Neutrophil Retention Research Articles

Use of autologous radiolabelled neutrophils to quantify lung neutrophil retention in healthy volunteers, experimental LPS-induced lung inflammation, and COPD

Background: There is a need for non-invasive imaging protocols to support early-phase studies in man that test the efficacy of drugs targeting neutrophilic inflammation. We aimed to quantify lung neutrophil accumulation and circulating biomarkers in healthy volunteers after saline or inhaled lipopolysaccharide (LPS) administration and in COPD patients with the use of radiolabelled neutrophils and single-photon-emission computed tomography (SPECT/CT). Methods: This was an exploratory study conducted at the Clinical Unit Cambridge (GSK) and Cambridge University Hospitals. 20 stable COPD patients (stage 2/3) and 18 healthy volunteers were studied. Healthy volunteers received either inhaled saline (n=6) or LPS (50 μg; n=12) 90 minutes or 180 minutes before injection of radiolabelled cells. Neutrophils were isolated using hetastarch red cell sedimentation and percoll-plasma gradients and labelled with 99mTechnetium-hexamethylpropyleneamine oxime. SPECT was performed over the thorax/upper abdomen at 45 minutes, 2 hours, 4 hours, and 6 hours post-injection of cells. Circulating IL-6 levels were measured. Pulmonary neutrophil clearance was determined using Patlak-Rutland analysis based on whole-lung volumes of interest drawn separately at each time-point. Parametric tests were applied. Ethics approval was received (14/EE/1273). Study registration number NCT02551614. Findings: We observed an increased rate (mean±1 SD) of accumulation of labelled neutrophils into the lungs of COPD patients (0.0022±0.00097 mL/min/mL lung-blood-distribution volume) and LPS-challenged subjects (0.0025±0.0008 mL/min/mL lung-blood-distribution volume) compared with saline-challenged subjects (0.0006±0.00025 mL/min/mL lung-blood-distribution volume; p=0.0003 and p<0.0001, respectively). Mean baseline IL-6 levels were elevated in COPD patients compared with healthy volunteers (1.453±1.058 pg/mL versus 0.48±0.24 pg/mL). LPS challenge had induced increased circulating IL-6 levels (7.54±2.72 pg/mL) 9 hours post-challenge. Interpretation: These data verify the use of inhaled LPS as a reliable, in vivo, model of lung neutrophilia and support the use of SPECT/CT and autologous radiolabelled neutrophils to quantify 'whole-lung' neutrophil clearance. This platform can be used to assess the efficacy of drugs that target lung neutrophil retention in COPD or after LPS challenge. Funding: GSK funded this study (number 201463). Conflicts of interest: We declare that we have no conflicts of interest.

ABIN1 Determines Severity of Glomerulonephritis via Activation of Intrinsic Glomerular Inflammation

Transcription factor NF-κB regulates expression of numerous genes that control inflammation and is activated in glomerular cells in glomerulonephritis (GN). We previously identified genetic variants for a NF-κB regulatory, ubiquitin-binding protein ABIN1 as risk factors for GN in systemic autoimmunity. The goal was to define glomerular inflammatory events controlled by ABIN1 function in GN. Nephrotoxic serum nephritis was induced in wild-type (WT) and ubiquitin-binding deficient ABIN1[D485N] mice, and renal pathophysiology and glomerular inflammatory phenotypes were assessed. Proteinuria was also measured in ABIN1[D485N] mice transplanted with WT mouse bone marrow. Inflammatory activation of ABIN1[D472N] (D485N homolog) cultured human-derived podocytes, and interaction with primary human neutrophils were also assessed. Disruption of ABIN1 function exacerbated proteinuria, podocyte injury, glomerular NF-κB activity, glomerular expression of inflammatory mediators, and glomerular recruitment and retention of neutrophils in antibody-mediated nephritis. Transplantation of WT bone marrow did not prevent the increased proteinuria in ABIN1[D845N] mice. Tumor necrosis factor-stimulated enhanced expression and secretion of NF-κB-targeted proinflammatory mediators in ABIN1[D472N] cultured podocytes compared with WT cells. Supernatants from ABIN1[D472N] podocytes accelerated chemotaxis of human neutrophils, and ABIN1[D472N] podocytes displayed a greater susceptibility to injurious morphologic findings induced by neutrophil granule contents. These studies define a novel role for ABIN1 dysfunction and NF-κB in mediating GN through proinflammatory activation of podocytes.

Aging Impairs Alveolar Macrophage Phagocytosis and Increases Influenza-Induced Mortality in Mice.

Influenza viral infections often lead to increased mortality in older people. However, the mechanisms by which aging impacts immunity to influenza lung infection remain unclear. We employed a murine model of influenza infection to identify these mechanisms. With aging, we found reduced numbers of alveolar macrophages, cells essential for lung homeostasis. We also determined that these macrophages are critical for influenza-induced mortality with aging. Furthermore, aging vastly alters the transcriptional profile and specifically downregulates cell cycling pathways in alveolar macrophages. Aging impairs the ability of alveolar macrophages to limit lung damage during influenza infection. Moreover, aging decreases alveolar macrophage phagocytosis of apoptotic neutrophils, downregulates the scavenging receptor CD204, and induces retention of neutrophils during influenza infection. Thus, aging induces defective phagocytosis by alveolar macrophages and increases lung damage. These findings indicate that therapies that enhance the function of alveolar macrophages may improve outcomes in older people infected with respiratory viruses.

Analysis of granulocytes and macrophages infiltrating a primary tumor site within the first 72 hours

Abstract While a great deal of work has been reported on innate and adaptive white blood cells infiltrating solid tumors, as well as the pre-metastatic niche, there is a paucity of information concerning the early innate immune response to primary tumors. This is particularly evident in the very early stages; before a solid tumor is palpable. Here we used a gelatin sponge model to investigate white blood cells infiltrating a tumor site 12–72 hours following injection. Initial studies focused on identifying the total number of infiltrating cells as well as the number of lymphocytes, macrophages and granulocytes. While 12 hours after injection there was no difference in the total number of cells infiltrating the tumor site versus a control site injected with saline, at 24 and 48 hours there was a small, yet significant increase in the number of cells at the tumor site. The majority of the cells to arrive at the tumor and saline injected sites within the first 12 hours were Gr1+F4/80+ (60%), and this population of cells decreased to 36% by 72 hours at both sites. These data contrasted with the F4/80+Gr1− cells which increased over time from about 10% at 12 hours to 25% at 72 hours. Moreover, two significant differences were noted between the tumor and saline sites. At 24 hours there were significantly more F4/80+Gr1-cells at the tumor site, and the number of Gr1+F4/80− cells remained higher at the tumor site over time. These data indicate early macrophage recruitment and neutrophil retention at the primary tumor site. Gene expression analysis by quantitative RT-PCR is underway to investigate additional differences in these cell populations.

Intravital Imaging of Neutrophil Recruitment Reveals the Efficacy of FPR1 Blockade in Hepatic Ischemia-Reperfusion Injury.

Neutrophils are considered responsible for the pathophysiological changes resulting from hepatic ischemia-reperfusion (I/R) injury, which is a complication of trauma, shock, liver resection, and transplantation. Recently, evidence is accumulating that formyl-peptide receptor (FPR) signaling constitutes an important danger signal that guides neutrophils to sites of inflammation. This study aimed to investigate dynamic neutrophil recruitment using two-photon laser-scanning microscopy (TPLSM) in response to FPR1 blockade during hepatic I/R. LysM-eGFP mice were subjected to partial warm hepatic I/R. They were pretreated with an FPR1 antagonist, cyclosporine H (CsH), or formyl peptide, fMLF. Liver was imaged after hepatic laser irradiation or I/R using the TPLSM technique. CsH treatment alleviated hepatic I/R injury, as evidenced by decreased serum transaminase levels, reduced hepatocyte necrosis/apoptosis, and diminished inflammatory cytokine, chemokine, and oxidative stress. In contrast, systemic administration of fMLF showed few effects. Time-lapse TPLSM showed that FPR1 blockade inhibited the accumulation of neutrophils in the necrotic area induced by laser irradiation in vivo. In the CsH-treated I/R group, the number and crawling velocity of neutrophils in the nonperfused area were lower than those in the control group. Meanwhile, FPR1 blockade did not affect monocyte/macrophage recruitment. Hepatic I/R promoted the retention of neutrophils and their active behavior in the spleen, whereas CsH treatment prevented their changes. Intravital TPLSM revealed that formyl-peptide-FPR1 signaling is responsible for regulating neutrophil chemotaxis to allow migration into the necrotic area in hepatic I/R. Our findings suggest effective approaches for elucidating the mechanisms of immune cell responses in hepatic I/R.

Homeostatic migration and distribution of innate immune cells in primary and secondary lymphoid organs with ageing.

Ageing of the innate and adaptive immune system, collectively termed immune senescence, is a complex process. One method to understand the components of ageing involves dissociating the effects of ageing on the cells of the immune system, on the microenvironment in lymphoid organs and tissues where immune cells reside and on the circulating factors that interact with both immune cells and their microenvironment. Heterochronic parabiosis, a surgical union of two organisms of disparate ages, is ideal for this type of study, as it has the power to dissociate the age of the cell and the age of the microenvironment into which the cell resides or is migrating. So far, however, it has been used sparingly to study immune ageing. Here we review the limited literature on homeostatic innate immune cell trafficking in ageing in the absence of chronic inflammation. We also review our own recent data on trafficking of innate immune subsets between primary and secondary lymphoid organs in heterochronic parabiosis. We found no systemic bias in retention or acceptance of neutrophils, macrophages, dendritic cells or natural killer cells with ageing in primary and secondary lymphoid organs. We conclude that these four innate immune cell types migrate to and populate lymphoid organs (peripheral lymph nodes, spleen and bone marrow), regardless of their own age and of the age of lymphoid organs.

A novel mechanism for enhanced neutrophil recruitment, retention, and podocyte damage in immune complex-mediated glomerular disease.

Abstract Glomerulonephritis (GN) is characterized by immune complex deposition and ensuing inflammation within the glomerulus of the kidney. Frank J. Dixon, a pioneer in immunologic kidney disease research, reported in 1965 that injection of nephrotoxic antibodies in rats and rabbits resulted in proteinuria and glomerular accumulation of neutrophils, and that depletion of circulating neutrophils reduced this immune-mediated glomerular damage and proteinuria. In the decades since, studies have confirmed a role for glomerular neutrophil accumulation in human GN, but the mechanisms for neutrophil recruitment and neutrophil-directed injury have not been resolved. We previously reported genetic variants for a NF-κB inhibitor ABIN1 as risks for GN. Others also report that glomerular ABIN1 gene expression is altered in GN in mice and humans. Here we present that transgenic disruption of ABIN1 function results in exacerbated podocyte injury, glomerular expression of inflammatory mediators, and glomerular recruitment and retention of neutrophils in a mouse model of acute antibody-mediated GN. Moreover, a novel inhibitor of neutrophil granule release (SNAP23 peptide) also attenuates podocyte injury in this model. These in vivo findings are supported by in vitro experiments showing that secretome from homologous ABIN1 mutant podocytes activates neutrophil chemotaxis and granule release and neutrophil granule contents specifically disrupts cytoskeletal organization of ABIN1 mutant podocytes. These studies unfold a role for ABIN1 dysfunction in a novel neutrophil-mediated mechanism of podocyte injury in GN and presents inhibition of neutrophil granule release as a promising novel therapeutic direction for kidney inflammation.

Endothelin‐receptor A (ETA) mediates enhanced agonist‐induced intracapillary neutrophil retention in lungs of sickle cell disease

Whether or not in an acute crisis, the vascular endothelium in patients with sickle cell disease (SCD) exhibits an activated pro‐inflammatory state, which undoubtedly influences the complex pathophysiology of the disease. Despite this significance, molecular mechanisms underlying such endothelial cell activation remain incompletely understood. Nonetheless, recent studies have implicated endothelin‐1 (ET‐1) as an important mediator for adverse vascular pathologies in patients with SCD. Here we investigated the role of ET‐1 and its receptors in the pro‐inflammatory lung endothelial cell activation in SCD. We first examined the presence of endothelin‐receptors A (ETA) and B (ETB) in rat and mouse pulmonary microvascular endothelial cells (PMVECs). Using sequence‐specific primers to target ETA and ETB, a single product of predicted size for each receptor was amplified from the cDNA from PMVECs; sequence analysis revealed that the cloned product was virtually identical to the previously reported ETA and ETB. RT‐PCR showed that direct adenylyl cyclase activation by forskolin (10 μmol/L), which was reported to increase ETA mRNA in smooth muscle cells, increased both ETA and ETB mRNA in PMVECs following a 6‐hr exposure. Further, RT‐PCR demonstrated an approximately 4‐fold increase in ETA and 6‐fold increase in ETB mRNA in PMVECs following chronic ET‐1 stimulation (20 nmol/L, 6 hr). This ET‐1‐induced increase in ETA and ETB mRNA was partially blocked by ETA antagonist BQ‐123 (1 μmol/L) but not ETB antagonist BQ‐788 (1 μmol/L), indicating that this endothelin receptor upregulation requires an ETA signaling pathway in PMVECs. Finally, we employed an ex vivo polymorphonuclear neutrophil (PMN)‐perfused lung model to determine whether the ET‐1‐ETA signaling pathway mediates an augmented agonist‐stimulated intracapillary neutrophil retention in SCD lungs. Lungs isolated from 12‐week transgenic humanized homozygous SCD (SCD−/−) and heterozygous (SCD+/−) mice were assessed for intravascular PMN sequestration using immunohistochemistry for leukocyte myeloperoxidase following either thrombin perfusion (3 unit/mL, 15 min) or hypoxic ventilation (5% O2, 30 min) followed by perfusion of non‐stimulated rat PMNs (1.0 × 105/mL, 20 min). Thrombin‐ and hypoxia‐stimulated intracapillary neutrophil retention was observed to be more pronounced in both groups of SCD−/− lungs. To examine which endothelin receptor pathway is involved in this process, we treated both groups with antagonists to either ETA, ambrisentan (20 mg/kg/i.p., 3 doses with each at a 24‐hr intervals), or ETA/B, bosentan (20 and 50 mg/kg/i.p., 3 doses with each at a 24‐hr intervals) prior to lung isolation. ETA or ETA/B blocking comparably reduced thrombin‐ and hypoxia‐stimulated intracapillary neutrophil retention in SCD−/− lungs, but had a minimal effect on SCD+/− lungs. Altogether, these results suggest site‐specific endothelial ETA expression and an important functional role of the ET‐1‐ETA signaling pathway in mediating an alveolar capillary endothelial pro‐inflammatory phenotype in SCD lungs.Support or Funding InformationHL‐117684 (to S. Meiler) and HL‐066299 (to S. Wu)

Human neutrophil antigen-3a antibodies induce neutrophil stiffening and conformational activation of CD11b without shedding of L-selectin.

HNA-3a antibodies induce severe transfusion-related acute lung injury (TRALI) in which neutrophils play a major role. As neutrophil passage through the pulmonary microvasculature is a critical step in the pathogenesis of TRALI, we investigated the impact of HNA-3a antibodies on two important factors that could impair granulocyte passage through lung capillaries: the elasticity of neutrophils and the expression and activation of adhesion molecules. The impact of HNA-3a antibodies on the elasticity of neutrophils was investigated using atomic force microscopy (AFM). Neutrophils were settled on poly-2-hydroxyethyl-methacrylate-coated glass slides before treatment with anti-HNA-3a plasma samples, control plasma, or control plasma containing formyl-methionyl-leucyl-phenylalanine (fMLP). Elasticity measurements were carried out in a temperature-controlled perfusion chamber using an atomic force microscopy (AFM) device. The impact of HNA-3a antibodies on the surface expression of total CD11b, activation of CD11b, and L-selectin (CD62L) shedding was investigated by flow cytometry. The functional impact of HNA-3a antibodies on neutrophil adhesion was assessed using fibrinogen-coated plates. HNA-3a antibodies induced stiffening of neutrophils (+24%-40%; p < 0.05) to a similar extent as fMLP. This effect was blocked by treatment of neutrophils with cytochalasin D. While total surface expression of CD11b and L-selectin on neutrophils was largely unaffected, HNA-3a antibodies induced alloantigen-specific activation of CD11b (+72%-107%; p < 0.05) and increased adhesion of neutrophils to fibrinogen. Accumulation of neutrophils in the pulmonary microvasculature during severe TRALI is likely mediated by increased rigidity and CD11b-mediated adhesion of neutrophils leading to retention of neutrophils.

Abstract 162: Immunomodulatory role of CRAMP (cathelicidin-related antimicrobial peptide) in prostate cancer

Abstract Prostate cancer (PCa) is the most commonly diagnosed cancer and the second leading cause of cancer deaths among males in the United States. Successful treatments for patients with metastatic PCa are limited suggesting a need for alternate targets. Recently, we identified that antimicrobial peptides in human (LL-37) and mouse (CRAMP) are positively correlated with PCa progression in humans and mice, respectively. As a host defense peptide, LL-37/CRAMP functions against microbial pathogens, and in chemotaxis of leukocytes at sites of inflammation. Preliminary studies pertaining to immunomodulation of PCa cell-derived CRAMP in situ indicated that CRAMP chemoattracts myeloid-derived suppressor cells (MDSC), a heterogeneous population of myeloid precursors that induce T cell suppression, to tumor microenvironment (TME) indicating the significance of CRAMP in pro-tumorigenic immune cell functions during PCa progression. The present study further evaluated role of CRAMP as an immunomodulator during PCa growth using transplantable mouse PCa cell line, TRAMP-C1, containing varying CRAMP expression in wild-type and CRAMP loss of function mouse (Cnlp-/-) models. Results of the study indicated targeted down-regulation of CRAMP in mouse PCa cells delays tumorigenesis in syngenic immunocompetent mouse model suggesting significance of CRAMP in PCa development. Whereas implantation of PCa cells abrogated of CRAMP resulted in retention of neutrophils and macrophages in spleen at significantly elevated levels, mice bearing CRAMP-expressing tumors displayed decreased number of neutrophils and macrophages in spleen, but increased infiltration toward TME. These results suggest tumor-derived CRAMP possibly mediates infiltration of neutrophils and macrophages toward TME from spleen. Whether the infiltrated neutrophils and macrophages correlate to pro-tumorigenic N2 and M2 cells, respectively, needs to be confirmed. Since host immune cells, mainly neutrophils, produce CRAMP and recruit additional innate effectors to inflammatory sites, we further analyzed whether host immune cell-derived CRAMP exert synergistic effects in PCa growth together with tumor-derived CRAMP by using Cnlp-/- mice. Results indicated not only comparable tumor growth kinetics, but also comparable levels of MDSC and neutrophils in TME between Cnlp-/- and wild-type mice, suggesting that tumor is a key source of CRAMP that functions as a chemoattractant of MDSC and neutrophils. Taken together, present study proposes that tumor-produced CRAMP functions as a chmoattractant for infiltration of pro-tumorigenic immune cells; MDSC, neutrophils, and macrophages that facilitate tumor growth. Citation Format: Ha-Ram Cha, Anandi Sawant, Jonathan Hensel, Carnella Lee, Selvarangan Ponnazhagan. Immunomodulatory role of CRAMP (cathelicidin-related antimicrobial peptide) in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 162. doi:10.1158/1538-7445.AM2014-162

β2-Adrenoreceptor blockade improves early posttrauma hyperglycemia and pulmonary injury in obese rats

Early hyperglycemia after trauma increases morbidity and mortality. Insulin is widely used to control posttrauma glucose, but this treatment increases the risk of hypoglycemia. We tested a novel method for early posttrauma hyperglycemia control by suppressing hepatic glycogenolysis via β2-adrenoreceptor blockade [ICI-118551 (ICI)]. We have shown that, after severe trauma, obese Zucker (OZ) rats, similar to obese patients, exhibit increased acute lung injury compared with lean Zucker (LZ) rats. We hypothesized that OZ rats exhibit a greater increase in early posttrauma glucose compared with LZ rats, with the increased posttrauma hyperglycemia suppressed by ICI treatment. Orthopedic trauma was applied to both hindlimbs in LZ and OZ rats. Fasting plasma glucose was then monitored for 6 h with or without ICI (0.2 mg·kg(-1)·h(-1) iv.) treatment. One day after trauma, plasma IL-6 levels, lung neutrophil numbers, myeloperoxidase (MPO) activity, and wet-to-dry weight ratios were measured. Trauma induced rapid hepatic glycogenolysis, as evidenced by decreased liver glycogen levels, and this was inhibited by ICI treatment. Compared with LZ rats, OZ rats exhibited higher posttrauma glucose, IL-6, lung neutrophil infiltration, and MPO activity. Lung wet-to-dry weight ratios were increased in OZ rats but not in LZ rats. ICI treatment reduced the early hyperglycemia, lung neutrophil retention, MPO activity, and wet-to-dry weight ratio in OZ rats to levels comparable with those seen in LZ rats, with no effect on blood pressure or heart rate. These results demonstrate that β2-adrenoreceptor blockade effectively reduces the early posttrauma hyperglycemia, which is associated with decreased lung injury in OZ rats.

PTU-130 Regulatory T Cells In Acute Liver Failure Are Functionally Intact And May Contribute To Retention Of Neutrophils In Areas Of Hepatic Necrosis

IntroductionAcute liver failure (ALF) is a sterile inflammation with a high mortality. The immunological response to acute liver injury is initiated by the infiltration of innate neutrophils and is followed...

Aryl hydrocarbon receptor attenuation of sub-chronic cigarette smoke-induced pulmonary neutrophilia is associated with retention of nuclear RelB and suppression of intercellular adhesion molecule-1 (CAM1P.233)

Abstract Cigarette smoke exposure is associated with chronic and enhanced pulmonary inflammation characterized by increased cytokine production and leukocyte recruitment to the lung. Although the aryl hydrocarbon receptor (AhR) is well-known to mediate toxic effects of environmental contaminants, the AhR has emerged as a suppressor of acute cigarette smoke-induced neutrophilia. As there is currently no information on the AhR prevention of lung inflammation due to varied and prolonged exposure regimes, we exposed control and AhR-/- mice to cigarette smoke for 2 weeks (sub-chronic exposure) utilizing low and high exposure protocols and evaluated pulmonary inflammation. Sub-chronic cigarette smoke exposure increased pulmonary neutrophilia dose-dependently in AhR-/- mice. There was no difference between smoke-exposed AhR+/- and AhR-/- mice in the expression of cytokines associated with neutrophil recruitment. However, expression of pulmonary intercellular adhesion molecule-1 (ICAM-1), an adhesion molecule involved in neutrophil migration and retention, was higher in AhR-/- mice. Nuclear RelB, an anti-inflammatory NFkB transcription factor, was significantly lower in sub-chronically exposed AhR-/- mice. These data support that absence of the AhR contributes to heightened pulmonary neutrophilia in response to on-going cigarette smoke exposure. Inter-individual variations in AhR expression may enhance the susceptibility to cigarette smoke induced diseases.

Aryl hydrocarbon receptor (AhR) attenuation of subchronic cigarette smoke-induced pulmonary neutrophilia is associated with retention of nuclear RelB and suppression of intercellular adhesion molecule-1 (ICAM-1).

Cigarette smoke is associated with chronic and enhanced pulmonary inflammation characterized by increased cytokine production and leukocyte recruitment to the lung. Although the aryl hydrocarbon receptor (AhR) is well-known to mediate toxic effects of manmade environmental contaminants, the AhR has emerged as a suppressor of acute cigarette smoke-induced neutrophilia by a mechanism involving the NF-κB protein RelB. Yet individuals who smoke often smoke for many years and vary in their cigarette consumption. As there is currently no information on the AhR prevention of lung inflammation, including neutrophilia, due to varied and prolonged exposure regimes, we exposed control and AhR(-/-) mice to cigarette smoke for 2 weeks (subchronic exposure) utilizing low and high exposure protocols and evaluated pulmonary inflammation. Subchronic cigarette smoke exposure significantly increased pulmonary neutrophilia dose-dependently in AhR(-/-) mice. Surprisingly, there was no difference between smoke-exposed AhR(+/-) and AhR(-/-) mice in the expression of cytokines associated with neutrophil recruitment. Expression of pulmonary intercellular adhesion molecule-1 (ICAM-1), an adhesion molecule involved in neutrophil migration and retention, was higher in pulmonary endothelial cells from AhR(-/-) mice. Although total lung RelB expression was increased by cigarette smoke, nuclear RelB was significantly lower in subchronically exposed AhR(-/-) mice. Inhibition of AhR activity by CH-223191 in endothelial cells potentiated ICAM-1 expression and prevented RelB nuclear translocation but had no effect on neutrophil adhesion. These data support that genetic absence of the AhR contributes to heightened pulmonary neutrophilia in response to ongoing cigarette smoke exposure. Interindividual variations in AhR expression may enhance the susceptibility to cigarette smoke-induced diseases.

Pulmonary retention of primed neutrophils: a novel protective host response, which is impaired in the acute respiratory distress syndrome

RationaleAcute respiratory distress syndrome (ARDS) affects over 200 000 people annually in the USA. Despite causing severe, and often refractory, hypoxaemia, the high mortality and long-term morbidity of ARDS results...

WHIM syndrome: presumptive diagnosis based on myelokathexis on bone marrow smear

The WHIM syndrome is a rare congenital immunodeficiency disorder characterized by human papillomavirus (HPV)-induced warts, hypogammaglobulinemia, bacterial infections and myelokathexis. Myelokathexis refers to abnormal retention of mature neutrophils in the bone marrow leading to severe neutropenia. We report the case of a 20 year old man presenting with chronic and severe neutropenia since early childhood without established diagnosis. He was addressed for chronic parodontal disease treatment. Examination of bone marrow smear showed morphological abnormalities of mature neutrophils strongly suggestive of myelokathexis. Diagnosis of WHIM syndrome was confirmed by molecular analysis: a nonsense mutation was identified in the gene encoding CXCR4, the CXCL12 (or SDF-1) chemokine receptor which notably controls cell adhesion to the marrow stroma and thereby regulates mature leukocytes release from the bone marrow. Treatment of the disease usually consists in prophylactic anti-infective measures including intravenous immunoglobulins administration, oral antibiotic prophylaxis and more recently HPV vaccination. Long term G-CSF therapy did not show any significant efficiency in preventing recurrent infections. The use of specific CXCR4 antagonist is being currently evaluated.

Resolvin D1 attenuates lipopolysaccharide induced acute lung injury through CXCL-12/CXCR4 pathway

BackgroundThe recruitment of neutrophils plays an important role in the progress of acute lung injury (ALI). Excessive neutrophils released from bone marrow accumulate in lung, release proinflammatory factors, and cause tissue damage. CXCL-12/CXCR4 is an important signaling pathway, which regulates the migration of bone marrow hematopoietic cells out of bone marrow and involves in neutrophil accumulation and retention in the inflammatory site. Resolvin D1 (RvD1) is a kind of lipid mediators, which can alleviate many inflammatory diseases. We hypothesized that RvD1 can alleviate lipopolysaccharide (LPS)-induced ALI through regulating CXCL-12/CXCR4 pathway. MethodsWe randomized mice into five groups: control group, RvD1 group, LPS group, LPS plus RvD1 group, and LPS plus AMD3100 group. ALI was established by intratracheal instillation of LPS. After 24 and 72 h, mice were sacrificed, and lung tissues were harvested for histologic analysis, wet-to-dry ratio, myeloperoxidase activity, and CXCL-12 expression. Bronchoalveolar fluid was collected for protein analysis, cytokines assay, and flow cytometry analysis. ResultsHistologic findings as well as wet-to-dry ratio, protein concentration, cytokines assay, neutrophil number, and myeloperoxidase activity confirmed that RvD1 and AMD3100 alleviated LPS-induced ALI. RvD1 decreased CXCL-12 messenger RNA expression in lung. However, RvD1 promoted CXCR4 expression in neutrophils in the initial stage of inflammation and reduced its level in the later stage. ConclusionsRvD1 protects LPS-induced ALI partially through regulating CXCL-12/CXCR4 pathway.

Genetics on a WHIM.

We initially described the WHIM syndrome based on the combination of Warts, Hypogammaglobulinaemia, Infections and Myelokathexis (neutrophil retention in the bone marrow). Translational research led to the discovery that this rare immunodeficiency disease is caused by a heterozygous mutation in the CXCR4 gene. Recently, Plerixafor has been suggested as a treatment for WHIM syndrome due to its efficacy as a CXCR4 antagonist, closing the translational research loop. In this review, we will focus on the clinical manifestations, pathophysiology, diagnosis and possible therapies for this rare entity.

The CXCR4 mutations in WHIM syndrome impair the stability of the T-cell immunologic synapse

AbstractWHIM (warts, hypogammaglobulinemia, infections, myelokathexis) syndrome is a rare disease characterized by diverse symptoms indicative of aberrantly functioning immunity. It is caused by mutations in the chemokine receptor CXCR4, which impair its intracellular trafficking, leading to increased responsiveness to chemokine ligand and retention of neutrophils in bone marrow. Yet WHIM symptoms related to adaptive immunity, such as delayed IgG switching and impaired memory B-cell function, remain largely unexplained. We hypothesized that the WHIM-associated mutations in CXCR4 may affect the formation of immunologic synapses between T cells and antigen-presenting cells (APCs). We show that, in the presence of competing external chemokine signals, the stability of T-APC conjugates from patients with WHIM-mutant CXCR4 is disrupted as a result of impaired recruitment of the mutant receptor to the immunologic synapse. Using retrogenic mice that develop WHIM-mutant T cells, we show that WHIM-mutant CXCR4 inhibits the formation of long-lasting T-APC interactions in ex vivo lymph node slice time-lapse microscopy. These findings demonstrate that chemokine receptors can affect T-APC synapse stability and allow us to propose a novel mechanism that contributes to the adaptive immune response defects in WHIM patients.

Reduction of body temperature governs neutrophil retention in hibernating and nonhibernating animals by margination

Hibernation consists of periods of low metabolism, called torpor, interspersed by euthermic arousal periods. During deep and daily (shallow) torpor, the number of circulating leukocytes decreases, although circulating cells, is restored to normal numbers upon arousal. Here, we show that neutropenia, during torpor, is solely a result of lowering of body temperature, as a reduction of circulating also occurred following forced hypothermia in summer euthermic hamsters and rats that do not hibernate. Splenectomy had no effect on reduction in circulating neutrophils during torpor. Margination of neutrophils to vessel walls appears to be the mechanism responsible for reduced numbers of neutrophils in hypothermic animals, as the effect is inhibited by pretreatment with dexamethasone. In conclusion, low body temperature in species that naturally use torpor or in nonhibernating species under forced hypothermia leads to a decrease of circulating neutrophils as a result of margination. These findings may be of clinical relevance, as they could explain, at in least part, the benefits and drawbacks of therapeutic hypothermia as used in trauma patients and during major surgery.