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What are the purposes of granulocytes in acute inflammation?

What are the purposes of granulocytes in acute inflammation?



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I heard the phrase

Neutrophilic leucocytes are kings in the acute inflammation.

Neutrophils are granulocytes, while leucocytes are not granulocytes. I think this statement refers to the fact that leucocytes have neutrophilic properties, instead of being neutrophils itself.

Acute inflammation players are, I think

Granulocytes, monocytes and leucocytes

while in chronic inflammation similar player but the main factor is macrophages so the phrase which I heard

Macrophages are the kings of chronic inflammation.

I think granulocytes are related to the Healing and Proliferation. Eosinophils for instance in the allergic responses of acute inflammation. I think granulocytes are phagocytosed by the macrophages and this way spread their functions through macrophages around. Probably, neutrophiles are attached to the leucocytes such that they spread their function through leucocytes (so called neutrophilic leucocytes).

Let's cover first this, since we can after understanding it go to the chronic one. What is the purposes of granulocytes in acute inflammation?


MattDMo's answer

Essentially, [neutrophilic granulocytes] are the first infiltrating cells to arrive at the site of injury at the beginning of acute inflammation (bacterial infection, environmental exposure, etc.).

which I agree with.


Granulocytes: Definition, Ranges, Causes & Immature cells

Granulocytes are the most common type of white blood cell. There are three types of white blood cells within the granulocyte �mily”. These cells are characterized by their enzyme granules, which develop in the cytoplasm.

When the immune system is under attack by an infection, an asthma attack, or an allergic reaction, the granulocyte releases the granules to fight against the problem.

Chemical signals either on the surface of, or released by, harmful and invading substances attract the granulocytes to the site of the issue. When drawn to the site, they fight the infection by releasing their granules.

Granulocytes develop in the bone marrow and live for a few days, and only circulate through the bloodstream in the last few hours of life.


White Blood Cell Production

White blood cells are produced within bones by bone marrow and some then mature in the lymph nodes, spleen, or thymus gland. Blood cell production is often regulated by body structures such as the lymph nodes, spleen, liver, and kidneys. The life span of mature leukocytes can be anywhere from a few hours to several days.

During times of infection or injury, more white blood cells are produced and sent into the blood. A blood test known as a white blood cell count or WBC is used to measure the number of white blood cells present in the blood. There are between 4,300-10,800 white blood cells present per microliter of blood in the average healthy person.

A low WBC count may be due to disease, radiation exposure, or bone marrow deficiency. A high WBC count may indicate the presence of an infectious or inflammatory disease, anemia, leukemia, stress, or tissue damage.


Inflammatory mediators

The inflammatory response is a combination of diverse chemical mediators from blood circulation, immune cells, and wounded tissue. These include vasoactive amines (histamine), peptides (bradykinin), and eicosanoids (leukotrienes).

Vasoactive amines

These are a group of compounds contains an amino acid to modify the pervasiveness of blood vessels. There are two effective molecules are histamine and serotonin. They are known as beginning inflammatory mediators stored in mast cells.

Histamine is made by mast cells generally. Basophils and platelets also produced it by the decarboxylation of histidine amino acid. It releases due to the number of stimulators such as tissue damage, Fc receptor binding of IgE antibody to the mast cell, C3a and C5a complement, etc. It widens blood vessels by tempting endothelial contraction and the development of interendothelial gaps.

It is a derivative of tryptophan present in platelets. It is also present in neurons and enterochromaffin cells. Platelets accumulation allow its release and function to narrow down blood vessels, neurotransmitter, etc. It functions in inflammation, opposite to histamine.

Bradykinin are nonapeptides, vasoactive in nature. They produced by the activity of the proteases enzyme. They are significant in blood pressure control and inflammatory reaction. It causes vasodilation of arteries and veins of gut, aorta, uterus, and urethra. It is now also known for its role in neuro-signaling and a controller of some kidney and vascular functions.

Eicosanoids

Eicosanoids obtain from polyunsaturated fatty acids by oxygenation of biolipid. They function in various physiological mechanisms and pathological responses such as allergy, fever, inhibition of inflammation, etc. Its signaling mechanism is alike to cytokines in the inflammatory response and pro-inflammatory component formation. Eicosanoids have significant subgroups of diverse compounds most noticeably prostaglandins, leukotrienes, lipoxin, thromboxane, eoxins, etc.

Prostaglandins

They have a significant role in inflammatory response generation. Their production increased in injured inflamed tissues and significant inflammation signs indicated by their output. Prostaglandin E2 is most plentifully made in the body with various functioning such as blood pressure control, immune reaction, fertility, etc. in the inflammatory process, they have an important character for generating basic signs of inflammation: redness, swelling, and pain. Erythema and swelling occur due to rapid blood flow by enlargement and increased pervasiveness of vessels. Pain is mediated due to the action of PGE2 on sensory neurons.

Leukotrienes

Leukotrienes belong to the eicosanoids family and use lipid signaling as autocrine or paracrine signaling. Their synthesis is complemented with histamine and prostaglandin production. They stimulate smooth muscle contraction and their excess amount causes inflammation in the bronchial lining and in allergic reactions. They have also chemotactic influence on neutrophils.

Thromboxane

Thromboxane are also produced from arachidonic acid metabolism. They obtain this name due to their clot formation activity known as thrombosis. The chief thromboxane types are thromboxane A2 and thromboxane B2. They are discharged from platelets but their secretion mechanism not understood. It has hypertensive nature and possesses vasoconstriction activity. And they promote platelet accumulation and formation of a clot.

Inflammatory cytokine

Cytokine are small protein released from cells having particular activity on the interaction between cells and it is a communicating module of cells. They self-action or neighboring action molecules and in some cases they function on secluded cells also. These cytokines include tumor necrotic factor, interferon-gamma, interleukin 1 (IL-1), IL-12, IL-8, and granulocyte-macrophage colony-stimulating factor.

Acute-phase protein

Acute-phase proteins (APP) generated as a component of innate immune response with variable serum concentration. These acute-phase proteins classified as negative and positive plasma concentrations.

Positive acute-phase proteins

Positive acute-phase protein is a sign of high inflammatory reaction. They are sub-grouped according to their level of raising concentration in the blood are major, moderate, and minor. Promptness and extent of these proteins contrast with different species.

Negative acute phase proteins

These proteins reduce in concentration by 25% in inflammatory reaction. The two chief negative acute-phase proteins are transferrin and albumin. The concentration of protein decreases slowly. The mechanism of reduction involves a number of responses such as the production of these proteins reduced, inflammatory cytokines induce less production, etc.


Core

Inflammation is the rapid response to injury of any vascularised living tissue in order to deliver various defensive materials such as white blood cells and proteins to the site of injury. The purpose of inflammation is to defend against infection and to clear damaged tissue.

The suffix '-itis' is used to indicate inflammation of an organ or tissue. For example, appendicitis is an inflammation of the appendix.

Acute inflammation is a type of inflammation which develops over the course of minutes, hours or days. If the inflammation lasts for weeks, months, or years and is associated with fibrotic changes then it is chronic inflammation.

Clinical Signs of Inflammation

There are 5 clinical signs of inflammation, which are as follows:

  • Rubor – Redness
  • Calor – Heat
  • Tumor – Swelling
  • Dolor – Pain
  • Functio laesa – Loss of function
    • This forces the person injured to immobilise the affect area which helps to reduce further damage.

    This process can be remembered through the memory aid – Raging Cows Trampled Down Fred, or Really Can't Tolerate Dead Languages.

    Causes of Acute Inflammation

    Common causes of acute inflammation include:

    • Microbial infections e.g. pyogenic (pus causing) organisms
    • Hypersensitivity reactions (in the acute phase)
    • Foreign bodies e.g. splinters, sutures, dirt
    • Tissue necrosis
    • Physical trauma
    • Chemical agents or radiation

    Stages of Acute Inflammation

    Acute inflammation is innate (present from birth) and stereotyped (happens the same way no matter the cause of the inflammation), and is controlled by chemical mediators.

    There are four stages of acute inflammation:

    1. Stimulus
    2. Vascular stage - slowing the circulation and forming exudate at the site of inflammation
    3. Cellular stage - the migration of neutrophils to the site
    4. Resolution or persistance

    Vascular Phase of Acute Inflammation

    The first phase of acute inflammation is the vascular phase, which occurs in the following steps:

    1. Vasoconstriction of arterioles (for a few seconds).
    2. Vasodilation of arterioles and then capillaries. This increases the blood flow to the affected area, resulting in heat and redness (calor and rubor).
    3. Increased permeability of local blood vessels allows proteins, cells and fluid to leave the blood vessel and enter the interstitial fluid causing swelling (tumor). This is called exudate
    4. The increased concentration of red blood cells in small vessels and increased blood viscosity leads to stasis within the vessel.

    This process is controlled by different chemical mediators. Within the first 30 minutes the main mediator, histamine, is released from mast cells, basophils, and platelets. Histamine acts to dilate vasculature, increase vascular permeability and stimulate pain (dolor). As the inflammatory response continues it is mediated by chemicals known as leukotrienes and bradykinins.

    Exudate is a collection of fluid, protein and cells that have left the blood vessel and formed in the interstitium. This exudate has several functions:

    • To deliver fibrin, inflammatory mediators and immunoglobulins to the site of damage.
    • Dilutes toxins to reduce the damage to tissues.
    • Increases lymphatic drainage to deliver antigens and pathogens to lymph nodes to initiate an immune reaction.

    Exudate Formation

    The fluid flow across the vessel walls is controlled by a combination of hydrostatic pressure (the pressure exerted by a fluid which forces fluid out) and oncotic pressure (osmotic pressure exerted by proteins which draws fluid in). These are known as Starling’s Forces.

    • If there is increased hydrostatic pressure, there is an increased flow of fluid out of the vessel.
    • If there is increased oncotic pressure within the vessel then there will be a reduction in the flow of fluid out of the vessel.
    • If there is an increased oncotic pressure in the interstitial fluid, there will be an increased flow of fluid out of the vessel as the oncotic pressure draws fluid out of the capillary.

    Diagram - The Starling forces and how they control the fluid movement into and out of the blood vessels

    SimpleMed original by Benjamin Norris

    Before exudate can form, the vasculature must become ‘leakier’ to allow proteins and fluid to cross more easily into the tissue space. Vascular leakage can occur due to the following mechanisms:

    • Endothelial contraction forming gaps between cells.
      • Mediated by histamine and leukotrienes.
      • Mediated by cytokines, interleukin-1, and TNF (tumour necrosis factor).
      • Reactive oxygen species and enzymes from leucocytes.
      • Mediated by VEGF.

      Exudate forms via the following sequence of events:

      1. Arteriolar dilation (but unchanged venous tone) increases hydrostatic pressure in the capillaries.
      2. Increased vessel permeability by the above mechanisms causes movement of protein into the interstitium. This increases the oncotic pressure in the tissue and decreases the oncotic pressure in the vessels.
      3. The changes in oncotic and hydrostatic pressures results in a net flow of fluid out of the vessels, leading to tissue oedema.

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      Types of Exudate

      • Pus/Abscess
        • Creamy/white as it is rich in neutrophils. This is typical of infections of pyogenic bacteria.
        • Red due to the presence of red blood cells. This indicates significant vascular damage.
        • Clear fluid with few leucocytes, suggesting no microorganisms. This exudate is produced in blisters and burns.
        • Significant deposition of fibrin. This can result in friction between serosal surfaces which can be heard as a rubbing sound on auscultation e.g. in pericarditis.

        The Role of Neutrophils in Acute Inflammation

        The primary type of white blood cell involved in inflammation is the neutrophil, which is a type of granulocyte. Neutrophils are normally only found in the blood and bone marrow, and their presence in tissue indicates a pathogenic organism or injury is present. They have a lifespan of 12-20 hours and cannot multiply.

        Diagram - The different granulocytes found in the body

        Creative commons source by OpenStax College [CC BY-SA 4.0 (https://creativecommons.org/licenses/by-sa/4.0)]

        To capture and kill a bacterium in the tissue space, a neutrophil must infiltrate the tissues. Neutrophils infiltrate the tissue and attach to bacteria by the following steps:

        1. Chemotaxis – neutrophils are attracted towards the site of injury by chemical attractants.
        2. Activation – neutrophils switch to a higher metabolic level and change shape to help them move towards the chemical attractant.
        3. Margination – neutrophils move towards the endothelial wall where they then roll along it until they become trapped. When they become trapped, they then crawl out of the vessel.
        4. Diapedesis – neutrophils relax the junctions between the endothelial cells so they can move across the endothelium. They also use collagenase to break down the basement membrane.
        5. Recognition Attachment – neutrophils recognise the bacterium via the opsonins which attach to the bacterium. Neutrophils then move towards and attach to the bacterium.
          1. Opsonins are substances which make it easier for phagocytes to recognise targets. Examples include complement compounds and antibodies.

          Chemotaxis is the directional movement towards a chemical attractant. Both exogenous and endogenous substances can be chemotactic for leucocytes (see summary table). Within five seconds of the chemotaxin binding to the cell surface receptors the leucocyte undergoes conformational change to ‘activate’ it, which makes the cell more able to stick to microorganisms.

          In most forms of acute inflammation neutrophils predominate in the inflammatory infiltrate during the first 6-24 hours. Neutrophils are replaced by monocytes in 24-48 hours.

          Phagocytosis

          Phagocytosis (cell eating) is the engulfment of solid particulate material by phagocytes, such as neutrophils. The process of phagocytosis is summarised in the diagram below.

          Diagram - The stages in phagocytosis

          Creative commons source by Graham Colm [CC BY-SA 4.0 (https://creativecommons.org/licenses/by-sa/4.0)]

          The membrane of the phagocyte forms a crater around the particle to be eaten. The edges then come together and the opposed plasma membranes fuse. The particle is then in a vacuole known as a phagosome. Lysosomes fuse with the phagosomes forming a phagolysosome. Chemicals are released that break down the engulfed particle. The debris are released by exocytosis.
          engulfed cells can be killed in one of two ways:

          • O2 dependent mechanisms – release of oxygen-derived free radicals into the phagosome. The mechanism of killing is known as the oxidative burst.
          • O2 independent mechanisms – uses enzymes to kill bacteria inside the phagolysosome e.g. proteases, lipases, nucleases.

          A Summary of Chemical Mediators of Inflammation

          Table - The different chemical mediators in the body and the functions they have during inflammation

          SimpleMed original by Benjamin Norris

          Local Complications of Acute Inflammation

          • Damage to normal tissue secondary to substances produced by neutrophils and released in phagocytosis
          • Obstruction of tubes, e.g. bile duct, intestine due to compression by surrounding swelling
          • Compression of vital structures secondary to swelling
          • Loss of fluid as exudate, e.g. burns
          • Pain and loss of function

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          Systemic Effects of Acute Inflammation

          • Fever
            • Pyrogenic substances are released that stimulate the hypothalamus to increase the body temperature e.g. IL-1 and TNF.
            • Prostaglandins can also cause fever. Prostaglandin synthesis is inhibited by aspirin so aspirin can be used to help reduce fevers.
            • IL-1 and TNF produce accelerated release of leucocytes from the bone marrow.
            • Macrophages and T lymphocytes also stimulate the further release of other leucocytes.
            • There is a change in the level of some plasma proteins as the liver changes its pattern of protein synthesis
            • The response causes decreased appetite, increased pulse rate, altered sleep patterns and changes in plasma concentration of acute phase proteins such as:
              • C-reactive protein (CRP) (clinically useful in assessing the level of inflammation)
              • Alpha-1 antitrypsin
              • Haptoglobin
              • Fibrinogen
              • Serum amyloid A protein
              • A clinical syndrome of circulatory failure, which can often be fatal.
              • Can happen if bacterial products or inflammatory mediators spread around the body in the blood stream, leading to body-wide inflammation.
              • In shock there is a dramatic drop in blood pressure caused by widespread vasodilation and increased vascular permeability in response to the bacterial products in the blood.

              What Can Happen After Acute Inflammation?

              There are several possibilities following acute inflammation, including persisting and become chronic inflammation with fibrous repair. The acute inflammation could continue along with chronic inflammation (e.g. an abscess). The best scenario for acute inflammation is resolution.

              With resolution the changes gradually reverse and vascular changes stop:


              Neutrophil subpopulations in disease

              As discussed earlier, it is becoming increasingly apparent that neutrophils are much more than just microbe-killing cells. They can display several phenotypes and perform a wide array of cellular functions. Several subsets of neutrophils are found in tissues under homeostatic conditions. We still have much to learn on how these different neutrophil subtypes are generated and recruited to tissues. In addition, various subsets of neutrophils with distinct properties are also detected in pathological conditions particularly in inflammation and in cancer (Silvestre-Roig et al., 2016 Yang et al., 2017).

              Neutrophil subpopulations in inflammation

              Neutrophils are the first cell type recruited to sites of inflammation. From there, they can switch phenotypes and generate various subpopulations with different cell functions. Neutrophils can also interact, directly, or via cytokines and chemokines, with other immune cells to modulate both innate and adaptive immune responses. There is not a complete understanding of these subpopulations of neutrophils, but some clear examples showing that bona fide inflammatory subsets occur are mentioned next.

              Upon infection with an antibiotic-resistant Staphylococcus aureus, two clear subsets of murine neutrophils can be observed. They differ in cytokine production, macrophage activation potential, expression of TLR, and expression of surface molecules. These subsets were named PMN-1 and PMN-2 (Tsuda et al., 2004). PMN-1 cells produce IL-12 classically activate macrophages, express TLR2/TLR4/TLR5/TLR8, and CD49d hi CD11b low . In contrast, PMN-2 cells produce IL-10 alternatively activate macrophages, express TLR2/TLR4/TLR7/TLR9, and CD49d low CD11b hi (Tsuda et al., 2004). In systemic inflammation condition, another subset of neutrophils is generated with low doses of endotoxin. These cells have a hypersegmented nucleus and display the phenotype CD62 low CD11b hi CD11c hi , which is similar to the one described for murine aged neutrophils (Casanova-Acebes et al., 2013 Zhang et al., 2015). Also, they are capable of inhibiting T lymphocytes by direct cell contact involving the integrin Mac1, and by local delivery of reactive oxygen species (ROS) (Pillay et al., 2012).

              In certain organs such as liver and adipose tissue, few neutrophils are detected in normal homeostatic conditions. However, upon an inflammatory state induced by experimental obesity, neutrophil numbers increase rapidly and a metabolic imbalance is slowly generated (Talukdar et al., 2012). First, neutrophils release elastase from azurophilic granules. This enzyme can destroy insulin receptor substrate 1 (IRS1) in adipocytes and hepatocytes, and in consequence induce insulin resistance and lipogenesis (Talukdar et al., 2012). Supporting a direct role for this neutrophil subtype in metabolic disorders was the observation that altered levels of elastase or its inhibitor (1-antitrypsin) are associated with metabolic syndrome and the onset of diabetes (Mansuy-Aubert et al., 2013).

              Neutrophil subpopulations induced by metabolic deregulation

              As mentioned before, neutrophil effector functions are markedly enhanced after priming. When certain metabolic functions are altered, neutrophils can be primed to present stronger pro-inflammatory responses.

              In hyperglycemia human and mouse neutrophils are primed to undergo NETosis (Wong et al., 2015). First, neutrophils in circulation respond to high glucose levels by releasing S100 calcium-binding proteins A8 (S100A8) and S100A9, which interact with the receptor for advanced glycation end products (RAGE), and induce macrophages and GMPs in the bone marrow to secrete G-CSF (Kraakman et al., 2017). In consequence, production of neutrophils is enhanced (Xiang et al., 2012). These new released neutrophils are primed for ROS production and formation of NETs (Wong et al., 2015). Similarly, during hypercholesterolemia, neutrophils showed a primed state characterized by elevated ROS production, increased release of myeloperoxidase (MPO) and increased expression of CD11b (Mazor et al., 2008). Together, these reports show that hyperglycemia and hyperlipidemia generate primed, proinflammatory neutrophils that may contribute to diabetes, adipose tissue inflammation, and cardiovascular inflammation.

              Neutrophil subpopulations and NET formation

              NETosis, the process for producing NETs can be activated by multiple types of microorganisms (Fuchs et al., 2007 Yipp et al., 2012). Yet, the capacity of neutrophils to undergo NETosis can vary with physiological states, suggesting a neutrophil diversity that could be clinically relevant. In fact, several reports indicate that NETs can influence thrombosis (Fuchs et al., 2010) and vascular inflammation (Kessenbrock et al., 2009 Chistiakov et al., 2015), cancer (Berger-Achituv et al., 2013 Garley et al., 2016) and autoimmunity (Stephenson et al., 2016). As mentioned before some metabolic conditions associated with states of chronic inflammation, can increase neutrophil predisposition to form NETs. Hence, neutrophils from diabetic patients (Wong et al., 2015) and from systemic lupus erythematosus (SLE) patients (Garcia-Romo et al., 2011 Villanueva et al., 2011) have been shown to be more prone to NET formation.

              Nowadays NETs have been described as a player of several pathophysiological processes, including vascular diseases, such as atherosclerosis and venous thrombosis (Qi et al., 2017 Bonaventura et al., 2018), and inflammatory pathologies, such as gout and pancreatitis (Hahn et al., 2016).

              Atherosclerosis is a cardiovascular disease accompanied by chronic vascular wall inflammation and endothelial cell dysfunction (Gisterå and Hansson, 2017). Hyperlipidemia can damage endothelial cells, promoting lipid deposition and plaque formation. This usually characterizes the onset of atherosclerosis. Hyperlipidemia and also hypercholesterolemia induce neutrophilia, which is positively associated with atherosclerotic plaque burden (Drechsler et al., 2010). Neutrophils attach themselves to atherosclerotic plaques, primarily through NETs formation, where cholesterol crystals function as danger signals, inducing IL-1β-mediated NETs release from neutrophils. Then, components of NETs, such as cathepsin G and cathelicidin-related antimicrobial peptide (CRAMP), can attract monocytes and macrophages to plaques (Döring et al., 2012 Wang et al., 2014). NETs can also regulate cytokine production from macrophages in atherosclerosis (Warnatsch et al., 2015), and induce endothelial dysfunction directly by activation and damage of endothelial cells (Knight et al., 2014). Furthermore, proteinases from NETs contribute to plaque instability (Hansson et al., 2015). These reports indicate that NETs directly participate in rupture of atherosclerotic plaques (Döring et al., 2017), which triggers platelet aggregation and fibrin deposition at the initial site of atherothrombosis. After plaque rupture, thrombin-activated platelets interact with neutrophils at the injured site inducing more formation of NETs (Stakos et al., 2015). Thus, neutrophils and NETs are major contributors to atherothrombosis. Different from arteries, thrombosis in veins is usually initiated by endothelial injury (Di Nisio et al., 2016) triggered by alteration in the blood flow or endothelial dysfunction (Xu et al., 2017). Subsequently, damaged endothelial cells secrete massive amounts of von Willebrand factor and P-selectin, which adhere to platelets and recruit leukocytes (Etulain et al., 2015 Michels et al., 2016). Platelets then interact directly with neutrophils and promote the production of NETs (Clark et al., 2007). NETs can also stimulate the activation of coagulation cascades (Brill et al., 2012) and not only platelet adhesion (Massberg et al., 2010), but also erythrocyte adhesion (Fuchs et al., 2010). Reciprocally, NETs induce endothelial cell activation through NET-derived proteases, histones, and defensins (Saffarzadeh et al., 2012), creating a positive feedback loop for thrombosis. These findings and studies in mice showing an association between the risk of venous thrombosis and high neutrophil counts (Ramacciotti et al., 2009), confirm that NETs make a substantial contribution to maintenance of venous thrombi.

              Another inflammation process in which NETs play an important role is pancreatitis. Acute pancreatitis is an inflammatory disorder of pancreas for which no specific treatment is available. Important risk factors of acute pancreatitis are formation of gallstones and alcohol abuse (Spanier et al., 2008). The most severe cases of the disease are associated with mortality, with acute respiratory distress syndrome being the most frequent cause of death in the early phase of the disease (Pandol et al., 2007). Obstruction of the pancreatic duct causes blockage of pancreatic secretion, which is accompanied by disorders in organelle function of pancreatic acinar cells (Gukovskaya et al., 2016). These disorders promote co-localization of zymogens-containing vesicles and lysosomes leading to formation of co-localization organelles. In co-localized organelles cathepsin B activates trypsinogen to trypsin (Halangk et al., 2000 Van Acker et al., 2002), which mediates acinar cell death and thus causing severe inflammation of the pancreas (Lankisch et al., 2015 Manohar et al., 2017). The destroyed tissue will eventually be replaced by fatty tissue, typical of chronic pancreatitis (Braganza et al., 2011), if the original acute pancreatitis is not resolved (Braganza et al., 2011). Neutrophils infiltrate the pancreatic parenchyma during this acute inflammatory response (Lankisch et al., 2015), and can augment trypsinogen activation via ROS. Both trypsin activation and pancreatic injury were reduced in NADPH oxidase-deficient mice(Gukovskaya et al., 2002). In addition, infiltrating neutrophils aggravate inflammation by releasing NETs in the pancreas and at the sites of systemic injury, namely, the lungs (Merza et al., 2015). NETs incubated in vitro with pancreatic acinar cells led to trypsin activation in these cells, degradation of the NETs by treatment with deoxyribonuclease (DNAse) abolished the trypsin activation, reduced local acinar damage, and systemic inflammation (Merza et al., 2015). Furthermore, neutrophils enter the pancreatic ducts and there they form large deposits of NETs, also known as aggregated NETs (aggNETs). AggNETs in turn obstruct secretory flow and thereby perpetuate inflammation (Leppkes et al., 2016). Therefore, NETs formed after an initial inflammatory stimulus become the activators of further inflammation in the pancreas.

              Contrary to the situations mentioned above, Gout is a disease where NETs appear to have a positive effect. Gout is an acute inflammatory reaction originated from precipitation of uric acid in the form of needle-shaped monosodium urate (MSU) crystals (So and Martinon, 2017). Aggregates of MSU crystals known as tophi, induce inflammation in the joints and tissues. Local immune cells such as macrophages and dendritic cells take up the crystals via phagocytosis. The MSU-containing phagosomes then fuse with lysosomes. The low pH in phagolysosomes causes a massive release of sodium and consequently raises intracellular osmolarity, which is balanced by passive water influx through aquaporins. This process dilutes intracellular sodium and potassium concentrations. The low potassium is a trigger for activation of NLRP3 inflammasomes (Schorn et al., 2011). Inflammasome activation by MSU crystals has been considered a response to �nger,” since damaged cells release urate and ATP into the environment (Busso and So, 2012). As a consequence of inflammasome activation, proIL-1β is cleaved to release active IL-1β and other pro-inflammatory cytokines (Kingsbury et al., 2011 So and Busso, 2014). Cytokine release then leads to a rapid and dramatic recruitment of neutrophils. Recruitment of neutrophils is further mediated by CXCR2, CXCL-8, CXCL-1, CXCL-2, and CXCL-3 (Terkeltaub et al., 1998). This neutrophil influx is accompanied by the infamously intense clinical symptoms of inflammation during an acute gout attack (So and Martinon, 2017). Due to the intense local inflammation, cytokines produced in large quantities can also enter the circulation, resulting in an acute phase response that can trigger fever and leukocytosis (Mauerr et al., 2015). Continuous recruitment of neutrophils to the site of inflammation results in very high neutrophil densities (Shah et al., 2007). After the neutrophil concentration in the tissue exceeds a certain threshold, NETs begin to aggregate and build aggNETs in which the MSU crystals are embedded in a mesh of DNA and proteins from neutrophil granules. As such, these aggNETs block MSU crystals and also trap and degrade pro-inflammatory mediators by serine proteases attached to the DNA fibers (Schauer et al., 2014). MSU crystal-induced aggNET formation is augmented by release of ATP and lactoferrin from activated neutrophils. The release of ATP during NETs formation is of high importance since extracellular nucleotides initiate anti-inflammatory clearance of dead cells by mononuclear phagocytes (Elliott et al., 2009). In addition, lactoferrin on NETs abrogates further recruitment of neutrophils and thus contributes to the anti-inflammatory action of NETs in highly infiltrated tissues (Bournazou et al., 2009). Clearly, in this case NETs have a positive anti-inflammatory effect.

              The inflammation processes described before show that NETs are much more than a simple anti-microbial tool and they can be produced in very different conditions of neutrophil activation. However, NETs are a double-edged sword. On the one hand, �” NETs are involved in stimulating inflammation, as shown in the obstruction of blood vessels and pancreatic ducts. On the other hand “good” NETs are able to contribute to the resolution of inflammation as shown in gout (Hahn et al., 2016).

              In addition, neutrophils primed by microbiota-derived products can form NETs more easily than neutrophils newly released from the bone marrow (Zhang et al., 2015). In contrast, the formation of NETs can be blocked by phagocytosis of small microorganisms via the C-type lectin receptor Dectin-1, which acts as a sensor of microorganism size (Branzk et al., 2014). Dectin-1 downregulates the translocation of neutrophil elastase (NE) to the nucleus. This protease promotes NETosis by degrading histones in the nucleus (Branzk et al., 2014). Also, neutrophils that phagocytose apoptotic cells, lose their capacity to up-regulate 㬢 integrins and to respond to activating stimuli that induce NETs formation (Manfredi et al., 2015). This could explain in part why in conditions in which phagocytosis of apoptotic cells is compromised, such as SLE and anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, a state of persistent inflammation is observed. These findings imply that heterogeneity in neutrophil function (for example for NETs formation) is also regulated by physiological signals.

              It is evident that infection and inflammation can regulate the appearance of neutrophil phenotypes with unique properties. Unfortunately, nowadays we can only (incompletely) describe the function of these neutrophil subtypes. It would become important to characterize these cells at the level of surface markers, functional responses, and transcriptional profiles in order to understand their role in multiple diseases.

              Neutrophil subpopulations in cancer

              The changes in neutrophil phenotype during cancer are perhaps the most impressive and best studied so far. Neutrophils play important and contradictory roles in cancer development, as reflected by several recent reviews (Sionov et al., 2015 Swierczak et al., 2015 Uribe-Querol and Rosales, 2015 Coffelt et al., 2016 Mishalian et al., 2017). In tumor-bearing mice, the number of circulating neutrophils increases along with tumor progression. Similarly, in patients with advanced cancer counts of neutrophils in blood are also increased. It is not clear how tumors can induce neutrophilia, but a common mechanism seems to be the production by tumors of cytokines that influence granulopoiesis, including G-CSF (McGary et al., 1995), IL-1, and IL-6 (Lechner et al., 2010). The presence of elevated numbers of neutrophils in the circulation is associated with poor outcome in several types of cancers (Schmidt et al., 2005). In addition, the presence of neutrophils in tumors also seems to be an indicator of poor outcome (Sionov et al., 2015). For this reason, the counts of neutrophils in blood in relation to other leukocytes have been suggested as a prognostic value in cancer. Therefore, the neutrophil to lymphocyte ratio (NLR) was introduced as a simple and inexpensive biomarker for many types of cancer (Peng et al., 2015 Faria et al., 2016). In general, the blood NLR is high in patients with more advanced or aggressive cancers (Guthrie et al., 2013), and correlates with poor survival of patients with many solid tumors (Paramanathan et al., 2014 Templeton et al., 2014). Despite the simplicity for using the NLR, it has not been accepted in many clinical settings. One reason for this is that neutrophilia can be the result of elevated granulopoiesis and as a consequence, it is not always a bad sign for cancer progression. Another reason is that neutrophilia does not correlate with poor clinical outcome in all types of cancer. In gastric cancer, for example a high NLR is indicative of positive prognosis (Caruso et al., 2002). This is indicative of the great plasticity neutrophils have. They can directly kill tumor cells and control cancer (Yan et al., 2014), but they can also acquire a pro-tumor phenotype and favor cancer (Fridlender and Albelda, 2012). Therefore, the exact role of neutrophils within the tumor is a controversial matter (Sionov et al., 2015 Uribe-Querol and Rosales, 2015).

              Myeloid-derived suppressor cells (MDSC)

              In several types of cancer, not only an increase in the number of neutrophils in blood is observed, but also an increase in immature myeloid cells (Brandau et al., 2013). These immature cells are at various stages of differentiation, accumulate in the spleen of tumor-bearing animals, and present an immunosuppressive phenotype that supports tumor progression (Nagaraj et al., 2010 Raber et al., 2014 Keskinov and Shurin, 2015). For this reason, they were named myeloid-derived suppressor cells (MDSC) (Peranzoni et al., 2010). These MDSC are a heterogeneous mixture of cells that can at least be divided into two subgroups: the granulocytic (G-MDSC) and the monocytic (Mo-MDSC) subgroups (Raber et al., 2014). The G-MDSC group resembles neutrophils. Hence, some researchers considerer them to be a bona fide phenotype of neutrophils (Pillay et al., 2013). However, the relationship among these cells is not clear since immature neutrophils do not have immunosuppressive properties (Solito et al., 2017), and neutrophils in the circulation are differentiated cells characterized by a lobulated nucleus (Pillay et al., 2013) while MDSCs are cells with clear immature morphology, including band or myelocyte-like nuclei (Pillay et al., 2013) (Figures ​ (Figures2, 2 , ​ ,4 4 ).

              Neutrophil subsets that can be separated through density gradient centrifugation. The bulk of mature (normal) neutrophils (PMN) are denser and separate at the bottom of the density gradient. These cells, named high-density neutrophils present the classical neutrophil morphology with a lobulated nucleus and many granules. In the upper part of the gradient, the less dense peripheral blood mononuclear cells (PBMC) are separated. Among these cells, low-density neutrophils can be found. They comprise immature and mature neutrophils with immunosuppressive properties. The immature population is also called granulocytic myeloid derived suppressor cells (G-MDSC).

              Murine neutrophils are defined as CD11b + Ly6G + cells (Daley et al., 2008). Murine G-MDSC are CD11b + and Ly6G + , thus they are considered neutrophils. In contrast Mo-MDSC express CD11b and Ly6C (Brandau et al., 2013 Keskinov and Shurin, 2015), making them more monocytic- than neutrophil-like cells. In humans the problem is more complex, since the Ly6G antigen does not exist. Human mature neutrophils are defined by the phenotype CD14 − CD15 + CD16 + CD66b + (Dumitru et al., 2012). MDSC share all these markers, making it impossible to differentiate these cells from mature neutrophils. An extended panel of six markers (adding CD11b, CD33, and HLA-DR) was used to evaluate human MDSC (Damuzzo et al., 2015). Mo-MDSC were described as CD11b + CD14 + CD15 − CD33 + CD66b + HLA-DR −/low while G-MDSC were described as CD11b + CD14 − CD15 + CD33 + CD66b + HLA-DR − (Brandau et al., 2013 Favaloro et al., 2014 Keskinov and Shurin, 2015). Although, these markers show differences between Mo-MDSC and G-MDSC, they do not allow for clear separation of these cells from neutrophils. At present, it is not possible to distinguish whether MDSC are indeed subpopulations of neutrophils or a separate cell type (Solito et al., 2017), but an international effort continues to find better ways for identification of these cells by flow cytometry (Mandruzzato et al., 2016).

              Low-density neutrophils (LDNs)

              An interesting subpopulation of neutrophils is the so-called low-density neutrophils (LDNs). Traditionally, neutrophils are purified by a Ficoll density gradient (Böyum, 1968 Garc໚-Garc໚ et al., 2013) where they appear at the bottom of the tube (high-density fraction), separated from mononuclear cells, which are found at the interphase of plasma and Ficoll (low-density fraction) (Figure ​ (Figure4). 4 ). In contrast, LDNs are found in the low-density fraction (Sagiv et al., 2015, 2016) (Figure ​ (Figure4). 4 ). Interestingly, the proportion of LDNs in the low-density fraction increases with tumor growth and progression (Mishalian et al., 2017), and includes cells with mature and immature neutrophil morphology (Sagiv et al., 2015) (Figure ​ (Figure4). 4 ). These LDNs are a subpopulation of neutrophils with characteristics and functions not well described.

              Although, LDNs were first reported in the blood of patients with SLE, rheumatoid arthritis, or rheumatic fever (Hacbarth and Kajdacsy-Balla, 1986), they attracted attention only recently because they seem to be associated with cancer (Brandau et al., 2011 Sagiv et al., 2015). These LDNs have been found in many other pathological conditions including sepsis (Morisaki et al., 1992), psoriasis (Lin et al., 2011), HIV infection (Cloke et al., 2012), asthma (Fu et al., 2014), ANCA-associated vasculitis (Grayson et al., 2015), and malaria (Rocha et al., 2015). In addition, LDNs have been reported in natural pregnancy (Ssemaganda et al., 2014). During pregnancy, downregulation of T cell functions is required to ensure materno-fetal tolerance. One way to inhibit T cell function is through the enzyme arginase, which depletes L-arginine, an essential amino acid required for proper expression of the T cell receptor CD3 ζ chain and for T cell proliferation (Rodriguez et al., 2004 Raber et al., 2012). Arginase activity is significantly increased in the peripheral blood of pregnant women and also in term placentae (Kropf et al., 2007). The source for arginase in these tissues was identified as LDNs with the phenotype CD15 + CD33 + CD66b + CD16 low (Ssemaganda et al., 2014). This phenotype is suggestive of a activated neutrophil (Fortunati et al., 2009), and is similar to the phenotype of G-MDSC (Favaloro et al., 2014 Keskinov and Shurin, 2015). Thus, situations of chronic inflammation and immunosuppression appear to induce neutrophil diversity. Very little is known about the function of these subpopulations of neutrophils. LDNs from SLE patients were also reported to readily form NETs (Villanueva et al., 2011), and because these NETs presented autoantigens, it has been suggested that LDNs in these patients are responsible for sustaining chronic inflammation leading to autoimmunity (Garcia-Romo et al., 2011 Khandpur et al., 2013). Similarly, it was recently reported that the number of CD66b + LDNs was markedly elevated in peritoneal cavity after abdominal surgery of gastric cancer (Kanamaru et al., 2018). These LDNs readily formed NETs that selectively attached cancer cells (Kanamaru et al., 2018). These NETs could then assist the clustering and growth of free tumor cells disseminated in the abdomen.

              The origin of LDNs remains unclear. Since LDNs are a mixture of cells with segmented or banded nuclei and myelocyte-like cells, one thought is that LDNs are immature neutrophils that are released from the bone marrow during chronic inflammation or immunosuppression (Denny et al., 2010 Carmona-Rivera and Kaplan, 2013). Another possibility is that these LDNs are activated neutrophils that have undergone degranulation and therefore they have a reduced density (Rocha et al., 2015 Deng et al., 2016). In mouse models of cancer, these LDNs seem to derive either from immature cells released by the bone marrow (Youn et al., 2008) or from normal-density neutrophils (Sagiv et al., 2015). It is important to notice that these LDNs are still not properly characterized. The immunosuppressive function of these cells has not been directly determined and the transition of mature (normal density) to LDNs appears to involve an increase in volume rather than degranulation (Sagiv et al., 2015). Thus, most likely these LDNs are not activated normal neutrophils. In addition, since SLE patients have chronic inflammation, it is unlikely that their LDNs present immunosuppressive activity. All these possibilities need to be further studied in the future. However, one serious limitation for the characterization of these cells is that there are not specific molecular markers that could distinguish among these possible neutrophils subpopulations.

              Tumor-associated neutrophils (TANs)

              The phenotypic changes of circulating neutrophils during tumor progression are also related to infiltration of neutrophils into tumors. Unfortunately, the relationship among immunosuppressive cells (MDSC), LDNs, high-density (normal) neutrophils, and tumor-associated neutrophils (TANs) is just beginning to be elucidated (Mishalian et al., 2013 Uribe-Querol and Rosales, 2015 Figure ​ Figure5 5 ).

              Neutrophils in the circulation display different phenotypes. Mature (normal) neutrophils (PMN) leave the bone marrow and display the classical pro-inflammatory and anti-tumor properties of these cells. It is thought that these PMN can migrate into tumors and display an anti-tumor (N1) phenotype. In tumor-bearing mice, immature neutrophils, such as band cells, also leave the bone marrow into the circulation. These “low-density” neutrophils include granulocytic myeloid derived suppressor cells (G-MDSC) and neutrophils with immunosuppressive properties. These cells can infiltrate tumors and display pro-tumor (N2) phenotype. Under the influence of transforming growth factor-beta (TGF-β, normal PMN can change into “low-density” neutrophils. The exact origin of recruited neutrophils is not known. Also, it is not clear if N1 cells can change into N2 cells and vice versa under the influence of the tumor microenvironment.

              Depending on the phenotype displayed by TANs in tumor-bearing mice, they have been classified as N1 or N2 (Fridlender et al., 2009). This classification is analogous to antitumor tumor-infiltrating macrophages (M1) or protumor macrophages (M2) (Galdiero et al., 2013). Murine N1 TANs are proinflammatory and antitumorigenic. In contrast, N2 TANs are protumorigenic (Fridlender et al., 2009). When tumor-bearing mice were treated to inhibit transforming growth factor-beta (TGF-β functions, the CD11b + Ly6G + neutrophils recruited to tumors were hypersegmented, more cytotoxic and more proinflammatory (N1). In contrast, in presence of TGF-β, had a protumor (N2) phenotype. Therefore, it seems that TGF-β within the tumor microenvironment induces a population of TANs with a protumor activity (Fridlender et al., 2009). This means that TANs can display an antitumor (N1) phenotype or a pro-tumor (N2) phenotype depending on the tumor microenvironment (Sionov et al., 2015). In addition, in tumor-bearing animals the LDNs increased progressively in circulation, were not cytotoxic, and had reduced expression of cytokines (Sagiv et al., 2015). The authors proposed that these immunosuppressive LDNs (G-MDSC) are the source of N2 TANs (Sagiv et al., 2015) (Figure ​ (Figure5). 5 ). This is consistent with the idea that some of the LDNs are indeed immature neutrophils (Sagiv et al., 2015). Also, in the same murine model mature neutrophils were capable of becoming LDNs upon treatment with TGF-β (Sagiv et al., 2015). This has been interpreted as normal mature neutrophils having the capacity to infiltrate tumors and being cytotoxic, but under the influence of TGF-β, being able to change into N2 cells (Mishalian et al., 2013) (Figure ​ (Figure5). 5 ). Although attractive, this hypothesis requires further evidence to be validated, since TGF-β was able to induce this change in tumor-bearing mice, but it had no effect on tumor-free mice (Sagiv et al., 2015).

              It is important to emphasize here that the paradigm of N1 and N2 TANs has been described only in murine models of cancer, and that the nature and function of TANs in the tumor microenvironment remains largely unknown, particularly with human tumors. There are only two reports on isolation of human TANs, but they describe controversial data on their immunosuppression capacity. In one report, TANs were isolated from digested human lung tumors (Eruslanov et al., 2014). These TANs had an activated phenotype (CD62L low CD54 hi ) and produced l proinflammatory cytokines. In consequence, these TANs stimulated T cell proliferation and interferon gamma (IFN-γ) production (Eruslanov et al., 2014). In another report, TANs were isolated from a colorectal tumor and found to present a typical neutrophil morphology. These TANs had the phenotype CD45 + Lin − HLADR − CD11b + CD33 + CD66b + , and were classified as G-MDSC (Wu et al., 2014). These TANs secreted arginase 1 (ARG1) and ROS, and inhibited proliferation of activated autologous T cells and IFN-γ production (Wu et al., 2014). Together these reports suggest that neutrophils are present in three subpopulations in cancer: normal high-density neutrophils, immature LDNs (G-MDSC), and large mature LDNs (Figure ​ (Figure5). 5 ). Early TANs (N1) are not immunosuppressive, but rather stimulate T cell responses (Eruslanov et al., 2014). Latter, the cells acquire an N2 phenotype and become immunosuppressive (Wu et al., 2014) (Figure ​ (Figure5 5 ).

              Immunogenic cell death

              Recently, another important role of neutrophils in anticancer therapy has been described. Immunogenic cell death can stimulate neutrophils to utilize cytotoxicity against residual live cancer cells after therapy. The concept of immunosurveillance explains how only the most immunoevasive or highly mutagenic neoplastic cells are able to generate clinically relevant tumors (Dunn et al., 2002 Schreiber et al., 2011). Yet, the immune system is capable of recognizing altered-self molecules in damaged cells through endogenously derived danger signals or alarmins (Bianchi, 2007 Garg et al., 2014). As a consequence of cell death, alarmins, collectively referred to as �mage-associated molecular patterns” (DAMPs), enhance sensing of dying cells by innate immune cells (Zitvogel et al., 2010 Garg et al., 2015b). Release of DAMPs can either be achieved in an unregulated fashion by necrosis (Aaes et al., 2016) or in a regulated fashion by immunogenic cell death, also called immunogenic apoptosis (Kepp et al., 2014 Garg et al., 2015a). Anti-cancer therapy-induced cancer cell death can be subdivided into three distinct types i.e., tolerogenic cell death, inflammatory cell death, and immunogenic cell death. The details of these types of cell death are beyond the scope of the present publication, but the reader is directed to some excellent recent reviews (Green et al., 2009 Garg et al., 2016 Garg and Agostinis, 2017). Briefly, immunogenic cell death is characterized by a defined discharge of DAMPs, type I interferon response, and the production of pathogen response-like chemokines (CXCL1, CCL2, and CXCL10) (Garg et al., 2017b) that together raise the immunogenic potential of dying cancer cells.

              Through immunogenic cell death, after some types of anti-cancer therapy (Galluzzi et al., 2012 Bezu et al., 2015 Garg et al., 2017a), a damaged cancer cell produces specific inflammatory chemokines to recruit neutrophils as first innate immune cells (Kolaczkowska and Kubes, 2013 Garg et al., 2017b). The damaged cancer cells also express two important �t me” signals on their membranes, namely, phosphatidylserine, and calreticulin. These signals trigger neutrophil phagocytosis of cancer cells and pro-inflammatory stimulation, leading to a change in neutrophil phenotype (Garg et al., 2017b). Neutrophils expressed a mature phenotype characterized by expression of CD86 hi MHC-II hi , and an activated phenotype characterized by IL-6 hi IL-1β hi IL-10 low expression (Garg et al., 2017b). As a result, neutrophils stimulated by immunogenic cell death showed cytotoxicity against residual live cancer cells (Garg et al., 2017b). Thus, neutrophils interacting with immunogenic apoptotic cells gain a pro-inflammatory profile, culminating into neutrophil dependent cytotoxicity against residual cancer cells.


              Prolonged neutrophil survival at necrotic sites is a fundamental feature for tissue recovery and resolution of hepatic inflammation

              Gustavo B. Menezes, Center for Gastrointestinal Biology, Departamento de Morfologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627—Belo Horizonte, Minas Gerais, 31270–901, Brazil.

              Center for Gastrointestinal Biology, Morphology Department, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil

              Center for Gastrointestinal Biology, Morphology Department, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil

              Center for Gastrointestinal Biology, Morphology Department, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil

              Center for Gastrointestinal Biology, Morphology Department, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil

              Center for Gastrointestinal Biology, Morphology Department, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil

              Center for Gastrointestinal Biology, Morphology Department, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil

              Center for Gastrointestinal Biology, Morphology Department, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil

              Center for Gastrointestinal Biology, Morphology Department, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil

              Center for Gastrointestinal Biology, Morphology Department, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil

              Center for Gastrointestinal Biology, Morphology Department, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil

              Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA

              Center for Gastrointestinal Biology, Morphology Department, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil

              Gustavo B. Menezes, Center for Gastrointestinal Biology, Departamento de Morfologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627—Belo Horizonte, Minas Gerais, 31270–901, Brazil.

              Abstract

              Neutrophils were classically described as powerful effectors of acute inflammation, and their main purpose was assumed to be restricted to pathogen killing through production of oxidants. As consequence, neutrophils also may lead to significant collateral damage to the healthy tissues, and after performing these tasks, these leukocytes are supposed to die within tissues. However, there is a growing body of evidence showing that neutrophils also play a pivotal role in the resolution phases of inflammation, because they can modulate tissue environment due to secretion of different kind of cytokines. Drug-induced liver injury (DILI) is a worldwide concern being one of the most prevalent causes of liver transplantation, and is well established that there is an intense neutrophil recruitment into necrotic liver during DILI. However, information if such abundant granulocyte infiltration is also linked to the tissue repairing phase of hepatic injury is still largely elusive. Here, we investigated the dynamics of neutrophil trafficking within blood, bone marrow, and liver during hepatic inflammation, and how changes in their gene expression profile could drive the resolution events during acetaminophen (APAP)-induced liver injury. We found that neutrophils remained viable during longer periods following liver damage, because they avidly patrolled necrotic areas and up-regulated pro-resolutive genes, including Tgfb, Il1r2, and Fpr2. Adoptive transference of “resolutive neutrophils” harvested from livers at 72 h after injury to mice at the initial phases of injury (6 h after APAP) significantly rescued organ injury. Thus, we provide novel insights on the role of neutrophils not only in the injury amplification, but also in the resolution phases of inflammation.

              Supplementary table 1. Sequence of primers used in this study.

              Supplementary table 2. Gene expression relative to control. Values are represented as 2-ΔΔCT ±SEM.

              Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.


              White blood cell

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              White blood cell, also called leukocyte or white corpuscle, a cellular component of the blood that lacks hemoglobin, has a nucleus, is capable of motility, and defends the body against infection and disease by ingesting foreign materials and cellular debris, by destroying infectious agents and cancer cells, or by producing antibodies.

              What is a white blood cell?

              A white blood cell, also known as a leukocyte or white corpuscle, is a cellular component of the blood that lacks hemoglobin, has a nucleus, is capable of motility, and defends the body against infection and disease. White blood cells carry out their defense activities by ingesting foreign materials and cellular debris, by destroying infectious agents and cancer cells, or by producing antibodies. Although white cells are found in the circulation, most occur outside the circulation, within tissues, where they fight infections the few in the bloodstream are in transit from one site to another. White cells are highly differentiated for their specialized functions, and they do not undergo cell division (mitosis) in the bloodstream however, some retain the capability of mitosis.

              What are the major classes of white blood cells?

              On the basis of their appearance under a light microscope, white cells are grouped into three major classes—lymphocytes, granulocytes, and monocytes—each of which carries out somewhat different functions. Lymphocytes, which are further divided into B cells and T cells, are responsible for the specific recognition of foreign agents and their subsequent removal from the host. Granulocytes, the most numerous of the white cells, rid the body of large pathogenic organisms such as protozoans or helminths and are also key mediators of allergy and other forms of inflammation. Monocytes, which constitute between 4 and 8 percent of the total number of white blood cells in the blood, move from the blood to sites of infection, where they differentiate further into macrophages.

              What is a healthy white blood cell count?

              A healthy adult human has between 4,500 and 11,000 white blood cells per cubic millimeter of blood. Fluctuations in white cell number occur during the day lower values are obtained during rest and higher values during exercise. An abnormal increase in the number of white cells is known as leukocytosis, whereas an abnormal decrease in number is known as leukopenia. White cell count may increase in response to intense physical exertion, convulsions, acute emotional reactions, pain, pregnancy, labour, and certain disease states, such as infections and intoxications. The count may decrease in response to certain types of infections or drugs or in association with certain conditions, such as chronic anemia, malnutrition, or anaphylaxis. In general, newborns have a high white blood cell count that gradually falls to the adult level during childhood.

              A healthy adult human has between 4,500 and 11,000 white blood cells per cubic millimetre of blood. Fluctuations in white cell number occur during the day lower values are obtained during rest and higher values during exercise. An abnormal increase in white cell number is known as leukocytosis, whereas an abnormal decrease in number is known as leukopenia. White cell count may increase in response to intense physical exertion, convulsions, acute emotional reactions, pain, pregnancy, labour, and certain disease states, such as infections and intoxications. The count may decrease in response to certain types of infections or drugs or in association with certain conditions, such as chronic anemia, malnutrition, or anaphylaxis.

              Although white cells are found in the circulation, most occur outside the circulation, within tissues, where they fight infections the few in the bloodstream are in transit from one site to another. As living cells, their survival depends on their continuous production of energy. The chemical pathways utilized are more complex than those of red blood cells and are similar to those of other tissue cells. White cells, containing a nucleus and able to produce ribonucleic acid (RNA), can synthesize protein. White cells are highly differentiated for their specialized functions, and they do not undergo cell division (mitosis) in the bloodstream however, some retain the capability of mitosis. On the basis of their appearance under a light microscope, white cells are grouped into three major classes—lymphocytes, granulocytes, and monocytes—each of which carries out somewhat different functions.

              Lymphocytes, which are further divided into B cells and T cells, are responsible for the specific recognition of foreign agents and their subsequent removal from the host. B lymphocytes secrete antibodies, which are proteins that bind to foreign microorganisms in body tissues and mediate their destruction. Typically, T cells recognize virally infected or cancerous cells and destroy them, or they serve as helper cells to assist the production of antibody by B cells. Also included in this group are natural killer (NK) cells, so named for their inherent ability to kill a variety of target cells. In a healthy person, about 25 to 33 percent of white blood cells are lymphocytes.

              Granulocytes, the most numerous of the white cells, rid the body of large pathogenic organisms such as protozoans or helminths and are also key mediators of allergy and other forms of inflammation. These cells contain many cytoplasmic granules, or secretory vesicles, that harbour potent chemicals important in immune responses. They also have multilobed nuclei, and because of this they are often called polymorphonuclear cells. On the basis of how their granules take up dye in the laboratory, granulocytes are subdivided into three categories: neutrophils, eosinophils, and basophils. The most numerous of the granulocytes—making up 50 to 80 percent of all white cells—are neutrophils. They are often one of the first cell types to arrive at a site of infection, where they engulf and destroy the infectious microorganisms through a process called phagocytosis. Eosinophils and basophils, as well as the tissue cells called mast cells, typically arrive later. The granules of basophils and of the closely related mast cells contain a number of chemicals, including histamine and leukotrienes, that are important in inducing allergic inflammatory responses. Eosinophils destroy parasites and also help to modulate inflammatory responses.

              Monocytes, which constitute between 4 and 8 percent of the total number of white blood cells in the blood, move from the blood to sites of infection, where they differentiate further into macrophages. These cells are scavengers that phagocytose whole or killed microorganisms and are therefore effective at direct destruction of pathogens and cleanup of cellular debris from sites of infection. Neutrophils and macrophages are the main phagocytic cells of the body, but macrophages are much larger and longer-lived than neutrophils. Some macrophages are important as antigen-presenting cells, cells that phagocytose and degrade microbes and present portions of these organisms to T lymphocytes, thereby activating the specific acquired immune response.

              Specific types of cells are associated with different illnesses and reflect the special function of that cell type in body defense. In general, newborns have a high white blood cell count that gradually falls to the adult level during childhood. An exception is the lymphocyte count, which is low at birth, reaches its highest levels in the first four years of life, and thereafter falls gradually to a stable adult level. See also blood cell formation.

              The Editors of Encyclopaedia Britannica This article was most recently revised and updated by Kara Rogers, Senior Editor.


              AP, acute pancreatitis NETs, neutrophil extracellular traps SIRS, systemic inflammatory response syndrome ALI, acute lung injury SAP, severe acute pancreatitis CDE, choline-deficient ethionine-supplemented G-CSF, granulocyte colony stimulating factor ROS, reactive oxygen species PMN, polymorphonuclear leukocyte LFA-1, lymphocyte function antigen-1 ICAM1, immunoglobulin-like cell adhesion molecule 1 JAM-C, junctional adhesion molecule-C rTEM, reverse transendothelial migration MMP-9, matrix metalloproteinase 9 MPO, myeloperoxidase NE, neutrophil elastase CTSG, Cathepsin G HBP, heparin-binding protein PAD4, peptidyl arginine deiminase 4 ANP, acute necrotizing pancreatitis DNase I, Deoxyribonuclease I IP6K1, platelet inositol hexaphosphatase 1 TRALI, transfusion-related acute lung injury CARS, compensatory anti-inflammatory response syndrome CDE, choline-deficient ethionine NSAIDs, non-steroidal anti-inflammatory drugs.

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              Keywords: neutrophil, pancreatitis, neutrophil granules, NETs, inflammation

              Citation: Wan J, Ren Y, Yang X, Li X, Xia L and Lu N (2021) The Role of Neutrophils and Neutrophil Extracellular Traps in Acute Pancreatitis. Front. Cell Dev. Biol. 8:565758. doi: 10.3389/fcell.2020.565758

              Received: 29 June 2020 Accepted: 22 December 2020
              Published: 21 January 2021.

              Roland Wohlgemuth, Lodz University of Technology, Poland

              Julia Mayerle, LMU Munich University Hospital, Germany
              Halesha Dhurvigere Basavarajappa, Beckman Research Institute, City of Hope, United States

              Copyright © 2021 Wan, Ren, Yang, Li, Xia and Lu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

              † These authors have contributed equally to this work and share first authorship


              Rare Disease Database

              Acquired agranulocytosis is a rare, drug-induced blood disorder that is characterized by a severe reduction in the number of white blood cells (granulocytes) in the circulating blood. The name granulocyte refers to grain-like bodies within the cell. Granulocytes include basophils, eosinophils, and neutrophils.

              Acquired agranulocytosis may be caused by a variety of drugs. However, among the drugs to which a patient may be sensitive are several used in the treatment of cancer (cancer chemotherapeutic agents) and others used as antipsychotic medications (e.g., clozapine). The symptoms of this disorder come about as the result of interference in the production of granulocytes in the bone marrow.

              People with acquired agranulocytosis are susceptible to a variety of bacterial infections, usually caused by otherwise benign bacteria found in the body. Not infrequently, painful ulcers also develop in mucous membranes that line the mouth and/or the gastrointestinal tract.

              Signs & Symptoms

              The first symptoms of acquired agranulocytosis are usually those associated with a bacterial infection such as general weakness, chills, fever, and/or extreme exhaustion. Symptoms that are associated with rapidly falling white blood cell levels (granulocytopenia) may include the development of infected ulcers in the mucous membranes that line the mouth, throat, and/or intestinal tract. Some people with these ulcers may experience difficulty swallowing due to irritation and pain.

              Granulocytopenia causes a concurrent decrease in the number of neutrophils in the circulating blood (neutropenia). As neutrophil levels decrease, the susceptibility of patients with acquired agranulocytosis to bacterial infections becomes even greater. Fevers and abnormal enlargement of the spleen (splenomegaly) are characteristic features of neutropenia. If neutropenia is not treated, bacterial infections can lead to life-threatening complications such as bacterial shock or bacterial contamination of the blood (sepsis.) (For more information on this disorder, choose “Neutropenia” as your search term in the Rare Disease Database.)

              Chronic acquired agranulocytosis generally progresses more slowly than acquired agranulocytosis. Canker sores in the mouth and chronic inflammation of the gums (gingivitis) may be recurring symptoms. Other systemic infections may recur regularly.

              Causes

              Acquired agranulocytosis is almost invariably caused by exposure to drugs and/or chemicals. Any chemical or drug that depresses the activity of the bone marrow may cause agranulocytosis. Some drugs cause this reaction in anyone given large enough doses. Other drugs may cause the reaction in one person but not in another (idiosyncratic). Clinicians do not understand why some people are susceptible to agranulocytosis and others are not.

              In some instances, the action of some drugs or chemicals suggests that the immune system is involved. In the case of gold, or anti-thyroid drugs, or quinidine, among others, antibodies are created that appear to break the granulocytes down.

              Other drugs that interfere with, or inhibit, granulocyte colony formation may induce agranulocytosis. Drugs with this characteristic include valproic acid, carbamazepine, and the beta-lactam antibiotics.

              A complicating factor is that several commonly used anti-cancer drugs are prone to cause agranulocytosis, thus interfering with treatment. The same may be said for several anti-psychotic medications.

              A variety of drugs can cause acquired agranulocytosis and neutropenia by destroying special cells in the bone marrow that later mature and become granulocytes (precursors). These drugs include phenytoin, pyrimethamine, methotrexate, and cytarabine. In rare cases of acute acquired agranulocytosis, destructive action of certain white blood cell antibodies (leukocyte isoantibodies) may be induced by certain drugs such as phenylbutazone, gold salts, sulfapyridine, aminopyrine, meralluride, and dipyrine.

              Affected Populations

              Acquired Agranulocytosis is a rare blood disorder that affects males and females in equal numbers. People who are taking certain medications such as cancer drugs, alkylating agents, anti-thyroid drugs, dibenzepin compounds, or other drugs can be at risk for this disorder.

              Related Disorders

              Symptoms of the following disorders can be similar to those of Acquired Agranulocytosis. Comparisons may be useful for a differential diagnosis:

              Chronic Granulomatous Disease is a very rare blood disorder that affects specialized white blood cells (i.e., neutrophils) and is characterized by widespread granular lesions in many areas of the body. People with this disorder have repeated infectious diseases, including abscesses of the liver respiratory infections and pneumonia and inflammation of the lymph nodes (suppurative lymphadenitis) and the spinal cord (osteomyelitis). Chronic infections may be evident in the liver, gastrointestinal tract, eyes, and/or brain. (For more information on this disorder, choose “Granulomatous Disease” as your search term in the Rare Disease Database.)

              Wegener’s Granulomatosis is a rare multisystem disease that is characterized by inflammation and degenerative changes in the blood vessels that serve the lungs and upper respiratory tract. Acute inflammation may also occur in the blood vessels of the kidneys (glomerulonephritis). The symptoms vary greatly among affected individuals, and the disease can be limited to one or more systems of the body. The first symptoms of Wegener’s Granulomatosis is usually those of an upper respiratory tract infection including nasal discharge, headache, and coughing accompanied by a mucous discharge. Other early symptoms may include fever, a general feeling of ill health, and weight loss. Symptoms that develop later in the course of the disease may affect the eyes, facial nerves, central nervous system, and/or heart. (For more information on this disorder, choose “Wegener’s Granulomatosis” as your search term in the Rare Disease Database.)

              Sarcoidosis is a rare disorder that affects many systems of the body. It is characterized by small round lesions (tubercles) of granular material. Symptoms vary depending on the severity of the disease. They may be absent, slight, or severe. Organ function may be impaired by active granulomatous disease or by fibrous changes that are associated with acute inflammation. The initial symptoms may include fever, weight loss, and/or joint pain. Persistent fever is especially common with liver (hepatic) involvement. Enlargement of lymph glands is also common and usually without symptoms. The lungs and the lymph glands between the lungs are frequently affected, and symptoms may include coughing and difficulty breathing. (For more information on this disorder, choose “Sarcoidosis” as your search term in the Rare Disease Database.)

              Leukemia is a group of malignant blood diseases affecting the white blood cells (leukocytes). These leukocytes play an important part in the body’s defenses against infection. Leukemia can affect both children and adults. Symptoms may include swollen lymph nodes, an enlarged spleen and liver, fevers, weight loss, paleness, fatigue, easy bruising, excessive bleeding, and/or repeated infections. (For more information on this disorder, choose “Leukemia” as your search term in the Rare Disease Database.)

              Myelofibrosis-Osteosclerosis is a rare disorder that is characterized by the growth of fibrous tissue in the bone marrow. This will result in anemia, generalized weakness, and fatigue due to low levels of red blood cells. Severe pain in the abdomen, bones, and joints may also occur. (For more information on this disorder, choose “Myelofibrosis” as your search term in the Rare Disease Database.)

              Diagnosis

              The diagnosis of acquired agranulocytosis is made by combining a thorough history with tests to confirm abnormally low levels of granulocytes in the circulating blood. Regular periodic blood testing is required for individuals who take drugs that place them at high risk for acquired agranulocytosis. In some cases (e.g., people who are taking clozapine), blood tests to monitor granulocyte levels are done on a weekly basis.

              Standard Therapies

              Filgrastim (Neupogen) has been designated an orphan drug and approved by the U.S. Food and Drug Administration (FDA) for the treatment of severe, chronic neutropenia and it has become a standard treatment for acquired agranulocytosis. Filgrastim is one of a class of colony-stimulating factors that does, indeed, stimulate the proliferation and differentiation of neutrophils. It is manufactured by Amgen, Inc., using recombinant DNA technology.

              The treatment of acquired agranulocytosis includes the identification and elimination of drugs or other agents that induce this disorder. Antibiotic medications may also be prescribed if there is a positive blood culture for the presence of bacteria or if a significant local infection develops.

              Treatment in adults with antibiotics should be limited to about 7-10 days since longer duration carries with it a greater risk of kidney (renal) complications and may set the stage for a new infection. When granulocyte levels return to a near normal range, fever and infections will generally subside.

              There is no definitive therapy that can stimulate bone marrow (myeloid) recovery. Corticosteroids are sometimes used to treat shock induced by overwhelming bacterial infection. However, these drugs are not recommended for the treatment of acute agranulocytopenia because they may mask other bacterial infections.

              People with abnormally low levels of immune factors in their blood (hypogammaglobulinemia) associated with acquired agranulocytosis are usually treated with infusions of gamma globulin.

              Mouth and throat ulcers associated with acquired agranulocytosis can be soothed with gargles of salt (saline) or hydrogen peroxide solutions. Anesthetic lozenges may also help to relieve irritation in the mouth and throat. Mouthwashes that contain the antifungal drug nystatin can be used to treat oral fungal infection (i.e., thrush or candida). A semi-solid or liquid diet may become necessary during episodes of acute oral and gastrointestinal inflammation. (For more information on this disorder, choose "Candidiasis" as your search term in the Rare Disease Database.)

              People with chronic granulocytopenia associated with acquired agranulocytosis need to be hospitalized during acute episodes of infection. These affected individuals should be taught to recognize the early symptoms and signs of acute infection and to seek immediate medical attention when necessary. The therapy for chronically affected individuals is similar to that for the acute form of the disease. People with chronic granulocytopenia, who take low-dose oral antibiotics on a rotating basis, must also be monitored for the infections caused by drug-resistant bacteria as well as infections with opportunistic organisms (e.g., fungi, cytomegalovirus). (For more information on this disorder, choose "Opportunistic Infections" as your search term in the Rare Disease Database.)

              Investigational Therapies

              Acquired agranulocytosis, granulocytosis, granulocytopenia, and other related blood disorders may be helped by new biotechnology drugs including granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage-CSF (GM-CSF). G-CSF and GM-CSF may stimulate the production and development of immature blood cells that later become granulocytes, ultimately increasing the number of granulocytes in the blood. These treatments are currently under investigation, and more studies are needed to determine the long-term safety and effectiveness of these factors for the treatment of acquired agranulocytosis.

              Information on current clinical trials is posted on the Internet at www.clinicaltrials.gov. All studies receiving U.S. government funding, and some supported by private industry, are posted on this government web site.

              For information about clinical trials being conducted at the NIH Clinical Center in Bethesda, MD, contact the NIH Patient Recruitment Office:

              For information about clinical trials sponsored by private sources, contact:

              Supporting Organizations

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                • Gaithersburg, MD 20898-8126
                • Phone: (301) 251-4925
                • Toll-free: (888) 205-2311
                • Website: http://rarediseases.info.nih.gov/GARD/
                • P.O. Box 1693
                • Brighton, MI 48116 USA
                • Phone: (877) 326-7117
                • Email: [email protected]
                • Website: http://www.neutropenianet.org
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                • Phone: (204) 489-8454
                • Toll-free: (800) 663-8876
                • Email: [email protected]
                • Website: http://www.neutropenia.ca
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                • Bethesda, MD 20892-0105
                • Phone: (301) 592-8573
                • Email: [email protected]
                • Website: http://www.nhlbi.nih.gov/

                References

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                Fauci AS, Braunwald E, Isselbacher KJ, et al. Eds. Harrison’s Principles of Internal Medicine. 14th ed.McGraw-Hill Companies. New York, NY 1998:354-55.

                Beers MH, Berkow R., eds. The Merck Manual, 17th ed. Whitehouse Station, NJ: Merck Research Laboratories 1999:931-34.

                Andres E, Kurtz JE, Maloisel F. Non-chemotherapy drug-induced agranulocytosis: experience of the Strasbourg teaching hospital. Clin Lab Haematol. 200224:99-106.

                Carlsson G, Fasth A. Infantile genetic agranulocytosis, morbus Kostmann: presentation of six cases from the original “Kostmann family” and a review. Acta Paediatr. 200190:757-64.

                Sheng WH, Hung CC, Chen YC et al. Antithyroid-drug-induced agranulocytosis complicated by life-threatening infections. QJM. 199992:455-61.

                Beauchesne MF, Shalansky SJ. Nonchemotherapy drug-induced agranulocytosis: a review of 118 patients treated with colony-stimulating factor. Pharmacotherpay. 199919:299-305.

                Guest I, Uetrecht J. Drugs that induce neutropenia/agranulocytosis may target specific components of the stromal cell extracellular matrix. Med Hypotheses. 199953:145-51.

                Hagg S, Rosenius S, Spigset O. Long-term combination treatment with clozapine and filgrastim in patients with clozapine-induced agranulocytosis. Int Clin Psychopharmacol. 200318:173-74.

                Joseph F, Younis N, Bowen-Jones D. Treatment of carbimazole-induced agranulocytosis and sepsis with colony-stimulating factor. Int J Clin Pract. 200357:145-46.

                Iverson S, Zahid N, Uetrecht JP. Predicting drug-induced agranulocytosis: Characterizing neutrophil-generated metabolites of a model compound, DMP 406, and assessing the relevance of an in vitro apoptosis assay for identifying drugs that may cause agranulocytosis. Chem Biol Interact. 200210:175-99.

                Andres E, Maloisel F, Kurtz JE, et al. Modern management of non-chemotherapy drug-induced agranulocytosis: a monocentric cohort study of 90 cases and review of the literature. Eur J Intern Med. 200213:324-28.

                Patel NC, Dorson PG, Bettinger TL. Sudden late onset of clozapine induced agranulocytosis. Ann Pharmacother. 200236:1012-15.

                Andres E, Kurtz JE, Martin-Hunyadi C, et al. Nonchemotherpay drug-induced agranulocytosis in elderly patients: the effects of granulocyte colony-stimulating factor. Am J Med. 200215:460-64.

                eMedicine – Agranulocytosis : Article by Ariel Distenfeld, MD

                Years Published

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