Mechanisms of toxicity mediated by neutrophil and eosinophil granule proteins

characterized by the presence of granules in the cytoplasm. Granules provide a safe storage site for granule proteins that play important roles in the immune function of granulocytes. Upon granulocytes activation, diverse proteins are released from the granules into the extracellular space and contribute to the ﬁ ght against infections. In this article, we describe granule proteins of both neutrophils and eosinophils able to kill pathogens and review their anticipated mechanism of antimicrobial toxicity. It should be noted that an excess of granules protein release can lead to tissue damage of the host resulting in chronic in ﬂ ammation and organ dysfunction.


Introduction
Granulocytes are polymorphonuclear (PMN) leukocytes that comprise neutrophils, eosinophils, and basophils, of which neutrophils are the most frequent type found in blood. These white blood cells are an essential part of the innate immune system and play important roles in fighting against infections, but, under pathological conditions, might also cause tissue injury. 1,2 Granules are formed during the differentiation and described as the hallmark of granulocytes that storage "pre-packed" proteins which can be released into the extracellular space upon stimulation. These diverse mediators, once released to the extracellular space, are able to encounter and destroy all types of microorganism, and, in the case of eosinophils and basophils, also known to orchestrate hypersensitivity reactions. 3 Granule proteins form together with a fibrous scaffold build of mitochondrial DNA (mtDNA) extracellular structures called extracellular traps (ETs) that can be formed by all three granulocyte types. ETs comprise an efficient mechanism of host defense through facilitating the binding and killing of invading bacteria and fungi in the extracellular space. Granule proteins with a high affinity for DNA are delivered into ETs and cause a high local concentration of antimicrobial proteins and simultaneously reduce the spread of harmful proteins such as proteinases to adjacent tissues. 4e10 The formation of granules (granulopoiesis) in neutrophils occurs sequentially during myeloid cell differentiation and is regulated by several myeloid transcription factors that are active at specific stages of neutrophil development. Four morphologically distinct granule populations can be distinguished: azurophilic (primary), specific (secondary), gelatinase (tertiary) and secretory granules. 11,12 Azurophilic granules are formed during the promyelocytic stage and represent the largest granules in neutrophils. They contain the most toxic mediators including elastase, myeloperoxidase (MPO), cathepsins as well as defensins, and contribute mainly to the antimicrobial effects of neutrophils. 13e16 In the metamyelocyte stage, specific granules emerge that are characterized by a high content of the glycoprotein lactoferrin. In addition, they also contain antimicrobial compounds, including neutrophil gelatinaseassociated lipocalin (NGAL), human cathelicidin (hCAP-18) and lysozyme. 16 The smallest granules, the gelatinase granules, are the last population formed during neutrophil maturation. They contain metalloproteases, including gelatinase and leukolysin, as well as of few antimicrobials. 16 These three classical granules are formed by budding from the Golgi. 16 In contrast, the secretory vesicles are formed through endocytosis at the end of neutrophil maturation, accordingly, they incorporate mainly plasma-derived proteins such as albumin. 16 Eosinophil granules are subdivided in primary and secondary granules. Primary granules contain Charcot-Leyden crystal protein and eosinophil peroxidase (EPO). Secondary granules are composed of matrix with a crystalline core. Eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN) and EPO are found in the matrix, meanwhile major basic protein (MBP) is restrained in the crystalline core. 17 Antimicrobial proteins (AMP) are ancient molecules also present in granules of both neutrophils and eosinophils evolved as part of host defense to eliminate pathogenic bacteria, fungi, parasites and viruses. 18 Their main modes of action consist of disruptions of bacterial membrane. Furthermore AMPs are able to vanish the electrochemical gradient of the membrane by pore formation or to interfere with DNA, protein or cell wall synthesis and inhibit protein folding after entering into the cytoplasm. 19 AMPs share some structural features: 1) smaller length than 60 amino acids, 2) often a net positive charge, 3) broad-spectrum antimicrobial activity at physiological conditions and 4) hydrophobic and hydrophilic amino acids clusters adopted in an amphipathic shape. 20 Granules of both neutrophils and eosinophils contain proteases which are important for physiological processes. However, their excessive, prolonged or inappropriate proteolytic activity is involved in the offset of diseases. 21 Proteases are subdivided in four classes based on their biochemistry of the active site: 1) serine proteases, 2) metalloproteases, 3) thiol-proteases and 4) aspartate proteases. Serine proteases and metalloproteases are most active at neutral pH thus are involved in extracellular protein digestion. 21 On the other hand, thiol-proteases and aspartate proteases are most active at acidic pH and participate in the degradation of intracellular proteins. 21 Peroxidases in granulocytes are members of the heme peroxidase superfamily. 22 Oxidants and reactive oxygen species (ROS) are required for many physiological processes, nonetheless their excessive production is involved in pathophysiological processes. Peroxidases are able to interact with H 2 O 2 and are required for its cytotoxicity. 23 In this article, we summarize our current understanding on the mechanisms of toxicity of neutrophil and eosinophil granule proteins ( Fig. 1, Table 1) as well as their regulation to inhibit their cytotoxic potential.

Primary granule proteins
Charcot-Leyden crystal (CLC) protein, also known as galectin 10, belongs to the galectin superfamily 24 and is only found in humans, but not in mouse eosinophils. 25 Unlike other galectin members, CLC does not have the ability to bind b-galactosidase 24 or any known mammalian glycan structures. 26 CLC is found to interact with glycosylated EDN and ECP, two human eosinophil granule cationic RNases, but induces no inhibitory function upon binding. Upon IFNg activation of eosinophils, CLC associates rapidly with CD63 positive secondary granules and EDN, suggesting a role of CLC in vesicular transport of cationic RNases. 26 The presence of CLC is required for eosinophil granulogenesis during differentiation. 26 Furthermore, CLC has been shown to be involved in inflammation in allergic, parasitic and other eosinophil-associated diseases and inflammatory reactions. 27 Eosinophil peroxidase (EPO, also known as EPX) catalyzes the two-electron redox reaction H 2 O 2 þ X À þ H þ / HOX þ H 2 O similar to other peroxidases. 28 Unlike for MPO, the physiologic substrate for EPO is still mainly uncertain. Three rather unusual substrates including Br À , NO À 2 and the pseudohalide thiocyanate (SCN À ) are involved in the oxidation by EPO and H 2 O 2 . 28 During degranulation, EPO can be released into large cytoplasmic vacuoles (phagosome) or directly onto the surface of a target. 29 Per-oxidaseeH 2 O 2 eiodide reaction leads to the iodination of protein and killing of bacteria. 30 EPO interacts with the superoxide generated by the NADPH oxidase to provide the bactericidal activity of eosinophils. 31 EPO exhibits cytotoxicity through the release of peroxidase-derived oxidants towards mammalian tumor cells, 32 HIV-1 33 and schistosomula of Schistosoma mansoni 34 when the peroxidase is combined with H 2 O 2 and a halide. Eosinophils adhere to schistosomula coated with antibody and complement. Upon release EPO binds to the surface of schistosomula. The binding itself is non-toxic but the presence of surfacebound EPO can enhance the eosinophil-mediated toxicity against the parasite. 35 Similar effect mediated by EPOeH 2 O 2 ehalide system occurs towards host endothelial cells and is observed in eosinophilic endocarditis. 36 Mycobacterium tuberculosis treated  with EPO exhibit alterations of the cell wall followed by cell wall fragmentation and cell lysis within 120 min, particularly in the presence of H 2 O 2 . 37 Binding of EPO to gram-negative bacteria membranes and subsequent inhibition of LPS and lipid A occurs in a haloperoxidase-independent manner. 38

Secondary granule proteins
Eosinophil granule proteins EDN/RNase 2 and ECP/RNase 3 are reported to be closely related proteins and part of RNase A superfamily. 39 RNase A superfamily itself is an important part of the human AMP family. 40 Members of the RNase A superfamily share a similar primary sequence and structural similarities such as 6e8 conserved cysteine residues forming characteristics distinct disulfide bonds and conserved histidines, as well as a lysine in the active center to catalyze the ribonuclease activity. 41,42 The ribonucleases are reported in mucosal secretions, in several types of immune cells as well as in major organs. 42 EDN exhibits ribonuclease activity against single stranded RNA viruses including respiratory syncytial virus, 43 hepatitis B virus 44 and HIV. 45 On the other hand, ECP has antibacterial and antiparasitic properties. 39,46e48 EDN has been shown to be less cationic and less cytotoxic than ECP and was originally described as a neurotoxin. 39 In contrast, ECP is uniquely expressed in eosinophils, more cationic and exhibits significant less ribonuclease activity compared to EDN or RNase A. 49 In fact, the cytotoxic activity of ECP is independent of its ribonuclease activity. 49 EDN displays the ability to penetrate the viral capsid thus gaining access to the viral RNA genome and inactivating extracellular virions by its ribonuclease activity. 50 Both EDN and ECP mediate cytotoxic effects on keratinocytes through the production of ROS and induction of apoptosis. 51 Additionally, ECP has been shown to promote keratinocyte cellematrix detachment and to upregulate MMP9 expression. 51 ECP is released in association with mitochondrial DNA in the context of forming eosinophil extracellular traps (EETs) which are able to bind and to kill bacteria in the extracellular space. 5,52,53 High-affinity binding of ECP to lipopolysaccharide (LPS) and peptidoglycan causes destabilizing of the bacterial cell wall and subsequently cell membrane depolarization. 54 Moreover, ECP-LPS interactions trigger LPS aggregation and neutralization of LPSstimulated TNF-a production. 55 ECP binds to the membrane phospholipid polar heads by electrostatic interactions. Following clustering of the liposomeeprotein complexes, liposome aggregation and destabilization of the lipid bilayer occurs that lead ultimately to membrane disruption and release of the liposome content. 56 ECP interacts preferably with anionic and zwitterionic phospholipids of cell membranes. The anionic lipids are exposed on the external side of microbial membranes but sequestered on cytoplasmic side of eukaryote host cell membrane explaining a potential mechanism of antibacterial properties. 57 Under in vitro condition, ECP was shown to form amyloid-like fibrils. The amyloid-type aggregates of ECP led to cell agglutination and bacteria clearance preceding the cell death. 58 Major basic protein 1 (MBP-1) is a cationic protein stored in the crystalline core of the eosinophil secondary granule. 59 MBP-1 is produced as a pre-protein which lacks toxicity owing to the presence of the N-terminal domain. Interestingly, the C-terminal region crystallizes to prevent intracellular toxicity when the pro-MBP-1 is cleaved upon granule maturation. Activation of eosinophils decreases the pH within the granules leading to the breakdown of the crystalline core and release of MBP-1 as an active cytotoxic protein. 59 Damage induced by MBP-1 is not selective, hence is directed against pathogen and mammalian target cells. 46,59 MBP-1 exhibit toxicity against bacteria, 46,48,59 schistosomula of S. mansoni 46 as well as host tissue associated with eosinophil infiltration such as intestine, spleen, skin, mucosa and peripheral blood mononuclear cells. 59,60 MBP-1 and EPO are able to cause degranulation of human eosinophils as potent as sIgA beads, a strong agonist for eosinophils. 61 Furthermore, MBP-1 enhances production of the proinflammatory cytokine IL-8. 61 MBP-1 is able to bind and to permeabilize membranes similar to cytotoxicity mediated by ECP that occurs through pore formation on bacterial cell membranes. 48,62 Furthermore, local perturbations of bacterial cell membrane are observed following MBP-1 treatment. 59 Moreover, MBP-1 is able to form amyloids that facilitate antimicrobial activity, 59 comparable to reports of ECP amyloid. 58 Accelerating the fiber formation by heparin decreases toxicity and is in line with the observation of non-toxic large MBP-1 amyloid accumulation in tissues. 59 The extracellular deposits are proposed to function as sequestration of toxic oligomers 59 that can recruit inflammatory cells, 60 including macrophages for the process of phagocytosis. 63 MBP and ECP were found attached to the DNA scaffold of EETs in vitro, 5 in colons of Crohn's disease patients, 5 allergic asthma, 64 acute atopic dermatitis, 52 bullous pemphigoid 52 and eosinophilic esophagitis. 65

Azurophilic granules
MPO is a heme-containing peroxidase of the peroxidase superfamily. In combination with H 2 O 2 and a halide, it catalyzes the formation of reactive oxygen intermediates. 66 MPO is released from azurophilic granules of activated neutrophils either into the phagolysosome or directly into the extracellular space. 21 Hypohalous acids formed by MPO exhibit antibacterial, antiviral as well as antifungal properties and are involved in chronic inflammation when produced in excess. 67 MPO is found in colocalization with mtDNA within NETs. 6 Serine proteases contain a catalytically essential serine residue at their active side. 21 They comprise the largest class of mammalian proteases and are mainly synthesized as inactive precursors. The exception are the three human leukocyte serine proteases elastase, cathepsin G and proteinase 3 that are stored in an active form within the azurophilic granules. 68 Neutrophil serine proteases exhibit antimicrobial activity against bacteria, yeast and fungi. 21 Furthermore, they are involved in several non-infections inflammatory diseases, including arthritis, 69 bullous pemphigoid, 70,71 chronic inflammatory lung diseases 72,73 and tissue damage following ischemia/reperfusion injury. 74 Cathepsin G-knockout mice are more susceptible to Gram-positive bacteria whereas mice lacking neutrophil elastase are more susceptible to Gram-negative bacteria and enterobacteria species. 75,76 Neutrophil elastase binds the bacterial membrane and directly degrades the outer membrane protein A of Escherichia coli or virulence factors of enterobacteria, resulting in the loss of bacterial integrity and cell lysis. 76,77 The human neutrophil serine proteases are upregulated on the cell surface upon activation. They mostly remain bound to the plasma membrane during exocytosis of the granules allowing the neutrophils to modulate their inflammatory response through the preservation of their catalytic activity. 78 Additionally, neutrophil serine proteases display extracellular activities. Serine proteinases are released in NET-forming neutrophils. 4,79 After exocytosis, proteinase 3 is required for proteolytic processing of the human cathelicidin to its active form LL-37. 80 Similarly, mature active IL-18 is released of epithelial cells after stimulation with IFN-g in combination of proteinase 3 and LPS without the activity of caspase-1. 81 In contrast, neutrophil elastase downregulates the biological activity of IL-18 through proteolysis. 82 Neutrophil elastase and cathepsin G abolish the pro-inflammatory effect of bacterial flagellin on epithelial cells through cleavage. 83 Interestingly, by cleaving caspase-8, cathepsin D is able to induce a proapoptotic death pathway in neutrophils themselves. 84,85 Moreover, neutrophil serine proteases are able to regulate chemokine activities by proteolytic modifications. For example, the activity and stability of CXCL8 and CXCL5 are enhanced following their N-terminal proteolytic modification by proteinase 3 and cathepsin G. 86,87 On the other hand, cleavage of CCL3, CXCL12 and CXCR4 results in lower chemotactic activity. 88,89 Recently, it has been shown that fibroblast activation protein e 1 alpha, a serine protease, is expressed in neutrophils and involved in the activation of NADPH oxidase. 90 In neutrophils, four small cationic peptides of a-defensins called human neutrophil peptides 1, 2, 3 and 4 (HNP1-4) are secreted upon activation and exhibit antibacterial, antiviral as well as antifungal activity. 91,92 Defensins are characterized by a b-sheet-rich fold, a high cathodal electrophoretic mobility and a framework of six disulphide-linked cysteines. Three subfamilies are identified: aand b-defensins that are formed by a triple-stranded b-sheet with a "defensin" fold and q-defensins. 93 HNP1 and HNP3 differ only at the first residue, while HNP2 is proteolytic processed of one of these two defensins. 94 HNP1 and HNP3 have been characterized as NETassociated proteins. 79 Defensins are produced as prepropeptides. First steps of maturation include removing of the signal peptide and packaging of the inactive propeptides in azurophilic granules. The full process occurs within the granules mediated by neutrophil elastase and proteinase 3. 95 HNPs are acting against Gram-positive and Gram-negative bacteria through membrane permeabilization. 21 Defensins decrease the membrane potential of target cells within minutes through the creation of small membrane channels followed by the permeabilization of the cells after a short lag period. Interestingly, cells can be rescued by removing the HNPcontaining media after 30 min, hence a second phase of injury and continuing presence of HNP is necessary for cell lysis. 91 HNP2 has been shown to form multimeric pores in lipid bilayers. 96 Defensin-mediated bactericidal activity occurs by outer membrane permeabilization following inner membrane permeabilization. 97 Synchronous inhibition of RNA, DNA and protein production, as well as loss of cellular metabolites and altered ionic cellular environment lead to loss of the bacterial colony-forming potential. 97 Defensins interfere and also disrupt the virus fusion to the host plasma membrane through inhibition of the interactions between viral glycoproteins and cellular receptors. 92 In contrast to antibacterial and antifungal mechanism, direct disruption of the virus lipid bilayer does not occur. Viral envelopes are mainly derived from host cell membranes helping the enveloped virus to escape the defensins. 92 Moreover, at sites of inflammations where defensins are present at high concentrations, they can bind serine proteinase inhibitors (serpins), particularly a1-proteinase inhibitor (a1-PI) and a1-antichymotrypsin (a1-ACT), to prevent their activity against serine proteases. 98 Within the phagolysosome of neutrophils, increased proteolytic activity of serine proteases through defensin-serpin binding may lead to enhanced phagocytic digestion of microbes. 98 Bactericidal/Permeability-Increasing Protein (BPI) belong to the lipid transfer/LPS binding protein family that is characterized by the binding of lipid substrates 99 as well as to the tubular-lipid binding protein (TULIP) family. 100 BPI exerts bactericidal activity against Gram-negative bacteria through the neutralization of LPS by binding to the lipid A moiety. 101 Penetration of the inner bacterial cell membrane induces membrane damage leading eventually to cell death. 102 BPI-mediated toxicity occurs in two stages. In an early, reversible sublethal phase, BPI acts on the outer bacterial membrane and induces growth arrest. Penetration of the cell membrane leads to a time-and pH-dependent lethal stage with the involvement of cytoplasmic membrane damage. 102 BPI is able to reduce LPS-mediated neutrophil stimulation, pyrogenicity and LPS-induced TNF-a production. 103,104 BPI enhances the interaction of bacterial outer membrane vesicles with dendritic cells (DCs) and is involved in the LPS uptake and antigen presentation by DCs. 105 In Gram-positive infections, binding of BPI to bacterial lipopeptides and lipoproteins, as well as lipoteichoic acid enhances their inflammatory effects. 106 The LPS-binding protein (LBP) moiety of the TULIP family binds to the same component of LPS on gram-negative bacterial membrane as BPI. However, LBP helps to initiate the host immune response by bringing small amounts of LPS to effector cells. 106,107 Secretory Leukoproteinase Inhibitor (SLPI), also known as antileukoprotease (ALP), is a endogenous serine protease inhibitor (serpin) produced by epithelial cells and some myeloid cells including neutrophils. 108 SLPI is mainly involved in the protection of local tissues against inflammatory consequences that are caused by proteases including cathepsin G, elastase and trypsin from neutrophils. 109 Furthermore, some studies propose antibacterial and antifungal capacity for SLPI. 110e112 While the proteinase inhibitory activities are shown to be located at the COOH-terminal domain, the antibacterial capacity against Gram-negative and Gram-positive bacteria depend on the NH 2 -terminal domain. 111 SLPI induces toxicity against E. coli by binding to DNA and mRNA leading to inhibition of translation. 110 Fungicidal activity of SLPI emerge against metabolically active Aspergillus fumigatus and Candida albicans. On the other hand, metabolically quiescent A. fumigatus has been reported to be resistant against SLPI. 112 Specific granule proteins LL-37 is expressed by immune cells including neutrophils, monocytes, mast cells, NK cells, B and T cells, as well as stem cells. 113 LL-37 is the mature peptide of 37 amino acids released from the C-terminus of the propeptide hCAP18. 114 hCAP18 is processed by extracellular proteolysis of the C-terminal end of the human cationic antimicrobial protein (hCAP). 115 hCAP is the only cathelicidin family member, one of the major AMP families, known in humans. They are comprised of a highly conserved N-terminal signal peptide e "cathelin domain" e and a structurally variable cationic AMP at the C-terminus. 116 LL-37 is active against the anionic membrane of Gram-positive and Gram-negative bacteria, similar to other cationic AMPs. The positive charge of the peptide enhances the binding to negatively charged bacterial membranes rather than mammalian cell membranes. 117 The importance of the electrostatic attraction for the antibacterial capacity is underlined by the fact that modification of the bacterial surface influences the susceptibility of the cells to LL-37. 118e120 LL-37 and its propeptide hCAP18 are released within NETs. LL-37 binds to the DNA scaffold of the extracellular traps, 121 reportedly leading to the loss of its antibacterial capacity. 122,123 Stephan et al. reported the internalization of LL-37:DNA complexes derived from NETs by macrophages enabling antimicrobial activity of LL-37 against intracellular pathogens. 124 Lactoferrin, also known as lactotransferrin, is a basic ironbinding glycoprotein of the secondary granules in neutrophils. 125,126 Leukocytes, macrophages, platelets and bacteria, as well as the gastrointestinal tract exhibit lactoferrin receptors. 125 Lactoferrin is found to be associated to NETs. 79 Lactoferrin exerts a bacteriostatic effect through iron sequestering inhibition of the bacterial proliferation. 127 Additionally, lactoferrin is reported to hold a bactericidal effect and inhibits the proliferation of fungi and viruses. 125 The protein modifies outer membrane of Gram-negative bacteria by binding and releasing LPS. This way, lactoferrin also enhances the entry of lysozyme into Gram-negative bacteria. 128 The modulation of bacterial cell membrane can be blocked by the addition of Ca 2þ and Mg 2þ . 129 Through its iron-binding ability, lactoferrin may interfere with cellular mechanisms including DNA and RNA synthesis, protein synthesis, expression of lymphocyte surface markers, immunoglobulin secretion and interleukin-2 expression. 125 Lactoferrin serves also as ligand for TLR4 of rheumatoid arthritis synovial fibroblasts stimulating increased expressions of inflammatory cytokines and chemokines. 130 Furthermore, lactoferrin has been reported to inhibit spontaneous apoptosis in human neutrophils if the iron saturation status is low. 126 Neutrophil gelatinase-associated lipocalin (NGAL, also known as lipocalin-2) is a member of the lipocalin family that is characterized by the ability of transporting small lipophilic substances, binding to specific cell-surface receptors and forming complexes with soluble macromolecules. 131 NGAL was first identified to be covalently associated with MMP-9. 132 Furthermore, NGAL-MMP-9 complex can be observed in a ternary complex with tissue inhibitor of metalloproteinase-1 (TIMP-1) that results in a 10-fold lower activity of MMP-9. 133 NGAL is able to bind to fMLP, 134 PAF 135 and leukotriene B4. 135 NGAL may also serve as a cofactor of microbial killing by other proteins through the binding to microbicidal, lipophilic ligands or modulating inflammatory responses induced by neutrophils. 136 Furthermore, NGAL has been found to bind to bacterial catecholate-type ferric siderophores preventing growth and spread of Myobacterium tuberculosis 137 and E. coli. 138 NGAL has also been identified in NETs and to be involved in their bactericidal effect in vitro. 139 Lysozyme is a conserved hydrolytic antimicrobial protein that is well known for its importance to host defense. Lysozyme is expressed in phagocytes, hepatocytes, and epithelial cells of mucosal surfaces as well as in body fluids such as blood, tears, urine, saliva and milk. 140 Almost 100 years ago, Fleming et al. observed the bactericidal effect mediated by lysozyme. 141 Lysozyme provokes bacterial cell lysis through targeted hydrolysis of the bacterial cell wall. 141 Lysozyme is identified as a NET-associated protein using a proteomic approach. 79 Lysozyme is supposed to contribute to the antimicrobial activity of NETs due to its action on the bacterial wall. 142 Matrix metalloproteases (MMPs) are characterized by their dependence on intrinsic Zn 2þ ions and extrinsic Ca 2þ . Their many function consist of degradation of all constitutes of the extracellular matrix. 68 Several MMPs are involved in the cleavage and inactivation of a 1 -antitrypsin, an inhibitor of neutrophil elastase, cathepsin G and proteinase 3. 143 Likewise, serine proteases are able to inactivate protease inhibitor like TIMP-1 144 and cystatin C 145 that serve as inhibitors of MMPs. Metalloproteinase-8 (MMP-8; also known as neutrophil collagenase or collagenase 2) is a potent collagenase highly expressed by neutrophils 146 as well as rheumatoid synovial fibroblasts, 147 endothelial cells, 147 activated macrophages, 148 smooth muscle cells, 149 bronchial epithelial cells, 148 mast cells 150 and chondrocytes. 151 MMP-8 are secreted in tuberculosis-associated inflammation driving the tissue destruction through degradation of the collagen. 152 MMP-8 mediates responsiveness of neutrophils to LPS through chemokine modification. 153 Cleavage of collagen by MMP-8 facilitates neutrophil migration to sites of inflammation and injury behind collagen-rich barriers. 146 Gelatinase granule proteins Metalloproteinase-9 (MMP-9; also known as gelatinase B) is a member of the MMP family and is secreted by endothelial cells, 154 monocytes/macrophages, 155 mast cells, 156 eosinophils 157 and neutrophils. 158 MMP-9 degrades collagen, elastin and gelatin of the extracellular matrix upon exocytosis. 159 In allergic asthmatic patients MMP-9 is released by neutrophils after exposure to allergens. 160 MMP-9 cleaves and inactivate SLPI at sites of inflammation preventing its binding to LPS and inhibition of neutrophil elastase activity. 161 Several MMPs including MMP-9 process human IL-1b and TNF precursor into its biologically active form regulating their appearance at inflammatory sites. 162,163 Leukolysin, also known as membrane-type 6 metalloproteinase (MT6-MMP, MMP25), is a GPI-anchored proteinase of the MMP family. 164 Leukolysin is expressed in leukocytes, 165 in SW480 colon carcinoma cells and several brain tumors including anaplastic astrocytomas and glioblastomas. 166 Leukolysin is the most efficient proteinase in the inactivation of a1-PI. a1-PI is found in high concentrations in interstital spaces and circulating plasma to protect tissue from neutrophil serine proteases. Action of leukolysin against a1-PI releases neutrophil elastase, proteinase 3 and cathepsin G from its inhibitor leading ultimately to tissue damage. 167

Conclusions
Granule proteins are secreted from eosinophils and neutrophils into the extracellular space upon activation and consist of a variety of important mediators that are crucial for the function of granulocytes within the innate immunity. The released granule proteins display a great variety of weapons and diverse antimicrobial mechanism that are directed against different kind of pathogens (Table 1). On the other hand, excessive production of granule proteins of eosinophils and neutrophils plays a significant role in the pathogenesis of diverse human diseases. These findings arise the possibility to use granule proteins as diagnostic marker and drug targets for the development of new therapeutics against infectious and chronic inflammatory diseases. The understanding of the functional consequences of the molecular interactions between DNA and granule proteins within NETs and EETs requires additional experimentation.