eliminate the harmful agent and restore the tissue. There are a variety of inflammatory mediators that can be found in the bloodstream during inflammatory responses or are produced at the site of inflammation by leukocytes and cells from injured tissues. The mediators derived from the circulation system are mostly synthesized by the liver. Circulating proteins are involved in three different interrelated systems: the complement, kinin, and coagulation systems. These systems are characterized by the activation of a cascade (a sequence of successive activations of molecules) to initiate an inflammatory response and kill microorganisms. On the other hand, cell-derived chemical mediators are found in intracellular granules of cells, which are readily released in response to an inflammatory stimulus (e.g., bacteria). Within this group are vasoactive amines (histamine and serotonin); arachidonic acid metabolites (prostaglandins, leukotrienes, and lipoxins), cytokines (tumor necrosis factor, interleukin-1, and chemokines); nitric oxide (NO); and others. The main inflammatory mediators produced by cells are listed in Table 1 and some of these mediators will be discussed below.
Table 1: Inflammatory mediators and their main functions. Adapted from (Juhn et al., 2008)
Vasoactive Amines
This class of molecules is responsible for blood vessel dilation (vasodilation) or constriction (vasoconstriction). Histamine is a vasoactive amine found in mast cells, basophils, epithelial cells, gastric mucosa, and neurons of the central nervous system. In the inflammatory process, histamine acts by promoting vasodilation and increased vascular permeability. These are important mechanisms associated with the formation of edema and hyperemia observed during inflammatory processes, especially allergies. For this reason, several anti-allergy medicines act by inhibiting the release of histamine (Lieberman, 2011). Serotonin, another vasoactive amine, is found in neurons in the central nervous system and the gastrointestinal tract. Although serotonin is best known for its action as a neurotransmitter in the central nervous system, it can also contribute to vasodilation and increased vascular permeability in inflammatory processes (Wu, Denna, Storkersen & Gerriets, 2019).
Arachidonic Acid Metabolites
Arachidonic acid is an essential fatty acid (lipid) constituent of cell membranes. Following irritation or tissue injury, arachidonic acid is released and metabolized by enzymes, i.e., cyclooxygenases (COXs), lipoxygenases (LOXs), and cytochrome P450 (CYP), leading to the formation of several inflammatory mediators, including prostaglandins and leukotrienes. These molecules play crucial roles in inflammation, inducing pain, vasodilation (swelling and redness), and fever (Funk, 2001).
Cytokines
Cytokines are small, secreted proteins released by cells. Cytokines are redundant in their activity, meaning similar functions can be stimulated by different cytokines. It is also common for different cell types to secrete the same cytokine or for a single cytokine to act on several different cell types (Zhang & An, 2007). Several cytokines have been described, and their functions may vary, displaying pro-inflammatory, anti-inflammatory, or chemoattractive activities (the ability to attract leukocytes and induce their migration) (Figure 7). There is abundant evidence that certain pro-inflammatory cytokines, such as interleukins-(IL)-1β, IL-6, and the Tumor Necrosis Factor alpha (TNF-α) are involved with inflammatory processes. TNF is a pro-inflammatory cytokine produced mainly by monocytes, macrophages, and T-lymphocytes. After trauma, surgical procedures, or infections, TNF-α is one of the earliest and most potent mediators of the inflammatory response to be released (Kalliolias & Ivashkiv, 2016). Interleukin-(IL)-1 is released by macrophages, monocytes, fibroblasts, epithelial cells, and others, and is one of the most important markers of pain associated with inflammatory responses (Cavalli et al., 2021). Chemokines are a family of small, soluble proteins that are part of the cytokine family. The primary function of chemokines is to act as chemoattractants, which means they attract leukocytes to sites of infection or damaged tissue. There are a large number of chemokines described so far, and they are classified into four main subfamilies based on the arrangement of their structure (amino acid sequences): C, CC, CXC, and CX3C chemokines. The classification is based on the number and spacing of cysteine (C) residues in their amino acid sequences (Hughes & Nibbs, 2018).
Nitric Oxide
Nitric oxide (NO) is a free radical (highly reactive molecules that can promote cell damage and deterioration in organs) that induces muscle relaxation of blood vessels leading to vasodilation. Due to their central role in inflammation, these mediators have become attractive targets for therapeutic interventions aimed at modulating inflammatory cell recruitment and inflammation in various diseases.
Anti-inflammatory and Pro-resolving Mediators
So far, we have discussed mediators that are involved in activating the inflammatory response, but several studies have shown mediators that can modulate inflammation as well, including anti-inflammatory cytokines (e.g., IL-4, IL-10). A successful inflammatory process relies on the production of mediators that actively influence its resolution (Serhan et al., 2007). Recent studies have shown that some endogenous mediators (produced during the inflammatory process) can be responsible for finely controlling the inflammatory response, leading to its end. The termination of inflammation is governed by molecules collectively referred to as pro-resolving mediators. Failure in the production of these molecules can lead to excessive tissue damage and chronic inflammation (Figure 8). There are many molecules that have been described as mediators of resolution, and new players are still being continuously discovered. These molecules act at key points of the inflammatory response, controlling the migration of leukocytes from the blood to the tissue, inducing the death of neutrophils, increasing phagocytosis, inducing the polarization of macrophages towards an anti-inflammatory profile, and reducing the production of pro-inflammatory mediators. Some examples are the specialized pro-resolving mediators (SPMs) (e.g., lipoxins, resolvins, protectins, and maresins), proteins, and peptides (e.g., annexin A1, adrenocorticotropic hormone, galectins), gaseous mediators (e.g., H2S and CO), as well as modulators of the central nervous system (acetylcholine and other neuropeptides) (Sugimoto, Vago, Perretti & Teixeira, 2019).
Conclusion
The inflammatory response is an intricate process that involves distinct cells and mediators that act in a coordinated way to combat the threat and initiate the healing process. An understanding of the inflammatory process requires knowledge of its main players. Here, important concepts regarding the inflammatory mediators, inflammatory cells, and their contribution to the inflammatory process were underlined. Inflammatory mediators work in a balanced way to initiate and maintain the inflammatory response, altering the local microenvironment and favoring the recruitment of inflammatory cells to the site of infection or injury. This complex process involves myriad players, including different triggers (e.g., pathogens), cell types, and chemical mediators, and the mechanisms involved in the inflammatory response are still being unraveled. Chronic inflammation and the development of various inflammatory diseases are marked by an imbalanced or prolonged release of inflammatory mediators and excessive leukocyte recruitment. Therefore, the modulation of the inflammatory response, tempered by the production and release of pro-resolving mediators, may be crucial for an effective and balanced response, preventing the development of diseases and many studies in this field are being conducted.
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