Elzbieta Kolaczkowska

Neutrophil recruitment and function in health and inflammation

Neutrophils have traditionally been thought of as simple foot soldiers of the innate immune system with a restricted set of pro-inflammatory functions. More recently, it has become apparent that neutrophils are, in fact, complex cells capable of a vast array of specialized functions. Although neutrophils are undoubtedly major effectors of acute inflammation, several lines of evidence indicate that they also contribute to chronic inflammatory conditions and adaptive immune responses. Here, we discuss the key features of the life of a neutrophil, from its release from bone marrow to its death. We discuss the possible existence of different neutrophil subsets and their putative anti-inflammatory roles. We focus on how neutrophils are recruited to infected or injured tissues and describe differences in neutrophil recruitment between different tissues. Finally, we explain the mechanisms that are used by neutrophils to promote protective or pathological immune responses at different sites.

Platelets and neutrophil extracellular traps collaborate to promote intravascular coagulation during sepsis in mice.

Neutrophil extracellular traps (NETs; webs of DNA coated in antimicrobial proteins) are released into the vasculature during sepsis where they contribute to host defense, but also cause tissue damage and organ dysfunction. Various components of NETs have also been implicated as activators of coagulation. Using multicolor confocal intravital microscopy in mouse models of sepsis, we observed profound platelet aggregation, thrombin activation, and fibrin clot formation within (and downstream of) NETs in vivo. NETs were critical for the development of sepsis-induced intravascular coagulation regardless of the inciting bacterial stimulus (gram-negative, gram-positive, or bacterial products). Removal of NETs via DNase infusion, or in peptidylarginine deiminase-4-deficient mice (which have impaired NET production), resulted in significantly lower quantities of intravascular thrombin activity, reduced platelet aggregation, and improved microvascular perfusion. NET-induced intravascular coagulation was dependent on a collaborative interaction between histone H4 in NETs, platelets, and the release of inorganic polyphosphate. Real-time perfusion imaging revealed markedly improved microvascular perfusion in response to the blockade of NET-induced coagulation, which correlated with reduced markers of systemic intravascular coagulation and end-organ damage in septic mice. Together, these data demonstrate, for the first time in an in vivo model of infection, a dynamic NET-platelet-thrombin axis that promotes intravascular coagulation and microvascular dysfunction in sepsis.

Expression profiles of matrix metalloproteinase 9 in teleost fish provide evidence for its active role in initiation and resolution of inflammation

Matrix metalloproteinase 9 (MMP‐9) belongs to a family of zinc‐dependent endopeptidases. As a consequence of its ability to cleave structural extracellular matrix molecules, mammalian MMP‐9 is associated with vital inflammatory processes such as leucocyte migration and tissue remodelling and regeneration. Interestingly, MMP‐9 genes have been identified in fish, but functional data are still limited and focus on the involvement of MMP‐9 in embryonic development, reproduction and post‐mortem tenderization. Here, we describe the involvement of MMP‐9 in the innate immunity of carp. In carp, MMP‐9 was most notably expressed in classical fish immune organs and in peritoneal and peripheral blood leucocytes, indicating a role of MMP‐9 in immune responses. In our well‐characterized zymosan‐induced peritonitis model for carp, we analysed expression of the MMP‐9 gene and the gelatinolytic levels of both pro‐ and activated forms of MMP‐9. The biphasic profile of MMP‐9 mRNA expression indicated involvement during the initial phase of inflammation and during the later phase of tissue remodelling. Also, in vitro stimulation of carp phagocytes with lipopolysaccharide or concanavalin A increased MMP‐9 gene expression, with a peak at 24 hr. The increase of MMP‐9 mRNA correlated with the peak of MMP‐9 gelatinolytic level in culture supernatants. These results provide evidence for an evolutionarily conserved and relevant role of MMP‐9 in the innate immune response.

Gelatinase B/MMP-9 as an inflammatory marker enzyme in mouse zymosan peritonitis: comparison of phase-specific and cell-specific production by mast cells, macrophages and neutrophils.

Neutrophil infiltration during zymosan peritonitis depends on matrix metalloproteinase-9 (MMP-9) activity as it is impaired both in MMP-9(-/-) and gelatinase inhibitor-treated animals. The producer cells of MMP-9 and their relative contribution are not known. The aim of this study was to identify and compare the cellular sources, timing and intensity of MMP-9 induction by zymosan in the murine peritoneal cavity. We detected MMP-9 mRNA in unstimulated peritoneal leukocytes and its levels increased after zymosan administration. To detect MMP-9 by flow cytometry, we selected and compared two specific monoclonal antibodies. We show that MMP-9 protein was absent in control peritoneal macrophages, whereas already at 30min of peritonitis almost all macrophages were producing the enzyme. Conversely, MMP-9 was constitutively present in unstimulated mast cells. Macrophages turned out to be prevalent MMP-9 producers in the early phase of peritonitis. During later stages macrophages kept the high expression of MMP-9 for at least 6h of inflammation. In contrast, the early phase expression of MMP-9 by neutrophils was limited albeit the highest percentage of MMP-9(+) neutrophils was observed at 2h but absolute numbers of the MMP-9 carrying neutrophils were low at that time. In contrast, during the late phase of peritonitis neutrophils became major producers of MMP-9 as they numerously infiltrated peritoneum. In conclusion, the study reports detection of MMP-9 at the single-cell level during peritonitis, demonstrates unexpectedly fast MMP-9 expression in macrophages and reveals quantitatively phase-specific contribution of mast cells, macrophages and neutrophils.

Imaging the dynamic platelet‐neutrophil response in sterile liver injury and repair in mice

Although platelets have been extensively studied in hemostasis and inflammation, their role is not well understood in sterile liver injury and repair. Using a thermally induced focal liver injury and repair model and multichannel spinning disk confocal microscopy allowed visualization of the dynamic behavior of platelets and neutrophils in this insult. Platelets instantaneously adhered to molecularly altered sinusoidal endothelium adjacent to the afflicted area, paving approximately 200 µm abutting the injury. Platelets remained adherent for at least 4 hours, but dissipated by 8 hours. The early recruitment occurred by GPIIbIIIa (CD41) and the later recruitment was dependent upon both GPIIbIIIa and GPIb (CD42B). Platelets did not occlude the vessels, but rather paved the altered endothelium. Endothelin‐induced vasoconstriction by hepatic stellate cells, and not platelet accumulation or coagulation, was responsible for temporarily restricted perfusion around the injury. Neutrophils crawled into the injury from significant distances through the sinusoids. The crawling neutrophils required the platelet‐paved endothelium given that very little neutrophil recruitment was noted in thrombocytopenic or CD41‐deficient mice. As platelets slowly dissipated, neutrophil recruitment was also halted. Previous work suggested that platelets binding to immobilized neutrophils induced neutrophil extracellular trap (NET) formation in response to infection as well as during thrombosis and other forms of sterile injury. In this model of neutrophils crawling on immobilized platelets, very few NETs were observed and no additional injury was noted. In fact, GPIIbIIIa‐deficient mice had delayed repair. Conclusion: In a liver model of sterile injury and repair, platelets play a critical role in forming a substratum and pave the way for neutrophils to enter the injured site for subsequent repair. (Hepatology 2015;62:1593–1605)

Inflammatory macrophages, and not only neutrophils, die by apoptosis during acute peritonitis.

The central paradigm says that during inflammation, after completing their function, granulocytes die apoptotically in periphery to avoid destruction of self-tissues. Here we aimed to investigate the kinetic aspect of inflammatory leukocyte apoptosis and verify whether apart from neutrophils also other inflammatory leukocytes numerously undergo apoptosis. We observed that in physiological conditions, less than 7% of either resident peritoneal macrophages or lymphocytes die apoptotically. The studies on a model of acute zymosan-induced peritoneal inflammation revealed that there are two waves of inflammatory leukocyte apoptosis. The first wave corresponds to the time of maximal neutrophil accumulation in peritoneum (6h) and the apoptotic death indeed concerns mostly neutrophils (over 30% of those cells), but also more macrophages die at this time (>10%). The second wave (at 3 days) concerns mostly macrophages (20% versus 3-6% for other populations) and coincides with the resolution of inflammation and the dominant presence of macrophages. In contrast, numbers of apoptotic T (1-3%) and B (approximately 5%) cells do not significantly change during the whole peritonitis. The two waves of apoptosis concur with an increase of caspase-8, -9 and -3 at the transcript and activity levels. The apoptosis inducer TNF-alpha is produced only during first hours while nitric oxide throughout all inflammation. Moreover, during the whole course of peritonitis the expression of pro-apoptotic Bax dominates over anti-apoptotic Bcl-2. In conclusion, we characterized kinetics of apoptotic death of inflammatory leukocytes during acute peritoneal inflammation and revealed that both phagocyte populations (neutrophils and macrophages) die numerously in peritoneum.

Enhanced early vascular permeability in gelatinase B (MMP-9)-deficient mice: putative contribution of COX-1-derived PGE2 of macrophage origin.

Increased vascular permeability leading to vascular leakage is a central feature of all inflammatory reactions and is critical for the formation of an inflammatory exudate. The leakage occurs because of gap formation between endothelial cells and breakdown of the basement membrane barriers. The present study aimed to investigate the role of gelatinase B [matrix metalloproteinase 9 (MMP‐9)], known to be involved in neutrophil exudation, in changes of vascular permeability at the early stages of acute zymosan peritonitis. We show that although MMP‐9 is being released already within the first minutes of peritonitis, its lack, induced pharmacologically or genetically, does not decrease but rather increases vasopermeability. In mice treated with an inhibitor of gelatinases (A and B), a tendency to increased vasopermeability existed, and in MMP‐9−/− mice [knockout (KO)], the difference was statistically significant in comparison with their controls. Moreover, in intact KO mice, significantly augmented production of prostaglandin E2 (PGE2) of cyclooxygenase 1 (COX‐1) origin was detected, and depletion of peritoneal macrophages, but not mast cells, decreased vasopermeability in KO mice. Thus, the increase of vasopermeability observed on KO mice is a result of the increased production of COX‐1‐derived PGE2 by peritoneal macrophages. We conclude that genetic deficiency in gelatinase B might lead to the development of a compensatory mechanism involving the COX pathway.

Neutrophil elastase activity compensates for a genetic lack of matrix metalloproteinase‐9 (MMP‐9) in leukocyte infiltration in a model of experimental peritonitis

Extracellular proteolysis of basement membranes and matrix is required for leukocyte diapedesis and migration to the inflammatory focus. Neutrophil elastase (NE) and matrix metalloproteinases (MMPs) are among the enzymes involved in these processes, as shown in mice genetically deprived of such enzymes. However, studies with MMP‐9−/− mice revealed that albeit neutrophil influx is impaired initially in these animals versus controls, neutrophilia is subsequently augmented during later stages of zymosan peritonitis. MMP‐9 as a MMP and NE as a serine protease belong to different enzyme classes. As MMP‐9 and NE are produced by neutrophils and have similar biological effects on matrix remodeling, it was evaluated whether enhanced NE activity might compensate for the lack of MMP‐9. In genetically uncompromised mice, two waves of NE expression and activity during zymosan peritonitis were observed in inflammatory neutrophils and macrophages at the time of influx of the respective cell populations into the peritoneum. Additionally, NE expression was associated with the activity of resident peritoneal mast cells and macrophages, as their depletion reduced NE activity. Most importantly, the NE mRNA and protein expression and activity were enhanced significantly in MMP‐9−/− mice during late stages of zymosan peritonitis. In addition, the application of a selective NE inhibitor restrained enhanced neutrophil accumulation significantly. In conclusion, during acute peritoneal inflammation, NE expression and activity increase gradually, facilitating leukocyte influx. Moreover, increased NE activity might compensate for a genetic lack of MMP‐9 (as detected in MMP‐9−/− mice), resulting in delayed accumulation of neutrophils during late zymosan peritonitis.

Altered apoptosis of inflammatory neutrophils in MMP-9-deficient mice is due to lower expression and activity of caspase-3

Matrix metalloproteinase 9 (MMP-9) is a Zn(2+)-dependent endopeptidase that degrades some of the components of basement membranes and extracellular matrix and thus participates in leukocyte infiltration during inflammation. In a model of zymosan peritonitis, neutrophil infiltration in MMP-deficient (MMP-9(-/-)) mice was significantly weaker at the time of their maximal influx in wild-type mice (6h). However, during the late stages of peritonitis (24h) an extended accumulation of neutrophils was observed in MMP-9(-/-)versus the wild-type mice. Recently, we reported that the ratio of apoptosis of inflammatory leukocytes is impaired in MMP-9(-/-) mice during late peritonitis and the process depends on COX-1-driven PGE(2). Here we scrutinized the alterations in apoptotic mechanisms by comparisons between MMP-9(-/-) and the wild-type mice. Altered apoptosis occurred only during late (24h) peritonitis and concerned only neutrophils, and not macrophages, mast cells or lymphocytes. Furthermore, expression and activity of caspases was altered in MMP-9(-/-) animals, delayed for caspase-8 and -9, and decreased in the case of caspase-3. Also the expression of Bax/Bcl-2 proteins was changed in MMP-9(-/-) mice. These changes, and in particular the impaired neutrophil apoptosis and weaker caspase-3 activity, were restored by the selective COX-1 inhibition. We conclude that in mice lacking MMP-9 the enhanced COX-1-PGE(2) decreases caspase-3 expression and activity leading to impaired apoptosis of inflammatory neutrophils resulting in abnormal accumulation of the cells at the inflammatory focus. The data also reinforce the notion that MMP-9 is a key enzyme in neutrophil biology.

Shedding light on vascular permeability during peritonitis: role of mast cell histamine versus macrophage cysteinyl leukotrienes

Abstract. The inflammatory response consists of sequential steps that are essentially the same whatever the cause and wherever the site. The main purpose of inflammation is to bring fluid, proteins, and cells from the blood into the damaged tissues. Therefore there are mechanisms that allow cells and proteins to gain access to extravascular sites, where and when they are needed if damage and infection has occurred. A critical process for formation of inflammatory exudate is an increase in permeability of local blood vessels. Vasopermeability changes can be usually attributed to mast cells and their mediators but recent studies reveal that also macrophages can be involved in this process. This short commentary discusses new data on cellular origin of major vasoactive mediators, and their receptors during peritoneal inflammation in mice.