Annexin A1 regulates neutrophil clearance by macrophages in the mouse bone marrow

Under homeostatic conditions, a proportion of senescent CXCR4(hi) neutrophils home from the circulation back to the bone marrow, where they are phagocytosed by bone marrow macrophages. In this study, we have identified an unexpected role for the anti-inflammatory molecule annexin A1 (AnxA1) as a critical regulator of this process. We first observed that AnxA1(-/-) mice have significantly increased neutrophil numbers in their bone marrow while having normal levels of GM and G colony-forming units, monocytes, and macrophages. Although AnxA1(-/-) mice have more neutrophils in the bone marrow, a greater proportion of these cells are senescent, as determined by their higher levels of CXCR4 expression and annexin V binding. Consequently, bone marrow neutrophils from AnxA1(-/-) mice exhibit a reduced migratory capacity in vitro. Studies conducted in vitro also show that expression of AnxA1 is required for bone marrow macrophages, but not peritoneal macrophages, to phagocytose apoptotic neutrophils. Moreover, in vivo experiments indicate a defect in clearance of wild-type neutrophils in the bone marrow of AnxA1(-/-) mice. Thus, we conclude that expression of AnxA1 by resident macrophages is a critical determinant for neutrophil clearance in the bone marrow.-Dalli, J., Jones, C. P., Cavalcanti, D. M., Farsky, S. H., Perretti, M., Rankin, S. M. Annexin A1 regulates neutrophil clearance by macrophages in the mouse bone marrow. FASEB J. 26, 387-396 (2012). www.fasebj.org

Regulation of Neutrophil Serine Proteases by Intracellular Serpins

Neutrophil granules contain serine proteases that are central components of the antimicrobial weapons of the innate immune system. Neutrophil proteases also contribute to the amplification and resolution of inflammatory responses through defined proteolytic cleavage of mediators, cell surface receptors, and extracellular matrix proteins. In the blood and at mucosal surfaces, neutrophil serine proteases are regulated by serpins found in plasma and by non-serpin secreted inhibitors. Distinct mechanisms leading to neutrophil cell death have been described for the granule serine proteases, neutrophil elastase, cathepsin G, and proteinase-3. Granule leakage in neutrophils triggers death pathways mediated by cathepsin G and proteinase-3, and both proteases are tightly regulated by their inhibitor SERPINB1 in a cell intrinsic manner. Although stored in the same types of granules, neutrophil elastase does not significantly contribute to cell death following intracellular release from granules into the cytoplasm. However, heterozygous mutations in ELANE, the gene encoding elastase, are the cause of severe congenital neutropenia, a life-threatening condition characterized by the death of neutrophils at an early precursor stage in the bone marrow. This chapter focuses on recent work exploring the biology of clade B intracellular serpins that inhibit neutrophil serine proteases and their functions in neutrophil homeostasis and serine protease control at sites of inflammation.

The SerpinB1 knockout mouse a model for studying neutrophil protease regulation in homeostasis and inflammation

SerpinB1 is a clade B serpin, or ov-serpin, found at high levels in the cytoplasm of neutrophils. SerpinB1 inhibits neutrophil serine proteases, which are important in killing microbes. When released from granules, these potent enzymes also destroy host proteins and contribute to morbidity and mortality in inflammatory diseases including emphysema, chronic obstructive pulmonary disease, cystic fibrosis, arthritis, and sepsis. Studies of serpinB1-deficient mice have established a crucial role for this serpin in Pseudomonas aeruginosa infection by preserving lung antimicrobial proteins from proteolysis and by protecting lung-recruited neutrophils from a premature death. SerpinB1⁻/⁻ mice also have a severe defect in the bone marrow reserve of mature neutrophils demonstrating a key role for serpinB1 in cellular homeostasis. Here, key methods used to generate and characterize serpinB1⁻/⁻ mice are described including intranasal inoculation, myeloperoxidase activity, flow cytometry analysis of bone marrow myeloid cells, and elastase activity. SerpinB1-knockout mice provide a model to dissect the pathogenesis of inflammatory disease characterized by protease:antiprotease imbalance and may be used to assess the efficacy of therapeutic compounds.

Neutrophil Elastase Increases Airway Epithelial Nonheme Iron Levels

Alpha-1-antitrypsin (A1AT) deficiency is characterized by increased neutrophil elastase (NE) activity and oxidative stress in the lung. We hypothesized that NE exposure generates reactive oxygen species by increasing lung nonheme iron. To test this hypothesis, we measured bronchoalveolar lavage (BAL) iron and ferritin levels, using inductively coupled plasma (ICP) optical emission spectroscopy and an ELISA, respectively, in A1AT-deficient patients and healthy subjects. To confirm the role of NE in regulating lung iron homeostasis, we administered intratracheally NE or control buffer to rats and measured BAL and lung iron and ferritin. Our results demonstrated that A1AT-deficient patients and rats postelastase exposure have elevated levels of iron and ferritin in the BAL. To investigate the mechanism of NE-induced increased iron levels, we exposed normal human airway epithelial cells to either NE or control vehicle in the presence or absence of ferritin, and quantified intracellular iron uptake using calcein fluorescence and ICP mass spectroscopy. We also tested whether NE degraded ferritin in vitro using ELISA and western analysis. We demonstrated in vitro that NE increased intracellular nonheme iron levels and degraded ferritin. Our results suggest that NE digests ferritin increasing the extracellular iron pool available for cellular uptake.

How hydrogen peroxide and CXCL8 modulate neutrophil recruitment "in vivo"

Tese de doutoramento, Ciências Biomédicas (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Medicina, 2014

SerpinB1 protects the mature neutrophil reserve in the bone marrow

SerpinB1 is among the most efficient inhibitors of neutrophil serine proteases--NE, CG, and PR-3--and we investigated here its role in neutrophil development and homeostasis. We found that serpinB1 is expressed in all human bone marrow leukocytes, including stem and progenitor cells. Expression levels were highest in the neutrophil lineage and peaked at the promyelocyte stage, coincident with the production and packaging of the target proteases. Neutrophil numbers were decreased substantially in the bone marrow of serpinB1(-/-) mice. This cellular deficit was associated with an increase in serum G-CSF levels. On induction of acute pulmonary injury, neutrophils were recruited to the lungs, causing the bone marrow reserve pool to be completely exhausted in serpinB1(-/-) mice. Numbers of myeloid progenitors were normal in serpinB1(-/-) bone marrow, coincident with the absence of target protease expression at these developmental stages. Maturation arrest of serpinB1(-/-) neutrophils was excluded by the normal CFU-G growth in vitro and the normal expression in mature neutrophils of early and late differentiation markers. Normal absolute numbers of proliferating neutrophils and pulse-chase kinetic studies in vivo showed that the bone marrow deficit in serpinB1(-/-) mice was largely restricted to mature, postmitotic neutrophils. Finally, upon overnight culture, apoptosis and necrosis were greater in purified bone marrow neutrophils from serpinB1(-/-) compared with WT mice. Collectively, these findings demonstrate that serpinB1 sustains a healthy neutrophil reserve that is required in acute immune responses.

Lack of cathelicidin processing in Papillon-Lefèvre syndrome patients reveals essential role of LL-37 in periodontal homeostasis.

BACKGROUND Loss-of-function point mutations in the cathepsin C gene are the underlying genetic event in patients with Papillon-Lefèvre syndrome (PLS). PLS neutrophils lack serine protease activity essential for cathelicidin LL-37 generation from hCAP18 precursor. AIM We hypothesized that a local deficiency of LL-37 in the infected periodontium is mainly responsible for one of the clinical hallmark of PLS: severe periodontitis already in early childhood. METHODS To confirm this effect, we compared the level of neutrophil-derived enzymes and antimicrobial peptides in gingival crevicular fluid (GCF) and saliva from PLS, aggressive and chronic periodontitis patients. RESULTS Although neutrophil numbers in GCF were present at the same level in all periodontitis groups, LL-37 was totally absent in GCF from PLS patients despite the large amounts of its precursor, hCAP18. The absence of LL-37 in PLS patients coincided with the deficiency of both cathepsin C and protease 3 activities. The presence of other neutrophilic anti-microbial peptides in GCF from PLS patients, such as alpha-defensins, were comparable to that found in chronic periodontitis. In PLS microbial analysis revealed a high prevalence of Aggregatibacter actinomycetemcomitans infection. Most strains were susceptible to killing by LL-37. CONCLUSIONS Collectively, these findings imply that the lack of protease 3 activation by dysfunctional cathepsin C in PLS patients leads to the deficit of antimicrobial and immunomodulatory functions of LL-37 in the gingiva, allowing for infection with A. actinomycetemcomitans and the development of severe periodontal disease.

Melanocyte homeostasis in vivo tolerates Rb1 loss in a developmentally independent fashion

There has been uncertainty regarding the precise role that the pocket protein Rb1 plays in murine melanocyte homeostasis. It has been reported that the TAT-Cre mediated loss of exon 19 from a floxed Rb1 allele causes melanocyte apoptosis in vivo and in vitro. This is at variance with other findings showing, either directly or indirectly, that Rb1 loss in melanocytes has no noticeable effect in vivo, but in vitro leads to a semi-transformed phenotype. In this study, we show that Rb1-null melanocytes lacking exon 19 do not undergo apoptosis and survive both in vitro and in vivo, irrespective of the developmental stage at which Cre-mediated ablation of the exon occurs. Further, Rb1 loss has no serious long-term ramifications on melanocyte homeostasis in vivo, with Rb1-null melanocytes being detected in the skin after numerous hair cycles, inferring that the melanocyte stem cell population carrying the Cre-mediated deletion is maintained. Consequently, whilst Rb1 loss in the melanocyte is able to alter cellular behaviour in vitro, it appears inconsequential with respect to melanocyte homeostasis in the mouse skin.

Stochastic modelling of T cell homeostasis for two competing clonotypes via the master equation

Stochastic models for competing clonotypes of T cells by multivariate, continuous-time, discrete state, Markov processes have been proposed in the literature by Stirk, Molina-París and van den Berg (2008). A stochastic modelling framework is important because of rare events associated with small populations of some critical cell types. Usually, computational methods for these problems employ a trajectory-based approach, based on Monte Carlo simulation. This is partly because the complementary, probability density function (PDF) approaches can be expensive but here we describe some efficient PDF approaches by directly solving the governing equations, known as the Master Equation. These computations are made very efficient through an approximation of the state space by the Finite State Projection and through the use of Krylov subspace methods when evolving the matrix exponential. These computational methods allow us to explore the evolution of the PDFs associated with these stochastic models, and bimodal distributions arise in some parameter regimes. Time-dependent propensities naturally arise in immunological processes due to, for example, age-dependent effects. Incorporating time-dependent propensities into the framework of the Master Equation significantly complicates the corresponding computational methods but here we describe an efficient approach via Magnus formulas. Although this contribution focuses on the example of competing clonotypes, the general principles are relevant to multivariate Markov processes and provide fundamental techniques for computational immunology.

Body temperature and its effect on leukocyte mobilization, cytokines and markers of neutrophil activation during and after exercise

We investigated the influence of rectal temperature on the immune system during and after exercise. Ten well-trained male cyclists completed exercise trials (90 min cycling at 60% VO(2max) + 16.1 - km time trial) on three separate occasions: once in 18 degrees C and twice in 32 degrees C. Twenty minutes after the trials in 32 degrees C, the cyclists sat for approximately 20 min in cold water (14 degrees C) on one occasion, whereas on another occasion they sat at room temperature. Rectal temperature increased significantly during cycling in both conditions, and was significantly higher after cycling in 32 degrees C than in 18 degrees C (P < 0.05). Leukocyte counts increased significantly during cycling but did not differ between the conditions. The concentrations of serum interleukin (IL)-6, IL-8 and IL-10, plasma catecholamines, granulocyte-colony stimulating factor, myeloperoxidase and calprotectin increased significantly following cycling in both conditions. The concentrations of serum IL-8 (25%), IL-10 (120%), IL-1 receptor antagonist (70%), tumour necrosis factor-alpha (17%), plasma myeloperoxidase (26%) and norepinephrine (130%) were significantly higher after cycling in 32 degrees C than in 18 degrees C. During recovery from exercise in 32 degrees C, rectal temperature was significantly lower in response to sitting in cold water than at room temperature. However, immune changes during 90 min of recovery did not differ significantly between sitting in cold water and at room temperature. The greater rise in rectal temperature during exercise in 32 degrees C increased the concentrations of serum IL-8, IL-10, IL-1ra, TNF-alpha and plasma myeloperoxidase, whereas the greater decline in rectal temperature during cold water immersion after exercise did not affect immune responses.

Characterization of inflammatory responses to eccentric exercise in humans

Eccentric exercise commonly results in muscle damage. The primary sequence of events leading to exercise-induced muscle damage is believed to involve initial mechanical disruption of sarcomeres, followed by impaired excitation-contraction coupling and calcium signaling, and finally, activation of calcium-sensitive degradation pathways. Muscle damage is characterized by ultrastructural changes to muscle architecture, increased muscle proteins and enzymes in the bloodstream, loss of muscular strength and range of motion and muscle soreness. The inflammatory response to exercise-induced muscle damage is characterized by leukocyte infiltration and production of pro-inflammatory cytokines within damaged muscle tissue, systemic release of leukocytes and cytokines, in addition to alterations in leukocyte receptor expression and functional activity. Current evidence suggests that inflammatory responses to muscle damage are dependent on the type of eccentric exercise, previous eccentric loading (repeated bouts), age and gender. Circulating neutrophil counts and systemic cytokine responses are greater after eccentric exercise using a large muscle mass (e.g. downhill running, eccentric cycling) than after other types of eccentric exercise involving a smaller muscle mass. After an initial bout of eccentric exercise, circulating leukocyte counts and cell surface receptor expression are attenuated. Leukocyte and cytokine responses to eccentric exercise are impaired in elderly individuals, while cellular infiltration into skeletal muscle is greater in human females than males after eccentric exercise. Whether alterations in intracellular calcium homeostasis influence inflammatory responses to muscle damage is uncertain. Furthermore, the effects of antioxidant supplements are variable, and the limited data available indicates that anti-inflammatory drugs largely have no influence on inflammatory responses to eccentric exercise. In this review, we compare local versus systemic inflammatory responses, and discuss some of the possible mechanisms regulating the inflammatory responses to exercise-induced muscle damage in humans.

Exercise-induced muscle damage, plasma cytokines and markers of neutrophil activation

Introduction: Unaccustomed eccentric exercise often results in muscle damage and neutrophil activation. We examined changes in plasma cytokines stress hormones, creatine kinase activity and myoglobin concentration, neutrophil surface receptor expression, degranulation, and the capacity of neutrophils to generate reactive oxygen species in response to in vitro stimulation after downhill running. Methods: Ten well-trained male runners ran downhill on a treadmill at a gradient of -10% for 45 min at 60% V̇O2max. Blood was sampled immediately before (PRE) and after (POST), 1 h (1 h POST), and 24 h (24 h POST) after exercise. Results: At POST, there were significant increases (P < 0.01) in neutrophil count (32%), plasma interleukin (IL)-6 concentration (460%), myoglobin (Mb) concentration (1100%), and creatine kinase (CK) activity (40%). At 1 h POST, there were further increases above preexercise values for neutrophil count (85%), plasma Mb levels (1800%), and CK activity (56%), and plasma IL-6 concentration remained above preexercise values (410%) (P < 0.01). At 24 h POST, neutrophil counts and plasma IL-6 levels had returned to baseline, whereas plasma Mb concentration (100%) and CK activity (420%) were elevated above preexercise values (P < 0.01). There were no significant changes in neutrophil receptor expression, degranulation and respiratory burst activity, and plasma IL-8 and granulocyte-colony stimulating factor concentrations at any time after exercise. Neutrophil count correlated with plasma Mb concentration at POST (r = 0.64, P < 0.05), and with plasma CK activity at POST (r = 0.83, P < 0.01) and 1 h POST (r = 0.78, P < 0.01). Conclusion: Neutrophil activation remains unchanged after downhill running in well-trained runners, despite increases in plasma markers of muscle damage.

Neutrophil activation, antioxidant supplements and exercise-induced oxidative stress

Neutrophils produce free radicals known as reactive oxygen species (ROS), which assist in the clearance of damaged host tissue. Tissue damage may occur during exercise due to muscle damage, thermal stress and ischaemia/reperfusion. When produced in excess, neutrophil-derived ROS may overwhelm the body's endogenous antioxidant defence mechanisms, and this can lead to oxidative stress. There is increasing evidence for links between oxidative stress and a variety of pathological disorders such as cardiovascular diseases, cancer, chronic inflammatory diseases and post-ischaemic organ injury. A small number of studies have investigated whether there is a link between neutrophil activation and oxidative stress during exercise. In this review, we have summarised the findings of these studies. Exercise promotes the release of neutrophils into the circulation, and some evidence suggests that neutrophils mobilised after exercise have an enhanced capacity to generate some forms of ROS when stimulated in vitro. Neutrophil activation during exercise may challenge endogenous antioxidant defence mechanisms, but does not appear to increase lipid markers of oxidative stress to any significant degree, at least in the circulation. Antioxidant supplements such as N-acetylcysteine are effective at attenuating increases in the capacity of neutrophils to generate ROS when stimulated in vitro, whereas vitamin E reduces tissue infiltration of neutrophils during exercise. Free radicals generated during intense exercise may lead to DNA damage in leukocytes, but it is unknown if this damage is the result of neutrophil activation. Exercise enhances the expression of inducible haem (heme)-oxygenase (HO-1) in neutrophils after exercise, however, it is uncertain whether oxidative stress is the stimulus for this response.