Neutrophil-like low-density granulocytes are elevated in patients with moderate to severe persistent asthma.

BACKGROUND: Elevations in neutrophil-like low-density granulocytes (LDGs) are observed in association with disease severity in some autoimmune and other disorders. This study evaluated whether a similar association with disease severity is observed in asthma. OBJECTIVE: To determine LDG levels in peripheral blood mononuclear cells of subjects with intermittent or mild persistent asthma, subjects with moderate persistent or severe persistent (SP) asthma, and control subjects without a history or allergy or asthma. METHODS: A brief medical history and physical examination, spirometry, and measurement of fraction of exhaled nitric oxide were performed. The LDGs were quantified by polychromatic flow cytometry. RESULTS: The LDGs displaying the same phenotype as those described previously for LDGs in other diseases were significantly elevated in peripheral blood mononuclear cells of subjects with moderate persistent or SP asthma. The LDGs comprised up to 39% of peripheral blood mononuclear cells, with elevated LDG levels most prevalent in subjects with SP asthma. The highest LDG levels were observed in 4 subjects with SP asthma. Fraction of exhaled nitric oxide levels and body mass were significantly increased in subjects with low LDG levels compared with control subjects, whereas fraction of exhaled nitric oxide levels and body mass were not elevated in subjects with moderate persistent or SP asthma and high LDG levels compared with control subjects. CONCLUSION: These findings identify a previously unrecognized association between LDG levels and asthma severity. Identification of the factor(s) responsible for the increased LDG levels in moderate persistent or SP asthma may provide a serum biomarker to aid in the identification of neutrophil-associated phenotypes of severe asthma.

Bioactive lysophosphatidylcholine 16:0 and 18:0 are elevated in lungs of asthmatic subjects.

PURPOSE: Asthma is a chronic inflammatory disease of the airways, and is associated with upregulation of phospholipase A2 (PLA2), the enzyme that hydrolyzes phosphatidylcholine, producing lysophosphatidylcholine (LPC) and free fatty acids. LPC is a lipid mediator with known pro-inflammatory and pro-atherogenic properties, and is believed to be a critical factor in cardiovascular diseases. We postulate that asthmatic subjects have an elevated content of LPC in the lung lining fluids. METHODS: Eight non-asthmatic controls and seven asthmatic subjects were recruited for broncho-alveolar lavage fluids (BALF) collection for analysis of LPC by high performance liquid chromatography-tandem mass spectrometry. RESULTS: LPC16:0 and LPC18:0 were significantly elevated in the BALF of asthmatics with impaired lung function characteristic of moderate asthma, but not mild asthma. The increased LPC content in BALF was accompanied by increased PLA2 activity. Furthermore, qRT-PCR analysis of the BALF cell fraction indicated increased secretory PLA2-X (sPLA2-X). CONCLUSIONS: The increased LPC content in the lung lining fluids is a potential critical lipid mediator in the initiation and/or progression of airway epithelial injury in asthma.

Loss of p120 catenin upregulates transcription of pro-inflammatory adhesion molecules in human endothelial cells.

P120 catenin (p120ctn) is an adherens junction protein recognized to regulate barrier function, but emerging evidence indicates that p120ctn may also exert control on other cellular functions such as transcriptional suppression of genes. We investigated the hypothesis that loss of p120ctn in human endothelial cells activates transcription of pro-inflammatory adhesion molecules. For study, siRNA targeted to p120ctn was transfected into brain microvascular (HBMECs) or pulmonary artery endothelial cells (HPAECs) for 24-120h, which depleted 50-80% of endogenous p120ctn. This loss of p120ctn resulted in increased promoter reporter activity of transcription factors, NFκB, AP-1, and Kaiso, as well as of target genes, MMP-1 and ICAM-1. Real-time RT-PCR analysis indicated that the mRNA for ICAM-1, VCAM-1, and E- and P-selectins were all upregulated during the period of 24-120h of p120ctn depletion, although the time-course and extent of the expression profiles differed. The upregulated mRNA of adhesion molecules corresponded with increased PMN adhesion to the EC surface and elevated ICAM-1 protein expression. We further explored the role of ERK1/2 as a potential signaling mechanism responsible for regulation of transcriptional activities by p120ctn. Results indicated that loss of p120ctn increased phosphorylated ERK1/2, and a MEK1 inhibitor (PD98059) prevented NFκB nuclear translocation. This implicates ERK1/2 in signaling the NFκB activation induced by p120ctn loss. The findings provide strong evidence that deficiency in p120ctn expression in endothelial cells is a potent stimulus for transcriptional upregulation of multiple adhesion molecules. We conclude that p120ctn functions to suppress transcription, which is an important and novel regulation in vascular endothelium.

HIV-1-infected peripheral blood mononuclear cells enhance neutrophil survival and HLA-DR expression via increased production of GM-CSF: implications for HIV-1 infection.

OBJECTIVE: HIV-1 bound to intact neutrophils efficiently infects activated peripheral blood mononuclear cells (PBMC). Here, we evaluated the effect of the local milieu created by activated PBMC before and after HIV-1 infection on neutrophil survival and HLA-DR expression, with emphasis placed on a role for GM-CSF. METHODS: PBMC of healthy adult individuals were activated by phytohemagglutinin (PHA) or anti-CD3/anti-CD28 and were subsequently cultured without (HIV-1⁻) or with HIV-1 (HIV-1+). The effects of the culture supernatants or recombinant GM-CSF on survival and HLA-DR expression by neutrophils of healthy adult individuals and of HIV-1-infected individuals were evaluated using flow cytometry. RESULTS: Conditioned medium from PHA-activated PBMC (HIV-1⁻ and HIV-1+) increased neutrophil survival and induced HLA-DR expression by neutrophils of healthy individuals in a GM-CSF dependent fashion. HIV-1 infection variably, but consistently, increased GM-CSF production by PHA-activated PBMC but not GM-CSF production by anti-CD3/anti-CD28-activated PBMC. The latter was correlated with a loss of CD3+GMCSF+ cells after infection. Neutrophils of elite controllers exhibited a diminished HLADR response to GM-CSF in culture, whereas neutrophils of HIV-1+ subjects having a low viral load on anti-retroviral therapy or subjects with a high viral load exhibited a range of HLA-DR responses. CONCLUSIONS: GM-CSF production within the mucosa or draining lymph nodes may promote HIV-1 infection by facilitating sustained contact between viable neutrophils with bound HIV-1 and CD4 lymphocytes. The minimal effect of GM-CSF on HLA-DR expression by neutrophils of elite controllers provides indirect support for this conclusion.

Cutting edge: CD49d+ neutrophils induce FcepsilonRI expression on lung dendritic cells in a mouse model of postviral asthma.

The increasing prevalence of atopy and asthma remains unexplained but may be due to infection with respiratory viruses. In support of this hypothesis, we showed that experimental asthma after viral infection in mice depended on type I IFN-driven upregulation of FcεRI on conventional dendritic cells (cDCs) in the lung. In this article, we demonstrate that FcεRI expression on lung cDCs depends on an unexpected activity of a CD49d(+) subset of polymorphonuclear neutrophils (PMNs) that are found in the lungs of wild-type C57BL6 but not mice deficient in type I IFNR. Expression of FcεRI depends in part on a CD11b-dependent interaction between PMNs and cDCs. This study demonstrates a PMN-cDC interaction in the lung that is necessary for the ability of viral infection to induce atopic disease.

Expression of the high affinity IgE receptor by neutrophils of individuals with allergic asthma is both minimal and insensitive to regulation by serum IgE.

We evaluated the hypothesis that serum IgE regulates neutrophil FcepsilonRI expression in the same manner as described for other FcepsilonRI+ cells. FcepsilonRI expression by neutrophils of 40 asthma subjects and 20 control subjects did not correlate with serum IgE levels, whereas FcepsilonRI expression by basophils of the same subjects showed a highly significant correlation. The level of FcepsilonRI expression by neutrophils of both asthma and control subjects was approximately 1% of that for basophil FcepsilonRI expression. IgE+ neutrophils were minimally detectable, and FcepsilonRI alpha-subunit was not detected in Western blots of neutrophil membranes and cytosol. The neutrophil FcepsilonRI did not support anti-IgE stimulated superoxide release or IgE-induced increase in neutrophil survival. We conclude that FcepsilonRI expression by neutrophils of both asthma patients and control individuals is minimal at best and that, if present, neutrophil FcepsilonRI expression, unlike that of other human FcepsilonRI+ cells, is not regulated by serum IgE.

A pH-stabilizing role of voltage-gated proton channels in IgE-mediated activation of human basophils.

Eosinophils and other phagocytes use NADPH oxidase to kill bacteria. Proton channels in human eosinophils and neutrophils are thought to sustain NADPH oxidase activity, and their opening is greatly enhanced by a variety of NADPH oxidase activators, including phorbol myristate acetate (PMA). In nonphagocytic cells that lack NADPH oxidase, no clear effect of PMA on proton channels has been reported. The basophil is a granulocyte that is developmentally closely related to the eosinophil but nevertheless does not express NADPH oxidase. Thus, one might expect that stimulating basophils with PMA would not affect proton currents. However, stimulation of human basophils in perforated-patch configuration with PMA, N-formyl-methionyl-leucyl-phenylalanine, or anti-IgE greatly enhanced proton currents, the latter suggesting involvement of proton channels during activation of basophils by allergens through their highly expressed IgE receptor (Fc epsilonRI). The anti-IgE-stimulated response occurred in a fraction of cells that varied among donors and was less profound than that to PMA. PKC inhibition reversed the activation of proton channels, and the proton channel response to anti-IgE or PMA persisted in Ca(2+)-free solutions. Zn(2+) at concentrations that inhibit proton current inhibited histamine release elicited by PMA or anti-IgE. Studied with confocal microscopy by using SNARF-AM and the shifted excitation and emission ratioing of fluorescence approach, anti-IgE produced acidification that was exacerbated in the presence of 100 microM Zn(2+). Evidently, proton channels are active in basophils during IgE-mediated responses and prevent excessive acidification, which may account for their role in histamine release.

HIV infection of mononuclear cells is calcium-dependent.

Strategies that prevent initial HIV infection of cells are greatly needed. In this study, we determined the requirement of divalent cations for HIV infection of and attachment to peripheral blood mononuclear cells (PBMC), which contain several types of HIV-infectable cells-CD4(+) T cells, monocytes and dendritic cells. EDTA, added only during PBMC exposure to HIV, reduced infection by an average of 92%. The reduction of infection by EDTA was accompanied by a reduction in HIV binding to PBMC; R5, X4 and dual-tropic HIV binding to PBMC were inhibited by >85%. EGTA similarly reduced HIV binding to PBMC, while addition of Ca(2+) or Mn(2+), but not Mg(2+), fully restored binding. Virus attachment was inhibited in a dose-dependent manner by trypsin treatment of PBMC, indicating protein involvement in HIV binding. In contrast, mannan or soluble ICAM-1 did not inhibit HIV binding to PBMC. These data indicate that a Ca(2+)-dependent cell-surface protein(s) is responsible for the majority of HIV attachment to and infection of PBMC. Further studies of this are likely to reveal novel strategies to prevent infection of PBMC.

Activation by inflammatory stimuli increases neutrophil binding of human immunodeficiency virus type 1 and subsequent infection of lymphocytes.

Resting neutrophils bind human immunodeficiency virus type 1 (HIV-1) and efficiently transfer infection to lymphocytes. The present study shows that a brief activation by inflammatory stimuli increases the neutrophil binding levels of both R5 and X4 isolates of HIV-1 at least twofold. The binding occurs independently of CD4, gp120, and incubation temperature and is observed with HIV-1 propagated either in lymphocytes or in HEK293 cells. Significantly, HIV-1 bound to the activated neutrophils accelerates the infection of activated lymphocytes compared to free HIV-1 or to HIV-1 bound to resting neutrophils. It is proposed that these events may contribute to the increased risk of HIV-1 transmission at sites of mucosal infection.

Eosinophil major basic protein stimulates neutrophil superoxide production by a class IA phosphoinositide 3-kinase and protein kinase C-zeta-dependent pathway.

Eosinophil major basic protein (MBP) is an effective stimulus for neutrophil superoxide (O(2)(-)) production, degranulation, and IL-8 production. In this study we evaluated the participation of phosphoinositide 3-kinase (PI3K) and PI3K-associated signaling events in neutrophil activation by MBP. Inhibition of PI3K activity blocked MBP-stimulated O(2)(-) production, but not degranulation or IL-8 production. Measurement of Akt phosphorylation at Ser(473) and Thr(308) confirmed that MBP stimulated PI3K activity and also demonstrated indirectly activation of phosphoinositide-dependent kinase-1 by MBP. Genistein and the Src kinase family inhibitor, 4-amino-5-(4-methyphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, inhibited MBP-stimulated phosphorylation of Akt. 4-Amino-5-(4-methyphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine also inhibited MBP-stimulated O(2)(-) production. MBP stimulated phosphorylation and translocation of the p85 subunit of class I(A) PI3K, but not translocation of the p110gamma subunit of class I(B) PI3K, to the neutrophil membrane. Inhibition of protein kinase Czeta (PKCzeta) inhibited MBP-stimulated O(2)(-) production. Measurement of phosphorylated PKCzeta (Thr(410)) and PKCdelta (Thr(505)) confirmed that PKCzeta, but not PKCdelta, is activated in MBP-stimulated neutrophils. The time courses for phosphorylation and translocation of the p85 subunit of class I(A) PI3K, activation of Akt, and activation of PKCzeta were similar. Moreover, inhibition of PI3K activity inhibited MBP-induced activation of PKCzeta. We conclude that MBP stimulates a Src kinase-dependent activation of class I(A) PI3K and, in turn, activation of PKCzeta in neutrophils, which contributes to the activation of NADPH oxidase and the resultant O(2)(-) production in response to MBP stimulation.

Temperature dependence of NADPH oxidase in human eosinophils.

The phagocyte NADPH oxidase helps kill pathogens by producing superoxide anion, O2-. This enzyme is electrogenic because it translocates electrons across the membrane, generating an electron current, Ie. Using the permeabilized patch voltage-clamp technique, we studied the temperature dependence of Ie in human eosinophils stimulated by phorbol myristate acetate (PMA) from room temperature to >37 degrees C. For comparison, NADPH oxidase activity was assessed by cytochrome c reduction. The intrinsic temperature dependence of the assembled, functioning NADPH oxidase complex measured during rapid temperature increases to 37 degrees C was surprisingly weak: the Arrhenius activation energy Ea was only 14 kcal mol(-1) (Q10, 2.2). In contrast, steady-state NADPH oxidase activity was strongly temperature dependent at 20-30 degrees C, with Ea 25.1 kcal mol(-1) (Q10, 4.2). The maximum Ie measured at 34 degrees C was -30.5 pA. Above 30 degrees C, the temperature dependence of both Ie and O2- production was less pronounced. Above 37 degrees C, Ie was inhibited reversibly. After rapid temperature increases, a secondary increase in Ie ensued, suggesting that high temperature promotes assembly of additional NADPH oxidase complexes. Evidently, about twice as many NADPH oxidase complexes are active near 37 degrees C than at 20 degrees C. Thus, the higher Q10 of steady-state Ie reflects both increased activity of each NADPH oxidase complex and preferential assembly of NADPH oxidase complexes at high temperature. In summary, NADPH oxidase activity in intact human eosinophils is maximal precisely at 37 degrees C.

Immobilized lactoferrin is a stimulus for eosinophil activation.

Eosinophils are strongly implicated in the pathogenesis of asthma, particularly in damage to the airway epithelial lining. We examined the potential for lactoferrin, a multifunctional glycoprotein present in the airway surface liquid, to activate eosinophils. Incubating eosinophils in tissue culture wells pretreated with 1-100 microg/ml human lactoferrin stimulated concentration-dependent superoxide production by eosinophils. The same concentrations of immobilized transferrin were without effect. The potency of immobilized lactoferrin was approximately one-third that of immobilized secretory IgA in the same experiments. In contrast, immobilized lactoferrin did not stimulate neutrophil superoxide production. Eosinophils bound lactoferrin as determined by flow cytometry and by binding of (125)I-labeled lactoferrin. Transferrin did not block binding of (125)I-labeled lactoferrin. Soluble lactoferrin, however, did not activate the eosinophils and did not block superoxide production stimulated by immobilized lactoferrin. Immobilized lactoferrin also stimulated release of eosinophil-derived neurotoxin and low levels of leukotriene C4 production; the latter was significantly enhanced in the presence of 100 pg/ml GM-CSF. GM-CSF also enhanced superoxide production and eosinophil-derived neurotoxin release stimulated by the lower concentrations of immobilized lactoferrin. Pretreatment of the lactoferrin with peptide N-glycosidase F or addition of heparin or chondroitin sulfate to the incubation contents had no or only a minimal effect on the activity of immobilized lactoferrin. These results demonstrate that lactoferrin adherent to the surface epithelium may contribute to the activation of eosinophils that infiltrate the airway lumen in eosinophil-associated disorders such as asthma.