Neutrophils and Emerging Targets for Treatment in Chronic Obstructive Pulmonary Disease

Mariska Meijer; Ger T Rijkers; Frans J van Overveld


Expert Rev Clin Immunol. 2013;9(11):1055-1068. 

In This Article

Clearing Infectious Agents


The first mechanism that neutrophils usually employ is that of phagocytosis. In this process, the microorganism is engulfed by the neutrophil. An important role in this process is played by Toll-like receptors (TLRs). More specifically, TLR2 triggers phagocytosis upon binding to membrane proteins on Gram-positive bacteria, whereas TLR4 does the same for Gram-negative bacteria. Moreover, these effects can be enhanced when complement factors as C3bi, which is recognized by β2-integrin MAC-1,[22] or antibodies bind to receptors on the neutrophil membrane.[4] If bacteria are opsonized with antibodies, they will bind to Fc receptors on the neutrophil membrane. In resting neutrophils, these are mainly CD32A, CD32C (which trigger phagocytosis and activation of oxidative burst) and CD14. The latter is the LPS receptor that transfers the bound complex to TLR4. Activation of the neutrophil by interferon leads to high-affinity binding by CD64 as well.[23] It has been suggested that opsonization by antibodies and complement factors does not have the same effect on the neutrophil. Though both will lead to phagocytosis, complement-opsonized particles will slowly 'sink in' to the cell, whereas binding of the antibodies to the Fcγ receptor will lead to extension of neutrophil pseudopods until they surround and eventually entrap the particle.[24] After the particle has been trapped, the phagosome must go through a series of changes to make it suitable for pathogen killing. This process is known as phagosome maturation and will be discussed further on.[22]

The exact mechanisms of phagosome formation in neutrophils are not known since neutrophils are rather resilient to the techniques that are often used to study this process in for example macrophages.[22] Most is known about the signals elicited by activation of the Fcγ receptor upon its phosphorylation. This is brought about by activated Src-family kinases, that are recruited toward the receptors as a result of receptor clustering by multivalent ligands.[22] As the receptor is phosphorylated, it becomes a docking site for several proteins, most importantly a tyrosine kinase known as Syk. This protein is crucial in Fc-mediated phagocytosis, as neutrophils from Syk-deficient mice are unable to ingest IgG-opsonized particles.[25] Syk activation leads to an entire cascade of events that eventually results in phagocytosis. In addition, this pathway leads to the polymerization of actin and the subsequent membrane remodeling without which particle ingestion would be impossible.[22]

Phagosome Maturation

After the phagosome has been formed, it must go through a series of events called phagosome maturation. According to the kiss-and-run hypothesis, this is a dynamic process of sequential events that collectively bring about tremendous changes to the contents and membrane of the phagosome.[22] The main components that are to be added are microbicidal enzymes, vacuolar ATPases and the NADPH oxidase complex. In neutrophils, this occurs via sequential fusions with early and late endosomes and finally lysosomes, thus yielding a phagolysosome. In contrast to macrophages, neutrophils differ in the contents of these endosomes. The vesicles and granules in neutrophils contain a highly specialized, heterogeneous and powerful mixture of microbicidal peptides and proteolytic enzymes. Four types of granules have been described and named after the order of development. First of all, primary granules, also known as azurophilic granules, contain, among others myeloperoxidase (MPO) and membrane-bound leukocyte sialoglycoprotein (CD43). Secondary granules, or specific granules, can be identified by their lactoferrin content and membrane-bound carcino-embryonic antigen-related cell adhesion molecule-8 (CD66b). Tertiary granules contain gelatinase but lack CD66b, thus setting them aside from specific granules. The fourth type of granules are secretory vesicles, containing albumin and expressing alkaline phosphatase and complement receptor type-1 (CD35) for C3b/C4b-coated particles on their membrane.[22]

One of the triggers for granule fusion with the phagosome is intracellular calcium. This is released from multiple sources, including the endoplasmic reticulum, which in turn activates the influx of extracellular calcium.[22,26] In addition, it might be released locally by the phagosome itself.[27] At high cytosolic calcium levels, a phospholipid-binding class of proteins known as annexins mediate the fusion of the phagosome with the granules.[28] The fixed order of granule fusion with the phagosome depends on increasing calcium levels and starts with secretory vesicles followed by gelatinase granules, specific granules and ends with the azurophillic granules, due to their different calcium thresholds for secretion.[29] The actin coat of the phagosome prevents the fusion to take place prematurely. Shortly after the phagosome is sealed, the actin coat is degraded allowing for the fusion to take place.[22] Before fusion, the granules become polarized, targeting it for fusion with the phagosome and no other structure. This process is independent of calcium and seems to be guided by microtubules.[30]

Granular Contents

After granular fusion, the granular contents start their work in killing pathogens. However, the total mix of proteins present in phagosomes is diverse with different proteins contributed by different types of granules (Figure 3). Besides granular proteins, the phagosome can also include ROS and RNS.

Figure 3.

Composition of neutrophil granules. Three types of neutrophil granules, secretory vesicles and their contents.

The formation of ROS in neutrophils is triggered by phagocytosis. They are formed by the NADPH phagosome oxidase (NADPH phox) enzyme complex during what is called the respiratory burst.[4] The NADPH phox complex is an electron transport chain. It forms ROS by transferring electrons from cytosolic NADPH to oxygen, thus forming directly the superoxide anion and subsequently hydrogen peroxide (H2O2).[30] The ROS are powerful bactericidal agents. Their effect is aided by RNS that are also formed during the respiratory burst. Nitric oxide is formed by inducible nitrogen oxide synthase and transformed to RNS by the NADPH phox complex. Although this double attack is very powerful, not all microorganisms are susceptible to it. Therefore, the granular proteins remain very important.[4]

Azurophillic Granules As mentioned before, one of the key proteins of azurophillic granules is MPO. MPO reacts with the H2O2 formed by the NADPH oxidase and increases its toxic potential through oxidation of chloride, tyrosine and nitrite. This leads to the formation of hypochlorous acid (HOCl), other chloride products, tyrosine radicals and reactive oxygen intermediates. All of these products are capable of attacking the surface membrane of pathogens.[31] A second class of proteins that can be found in these granules are alpha-defensins. So far, four different types have been identified, human neutrophil peptide 1–4.[32] All of these peptides are found in the azurophillic granules and can kill a broad range of pathogens, including many bacteria, fungi, enveloped viruses as well as protozoa by creating pores in their membranes.[33–36] Furthermore, upon exocytosis alpha-defensins cause chemotaxis of CD4+ and CD8+ T-cells, mast cells and monocytes to the site of infection.[37–39]

Serprocidins, also known as neutrophil serine proteases (NSPs), are serine proteases with microbicidal activity. They form the third component of azurophillic granules.[40] Three different serprocidins are present: proteinase-3, cathepsin-G and neutrophil elastase (NE).[41–43] In order to prevent excessive harm, inhibitors of these proteins can be found in the plasma and a deficiency of one of these proteins, α1-antitrypsin (A1AT), which inhibits NE, is believed to play a role in the pathogenesis of pulmonary emphysema.[40,44] Finally, a fourth component, the serine protease-related azurocidin, has a role in chemotaxis of monocytes, fibroblasts and T-cells.[44]

Specific Granules &Gelatinase Granules The distinction between specific and gelatinase granules is more a functional than a developmental one, as they develop as a continuum yet differ in their contents.[40] Specific granules are larger and contain more antibiotic substances. However, gelatinase granules are released more easily.[45] It has therefore been suggested that specific granules are mostly involved in antimicrobial activity, whereas gelatinase granules are more important in earlier stages, including extravasation.[40]

An important constituent of gelatinase granules is the so-called natural resistance-associated macrophage protein 1 (Nramp1). Upon granular fusion, Nramp1 is inserted in the phagosomal membrane where it acts as a divalent cation transporter.[45] It is hypothesized that the function of Nramp1 is to deprive microorganisms in the phagosome of divalent metals such as Fe2+, Mn2+ and Zn2+, which are thought to be essential for microbial functioning.[46] Furthermore, gelatinase granules contain two matrix metalloproteases (MMPs), being gelatinase (MMP-9) and leukolysin (MMP-25).[47,48] The latter can also be found in secretory vesicles, the plasma membrane and specific granules, although the gelatinase granules contain the largest fraction.[48] Stored as inactive preforms, the metalloproteases are activated by proteolysis after exocytosis. Together, they are capable of degrading structural components of the extracellular matrix, including collagens, fibronectin, proteoglycans, laminin and gelatin. Furthermore, they are believed to degrade the vascular basement membrane during neutrophil extravasation.[49]

Specific granules contain a third MMP, known as neutrophil collagenase or MMP-8.[50] This MMP aids the other two in the above-mentioned effects. Furthermore, specific granules contain lactoferrin, which, like Nramp1, impairs bacterial growth by sequestering iron.[51,52] It is also capable of binding to LPS on bacterial cell membranes causing irreversible damage through the oxidized iron, which eventually leads to lysis of the bacterial cell.[53] Another component of specific granules that depletes iron from the phagosome, is neutrophil gelatinase-associated lipocalin.[54]

Another component of specific granules is human cathelicidin (hCAP-18). hCAP-18 can exert antibacterial effects as well as induce chemotaxis of more neutrophils, T-cells and monocytes to the site of infection.[55,56] Finally, specific granules are the primary source of lysozyme in neutrophils, although this enzyme can be found in all granules.[57] Lysozyme binds lipopolysaccharides (LPS) of the bacterial wall and thereby can reduce LPS-induced cytokine production, thus contributing to reduction in mortality in septic shock.[58]

Neutrophil Extracellular Traps Recently, it was discovered that neutrophils are able to release their nuclear DNA in the form of an extracellular web, known as NETs.[3] Combined with the DNA are granular antimicrobial proteins, adding to the antimicrobial properties of NETs. However, a standardized procedure to quantify NETs has still to be developed. In addition, NET formation has been mainly associated with autoinflammatory diseases instead of chronic inflammation. However, the presence of NETs has been demonstrated in the lungs of patients suffering from acute lung injury.[59,60]