Larry L. Thomas

Stimulus-specific regulation of chemokine expression involves differential activation of the redox-responsive transcription factors AP-1 and NF-kappaB.

The promoters of the IL‐8, MCP‐1, and RANTES genes contain binding sites for the redox‐responsive transcription factors AP‐1 and NF‐κB, which have been shown to be important for their expression. In this overview, we present evidence from our laboratories that the stimulus‐specific regulation of these chemokines by the reactive oxidant H2O2, the proinflammatory cytokine TNF‐α, and respiratory syncytial virus (RSV) is mediated in a cell type‐specific manner involving different patterns of AP‐1 and NF‐κB binding activity. Our results demonstrate that H2O2 induction of IL‐8 gene expression is linked with the selective binding of AP‐1 to the IL‐8 promoter, whereas TNF‐α and RSV induction of IL‐8 correlates with the activation of NF‐κB binding. We propose that the differential activation and binding of inducible transcription factors to the promoter regions of chemokine genes provides a critical regulatory mechanism by which the CXC and CC chemokines can be selectively expressed in a cell type‐specific and stimulus‐specific manner. Such a regulatory mechanism of differential chemokine expression could critically influence the site‐ specific recruitment of distinct subsets of leukocytes to sites of inflammation and injury. J. Leukoc. Biol. 65: 291–298; 1999.

Interactions between NADPH oxidase‐related proton and electron currents in human eosinophils

1 Proton and electron currents in human eosinophils were studied using the permeabilized‐patch voltage‐clamp technique, with an applied NH4+ gradient to control pHi. 2 Voltage‐gated proton channels in unstimulated human eosinophils studied with the permeabilized‐patch approach had properties similar to those reported in whole‐cell studies. 3 Superoxide anion (O2−) release assessed by cytochrome c reduction was compared in human eosinophils and neutrophils stimulated by phorbol myristate acetate (PMA). PMA‐stimulated O2 release was more transient and the maximum rate was three times greater in eosinophils. 4 In PMA‐activated eosinophils, the H+ current amplitude (IH) at +60 mV increased 4.7‐fold, activation was 4.0 times faster, deactivation (tail current decay) was 5.4 times slower, the H+ conductance‐voltage (gH‐V) relationship was shifted ‐43 mV, and diphenylene iodinium (DPI)‐inhibitable inward current reflecting electron flow through NADPH oxidase was activated. The data reveal that PMA activates the H+ efflux during the respiratory burst by modulating the properties of H+ channels, not simply as a result of NADPH oxidase activity. 5 The electrophysiological response of eosinophils to PMA resembled that reported in human neutrophils, but PMA activated larger proton and electron currents in eosinophils and the response was more transient. 6 ZnCl2 slowed the activation of H+ currents and shifted the gH‐V relationship to more positive voltages. These effects occurred at similar ZnCl2 concentrations in eosinophils before and after PMA stimulation. These data are compatible with the existence of a single type of H+ channel in eosinophils that is modulated during the respiratory burst.

Activation of NADPH oxidase-related proton and electron currents in human eosinophils by arachidonic acid.

1 Effects of arachidonic acid (AA) on proton and electron currents in human eosinophils were studied using the permeabilized‐patch voltage‐clamp technique, using an applied NH4+ gradient to control pHi. 2 Superoxide anion (O2−) release was assessed by cytochrome c reduction in human eosinophils. Significant O2− release was stimulated by 5‐10 μm AA. 3 AA activated diphenylene iodinium (DPI)‐inhibitable inward current reflecting electron efflux through NADPH oxidase. These electron currents (Ie) were elicited in human eosinophils at AA concentrations (3‐10 μm) similar to those that induced O2− release. 4 The voltage‐gated proton conductance (gH) in eosinophils stimulated with AA was profoundly enhanced: H+ current amplitude (IH) increased 4.6 times, activation was 4 times faster, and the H+ conductance‐voltage (gH‐V) relationship was shifted to substantially more negative voltages. The electrophysiological effects of AA resembled those reported for PMA, except that AA did not consistently slow τtail (deactivation of H+ currents). 5 The stimulation of both proton and electron currents by AA was reversible upon washout. Repeated exposure elicited repeated responses. The activation of H+ currents by AA was dissociable from its activation of NADPH oxidase; H+ currents were enhanced at low concentrations of AA that did not elicit detectable Ie or when NADPH oxidase was inhibited by DPI. 6 Most of the effects of AA on H+ currents qualitatively resemble those reported in whole‐cell studies, reflecting a more direct action than PMA. The results are compatible with AA being an immediate activator of both NADPH oxidase and proton channels in human eosinophils.

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 (FcεRI). 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 Ca2+-free solutions. Zn2+ 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 μM Zn2+. Evidently, proton channels are active in basophils during IgE-mediated responses and prevent excessive acidification, which may account for their role in histamine release.

A NOVEL AND HIGHLY DIVERGENT HOMOLOG OF HUMAN EOSINOPHIL GRANULE MAJOR BASIC PROTEIN

Eosinophils are important effector cells in defense against helminth infection and in allergic diseases. To identify novel eosinophil proteins, large scale sequencing of a cDNA library prepared from interleukin-5-stimulated umbilical cord precursor cells was performed, and the major genes expressed by maturing eosinophils were determined. This resulted in the identification of a cDNA with 64% identity to human prepro-major basic protein (hprepro-MBP). This cDNA was designated hprepro-MBP homolog (hprepro-MBPH). Interestingly, the calculated pI values for hMBPH and hMBP differed by >100-fold, with pI values of 8.7 and 11.4, respectively. Given this pronounced basicity difference, the homolog transcript’s abundance (1.1%), and MBP’s critical role in eosinophil biological activity, we further characterized the homolog. Reverse transcription-polymerase chain reaction detected transcription of hprepro-MBPH in bone marrow only, and this result was confirmed by analysis of a large cDNA data base (electronic Northern). hMBPH was isolated from human eosinophil granule lysates, and its identity was verified by amino acid sequencing and by mass spectrometry. Analyses of the biological activities showed that hMBPH had effects similar to hMBP in cell killing and neutrophil (superoxide anion production and interleukin-8 release) and basophil (histamine and leukotriene C4 release) stimulation assays, but usually with reduced potency. Overall, this novel homolog’s unique physical properties indicated that the high net positive charge of hMBP is important but not essential for biological activity.

Human basophils selectively express the FcγRII (CDw32) subtype of IgG receptor

Abstract The role of IgG antibody in the sensilization of human basophils and mast cells to antigen is uncertain. To help resolve this uncertainty, we characterized by two-color fluorometric analysis the Fc receptors for IgG (FcγR) on human basophils. Basophil-containing mononuclear cell fractions of atopic and nonatopic adult volunteers were incubated sequentially with fluorescein isoihiocvanate-conjugated murine monoclonal IgE and biotinylated monoclonal antibodies (MAb) that bind specifically to the different FcγR subtypes. Binding of biotinylated MAbs was visualized after subsequent incubation with phvcoerythrin-strepavidin conjugate. Basophils did not react with a murine monomeric IgG2a, which binds specifically through its Fc to the high-affinity FcγRI (CD64), or with MAbs specific for the low-affinity FcγRIII (CD/6). However, basophils reacted with a MAb specific for the low-affinity FcγRII (CDw32). The profile of basophil FcγR expression was not altered after brief IgE-mediated activation. In addition, pretreatment with gamma interferon, which induced expression of FcγRI (CD64) on neutrophils, did not induce FcγRI expression on basophils. These results indicate that the FcγR present on basophils is exclusively of the FcγRII (CDw32) subtype. The absence of the high-affinity FcγRI (CD64) suggests that antigenic sensitization of basophils by monomeric IgG does not occur.

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°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°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°C, with Ea 25.1 kcal mol−1 (Q10, 4.2). The maximum Ie measured at 34°C was −30.5 pA. Above 30°C, the temperature dependence of both Ie and O2− production was less pronounced. Above 37°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°C than at 20°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°C.

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

Eosinophil major basic protein (MBP) is an effective stimulus for neutrophil superoxide (O2−) 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 O2− production, but not degranulation or IL-8 production. Measurement of Akt phosphorylation at Ser473 and Thr308 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 O2− production. MBP stimulated phosphorylation and translocation of the p85 subunit of class IA PI3K, but not translocation of the p110γ subunit of class IB PI3K, to the neutrophil membrane. Inhibition of protein kinase Cζ (PKCζ) inhibited MBP-stimulated O2− production. Measurement of phosphorylated PKCζ (Thr410) and PKCδ (Thr505) confirmed that PKCζ, but not PKCδ, is activated in MBP-stimulated neutrophils. The time courses for phosphorylation and translocation of the p85 subunit of class IA PI3K, activation of Akt, and activation of PKCζ were similar. Moreover, inhibition of PI3K activity inhibited MBP-induced activation of PKCζ. We conclude that MBP stimulates a Src kinase-dependent activation of class IA PI3K and, in turn, activation of PKCζ in neutrophils, which contributes to the activation of NADPH oxidase and the resultant O2− production in response to MBP stimulation.

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.

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.