The mouse has become an important model for immunological studies including innate immunity. Creating transgenic mice offers unique possibilities to study gene-function relationships. However, relatively little is known about the physiology of neutrophils from wild-type mice. Do they behave like human neutrophils, or are there species-specific differences that need to be considered when extrapolating results from mice to humans? How do we isolate neutrophils from mice? For practical reasons, many studies on mouse neutrophils are done with bone marrow cells. However, human bone marrow neutrophils appear to be heterogeneous and functionally immature. We have isolated and compared neutrophils from mouse bone marrow and from peripheral blood obtained by tail bleeding. Using the same Percoll density gradient for both preparations, we have obtained morphologically mature neutrophils from bone marrow and blood. Both cell populations responded to formylmethionyl-leucyl-phenylalanine (fMLF) with primary and secondary granule release and superoxide production. Quantitative analysis of our data revealed minor differences between cells from bone marrow and blood. Superoxide production and primary granule release were stimulated at lower fMLF concentrations in blood neutrophils. However, the amplitude and the kinetics of maximal responses were similar. The principal difference was the lifespan of the two cell populations. Bone marrow cells survived significantly longer in culture, which may suggest that they are receiving antiapoptic signals that are absent in the blood. Our data suggest that mice have a large reservoir of functionally competent neutrophils in their bone marrow. This reservoir may be needed to replace circulating neutrophils rapidly during infection.

Neutrophil elastase (NE) activity is increased in many diseases. Other families of proteases, including cathepsins and matrix metalloproteases (MMPs), are also present at elevated levels in similar disease conditions. We postulated that NE could induce expression of cathepsins and MMPs in human macrophages. NE exposure resulted in macrophages, producing significantly greater amounts of cathepsin B and latent and active MMP-2. Cathepsin B and MMP-2 activities were decreased in Pseudomonas-infected NE knockout mice compared with wild-type littermates. We also demonstrate that NE can activate NF-κB in macrophages, and inhibition of NF-κB resulted in a reduction of NE-induced cathepsin B and MMP-2. Also, inhibition of TLR-4 or transfection of macrophages with dominant-negative IL-1R-associated kinase-1 resulted in a reduction of NE-induced cathepsin B and MMP-2. This study describes for the first time a novel hierarchy among proteases whereby a serine protease up-regulates expression of MMPs and cathepsins. This has important implications for therapeutic intervention in protease-mediated diseases.

A variety of immune parameters are modified during and after a spaceflight. The effects of spaceflights on cellular immunity are well documented; however, little is known about the effects of these flights on humoral immunity. During the Genesis space experiment, two adult Pleurodeles waltl (urodele amphibian) stayed 5 mo onboard Mir and were subjected to oral immunization. Animals were killed 10 days after their return to earth. IgM and IgY heavy-chain transcripts in their spleens were quantified by Northern blotting. The use of the different VH families (coding for antibody heavy-chain variable domains) in IgM heavy chain transcripts was also analyzed. Results were compared with those obtained with ground control animals and animals reared in classical conditions in our animal facilities. We observed that, 10 days after the return on earth, the level of IgM heavy-chain transcription was normal but the level of IgY heavy-chain transcription was at least three times higher than in control animals. We also observed that the use of the different VH families in IgM heavy-chain transcripts was modified by the flight. These data suggest that the spaceflight affected the antibody response against the antigens contained in the food.

BackgroundIn acutely injured lungs, massively recruited polymorphonuclear neutrophils (PMNs) secrete abnormally neutrophil elastase (NE). Active NE creates a localized proteolytic environment where various host molecules are degraded leading to impairment of tissue homeostasis. Among the hallmarks of neutrophil-rich pathologies is a disrupted epithelium characterized by the loss of cell-cell adhesion and integrity. Epithelial-cadherin (E-cad) represents one of the most important intercellular junction proteins. E-cad exhibits various functions including its role in maintenance of tissue integrity. While much interest has focused on the expression and role of E-cad in different physio- and physiopathological states, proteolytic degradation of this structural molecule and ensuing potential consequences on host lung tissue injury are not completely understood.MethodsNE capacity to cleave E-cad was determined in cell-free and lung epithelial cell culture systems. The impact of such cleavage on epithelial monolayer integrity was then investigated. Using mice deficient in NE in a clinically relevant experimental model of acute pneumonia, we examined whether degraded E-cad is associated with lung inflammation and injury and whether NE contributes to E-cad cleavage. Finally, we checked for the presence of both degraded E-cad and NE in bronchoalveolar lavage samples obtained from patients with exacerbated COPD, a clinical manifestation characterised by a neutrophilic inflammatory response.ResultsWe show that NE is capable of degrading E-cad in vitro and in cultured cells. NE-mediated degradation of E-cad was accompanied with loss of epithelial monolayer integrity. Our in vivo findings provide evidence that NE contributes to E-cad cleavage that is concomitant with lung inflammation and injury. Importantly, we observed that the presence of degraded E-cad coincided with the detection of NE in diseased human lungs.ConclusionsActive NE has the capacity to cleave E-cad and interfere with its cell-cell adhesion function. These data suggest a mechanism by which unchecked NE participates potentially to the pathogenesis of neutrophil-rich lung inflammatory and tissue-destructive diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-016-0449-x) contains supplementary material, which is available to authorized users.

Pulmonary surfactant protein D (SP-D)5 is a collagenous C-type lectin (collectin) that plays important, if not critical, roles in antimicrobial host defense, inflammatory regulation, and surfactant homeostasis (1-3). Although SP-D is primarily synthesized by respiratory epithelial cells and secreted in the alveolar spaces, it is also expressed at mucosal surfaces and other extrapulmonary sites, including the gastrointestinal and genitourinary tracts, where it could play similar roles in inflammatory and immune regulation (4).SP-D consists of one or more trimeric subunits, each with four structurally distinct domains (5, 6). The N-terminal domains mediate the association of subunits and contain conserved cysteine residues (Cys 15 and Cys 20 of the mature protein) that participate in intersubunit cross-links. By contrast, the C-terminal lectin domains mediate binding to microbial cell wall glycoconjugates, such as lipopolysaccharides (LPS), organic particulate antigens, nucleic acids, and specific cellular receptors (5,7,8). The intervening collagenous and neck domains maintain the trimeric structure of SP-D subunits and ensure an appropriate spatial distribution of the terminal lectin domains. Although the trimerization of CRDs is necessary for high affinity binding, higher order oligomerization of trimeric subunits is required for aggregation and bridging interactions of particulate ligands and effects on surfactant metabolism.Acute inflammation is characteristically accompanied by the recruitment and activation of neutrophils. SP-D can directly interact with neutrophils and modulate the antimicrobial functions of neutrophils in vitro (9, 10); it can also modestly enhance macrophage uptake of apoptotic neutrophils (11). SP-D-deficient mice show an exaggerated neutrophil response to viral and bacterial challenge (12,13). Recently, we demonstrated degradation of SP-D by neutrophil serine proteases (NSPs) in vitro and in vivo (14). Notably, all three granule-associated NSPs were able to cleave at specific sites within the functionally important lectin domain, abrogating carbohydrate recognition and bacterial aggregation. Together, these observations suggest a complex interplay between neutrophils and SP-D at sites of acute inflammation in vivo.In addition to proteases, neutrophil granules contain potent oxidant-generating enzymes, and recruited neutrophils are the major source of oxidants in the setting of acute inflammation