Vesa-Matti Loitto

Neutrophil leukocyte motility requires directed water influx

The ability of neutrophils to sense and move to sites of infection is essential for our defense against pathogens. For motility, lamellipodium extension and stabilization are prerequisites, but how cells form such membrane protrusions is still obscure. Using contrast‐enhanced video microscopy and Transwell® assays, we show that water‐selective aquaporin channels regulate lamellipodium formation and neutrophil motility. Addition of anti‐aquaporin‐9 antibodies, HgCl2, or tetraethyl ammonium inhibited the function(s) of the channels and blocked motility‐related shape changes. On human neutrophils, aquaporin‐9 preferentially localized to the cell edges, where N‐formyl peptide receptors also accumulated, as assessed with fluorescence microscopy. To directly visualize water fluxes at cell edges, cells were loaded with high dilution‐sensitive, self‐quenching concentrations of fluorophore. In these cells, motile regions always displayed increased fluorescence compared with perinuclear regions. Our observations provide the first experimental support for motility models where water fluxes play a pivotal role in cell‐volume increases accompanying membrane extensions.

Rotavirus stimulates release of serotonin (5-HT) from human enterochromaffin cells and activates brain structures involved in nausea and vomiting.

Rotavirus (RV) is the major cause of severe gastroenteritis in young children. A virus-encoded enterotoxin, NSP4 is proposed to play a major role in causing RV diarrhoea but how RV can induce emesis, a hallmark of the illness, remains unresolved. In this study we have addressed the hypothesis that RV-induced secretion of serotonin (5-hydroxytryptamine, 5-HT) by enterochromaffin (EC) cells plays a key role in the emetic reflex during RV infection resulting in activation of vagal afferent nerves connected to nucleus of the solitary tract (NTS) and area postrema in the brain stem, structures associated with nausea and vomiting. Our experiments revealed that RV can infect and replicate in human EC tumor cells ex vivo and in vitro and are localized to both EC cells and infected enterocytes in the close vicinity of EC cells in the jejunum of infected mice. Purified NSP4, but not purified virus particles, evoked release of 5-HT within 60 minutes and increased the intracellular Ca2+ concentration in a human midgut carcinoid EC cell line (GOT1) and ex vivo in human primary carcinoid EC cells concomitant with the release of 5-HT. Furthermore, NSP4 stimulated a modest production of inositol 1,4,5-triphosphate (IP3), but not of cAMP. RV infection in mice induced Fos expression in the NTS, as seen in animals which vomit after administration of chemotherapeutic drugs. The demonstration that RV can stimulate EC cells leads us to propose that RV disease includes participation of 5-HT, EC cells, the enteric nervous system and activation of vagal afferent nerves to brain structures associated with nausea and vomiting. This hypothesis is supported by treating vomiting in children with acute gastroenteritis with 5-HT3 receptor antagonists.

Aquaporin 9 phosphorylation mediates membrane localization and neutrophil polarization.

Neutrophils are of prime importance in the host innate defense against invading microorganisms by using two primary mechanisms—locomotion toward and phagocytosis of the prey. Recent research points to pivotal roles for water channels known as AQPs in cell motility. Here, we focused on the role of AQP9 in chemoattractant‐induced polarization and migration of primary mouse neutrophils and neutrophil‐like HL60 cells. We found that AQP9 is phosphorylated downstream of fMLFR or PMA stimulation in primary human neutrophils. The dynamics of AQP9 were assessed using GFP‐tagged AQP9 constructs and other fluorescent markers through various live‐cell imaging techniques. Expression of WT or the phosphomimic S11D AQP9 changed cell volume regulation as a response to hyperosmotic changes and enhanced neutrophil polarization and chemotaxis. WT AQP9 and S11D AQP9 displayed a very dynamic distribution at the cell membrane, whereas the phosphorylation‐deficient S11A AQP9 failed to localize to the plasma membrane. Furthermore, we found that Rac1 regulated the translocation of AQP9 to the plasma membrane. Our results show that AQP9 plays an active role in neutrophil volume regulation and migration. The display of AQP9 at the plasma membrane depends on AQP9 phosphorylation, which appeared to be regulated through a Rac1‐dependent pathway.

Nitric oxide induces dose‐dependent CA2+ transients and causes temporal morphological hyperpolarization in human neutrophils

We exposed adherent neutrophils to the nitric oxide (NO)‐radical donors S‐nitroso‐N‐acetylpenicillamine (SNAP), S‐nitrosoglutathione (GSNO), and sodium nitroprusside (SNP) to study the role of NO in morphology and Ca2+ signaling. Parallel to video imaging of cell morphology and migration in neutrophils, changes in intracellular free Ca2+ ([Ca2+]i) were assessed by ratio imaging of Fura‐2. NO induced a rapid and persistent morphological hyperpolarization followed by migrational arrest that usually lasted throughout the 10‐min experiments. Addition of 0.5–800 μM SNAP caused concentration‐dependent elevation of [Ca2+]i with an optimal effect at 50 μM. This was probably induced by NO itself, because no change in [Ca2+]i was observed after treatment with NO donor byproducts, i.e. D‐penicillamine, glutathione, or potassium cyanide. Increasing doses of SNAP (≥200 μM) attenuated the Ca2+ response to the soluble chemotactic stimulus formyl‐methionyl‐leucyl‐phenylalanine (fMLP), and both NO‐ and fMLP‐induced Ca2+ transients were abolished at 800 μM SNAP or more. In kinetic studies of fluorescently labeled actin cytoskeleton, NO markedly reduced the F‐actin content and profoundly increased cell area. Immunoblotting to investigate the formation of nitrotyrosine residues in cells exposed to NO donors did not imply nitrosylation, nor could we mimic the effects of NO with the cell permeant form of cGMP, i.e., 8‐Br‐cGMP. Hence these processes were probably not the principal NO targets. In summary, NO donors initially increased neutrophil morphological alterations, presumably due to an increase in [Ca2+]i, and thereafter inhibited such shape changes. Our observations demonstrate that the effects of NO donors are important for regulation of cellular signaling, i.e., Ca2+ homeostasis, and also affect cell migration, e.g., through effects on F‐actin turnover. Our results are discussed in relation to the complex mechanisms that govern basic cell shape changes, required for migration. J. Cell. Physiol. 182:402–413, 2000.

Assessment of neutrophil N‐formyl peptide receptors by using antibodies and fluorescent peptides

Enrichment of chemoattractant receptors on the neutrophil surface has been difficult to assess, primarily because of limitations in sensitivity of visualization. Using an ultrasensitive, cooled charge‐coupled device camera, we investigated spatial‐temporal relationships between N‐formyl peptide receptor distribution and directional motility of human neutrophils. Live cells were labeled with fluorescent receptor ligands, i.e., fluoresceinated tert‐butyl‐oxycarbonyl‐Phe‐(d)‐Leu‐Phe‐(d)‐Leu‐Phe‐OH (Boc‐FLFLF) and formyl‐Nle‐Leu‐Phe‐Nle‐Tyr‐Lys (fnLLFnLYK), while fixed cells were labeled with either fluorescent peptides or monoclonal antibodies. Double labeling of receptors and filamentous actin (F‐actin) was done to investigate possible colocalization. N‐Formyl peptide receptors on unstimulated cells were randomly distributed. However, on polarized neutrophils, the receptors accumulated toward regions involved in motility and distributed nonuniformly. In fixed neutrophils, antibody‐labeled receptors colocalized with the F‐actin‐rich leading edge whereas peptide‐labeled receptors lagged behind this region. We suggest that neutrophils use an asymmetric receptor distribution for directional sensing and sustained migration. A separation between receptors labeled with peptides and those labeled with antibodies reflects two functionally distinct receptor populations at the membrane of motile neutrophils.

Boar spermatozoa successfully predict mitochondrial modes of toxicity: Implications for drug toxicity testing and the 3R principles

Replacement of animal testing by in vitro methods (3-R principles) requires validation of suitable cell models, preferably obtained non-invasively, defying traditional use of explants. Ejaculated spermatozoa are highly dependent on mitochondrial production and consumption of ATP for their metabolism, including motility display, thus becoming a suitable model for capturing multiple modes of action of drugs and other chemicals acting via mitochondrial disturbance. In this study, a hypothesis was tested that the boar spermatozoon is a suitable cell type for toxicity assessment, providing a protocol for 3R-replacement of animals for research and drug-testing. Boar sperm kinetics was challenged with a wide variety of known frank mito-toxic chemicals with previously shown mitochondrial effects, using a semi-automated motility analyser allied with real-time fluorescent probing of mitochondrial potential (MitoTracker & JC-1). Output of this sperm assay (obtained after 30 min) was compared to cell viability (ATP-content, data obtained after 24-48 h) of a hepatome-cell line (HepG2). Results of compound effects significantly correlated (P<0.01) for all sperm variables and for most variables in (HepG2). Dose-dependent decreases of relative ATP content in HepG2 cells correlated to sperm speed (r=0.559) and proportions of motile (r=0.55) or progressively motile (r=0.53) spermatozoa. The significance of the study relies on the objectivity of computerized testing of sperm motility inhibition which is comparable albeit of faster output than somatic cell culture models. Sperm suspensions, easily and painlessly obtained from breeding boars, are confirmed as suitable biosensors for preclinical toxicology screening and ranking of lead compounds in the drug development processes.

Lymphocytes eject interferogenic mitochondrial DNA webs in response to CpG and non-CpG oligodeoxynucleotides of class C

Significance Release of pathogen- and danger-associated molecular patterns (PAMPs and DAMPs) contributes to inflammatory responses and antiviral signaling. Mitochondrial DNA (mtDNA) is a potent DAMP molecule observed in blood circulation of trauma, autoimmune, HIV, and certain cancer patients. Here, we report a previously unrecognized lymphocyte feature that CpG and non-CpG oligodeoxynucleotides of class C promptly induce release of mtDNA as extracellular web-like structures. Lymphocyte mtDNA webs provoked antiviral type I IFN production in peripheral blood mononuclear cells but were devoid of bactericidal proteins. Notably, cells remained viable after the release. Our findings imply an alternative role for lymphocytes in antiviral signaling by utilizing their mtDNA as a rapid signaling molecule to communicate danger. Circulating mitochondrial DNA (mtDNA) is receiving increasing attention as a danger-associated molecular pattern in conditions such as autoimmunity, cancer, and trauma. We report here that human lymphocytes [B cells, T cells, natural killer (NK) cells], monocytes, and neutrophils derived from healthy blood donors, as well as B cells from chronic lymphocytic leukemia patients, rapidly eject mtDNA as web filament structures upon recognition of CpG and non-CpG oligodeoxynucleotides of class C. The release was quenched by ZnCl2, independent of cell death (apoptosis, necrosis, necroptosis, autophagy), and continued in the presence of TLR9 signaling inhibitors. B-cell mtDNA webs were distinct from neutrophil extracellular traps concerning structure, reactive oxygen species (ROS) dependence, and were devoid of antibacterial proteins. mtDNA webs acted as rapid (within minutes) messengers, priming antiviral type I IFN production. In summary, our findings point at a previously unrecognized role for lymphocytes in antimicrobial defense, utilizing mtDNA webs as signals in synergy with cytokines and natural antibodies, and cast light on the interplay between mitochondria and the immune system.