Kita, Hirohito

ABSTRACTStaphylococcus aureusis widely prevalent in the nasopharynges of healthy individuals (carriers) but can also cause serious infections.S. aureuscan elaborate a variety of superantigen exotoxins in “carrier” or “pathogenic” states.Streptococcus pyogenescan also colonize the nasopharynx and elaborate superantigens. Unlike the acute effects of superantigen exotoxins absorbed through the gut or vaginal mucosa, little is known regarding the pathogenesis of superantigens entering through the intranasal route. In the current study, we evaluated the local and systemic effects of staphylococcal enterotoxin B (SEB) and streptococcal pyrogenic exotoxin A (SPEA) delivered through the intranasal route. Superantigens were administered intranasally on multiple occasions, and experimental animals were sacrificed on day 8 for experimental analyses. SEB-induced airway inflammation was more pronounced for HLA-DR3 transgenic mice than for BALB/c mice, consistent with bacterial superantigens binding more efficiently to human than murine major histocompatibility complex class II. The nature of the airway inflammation in HLA-DR3 mice was determined by the concentration of SEB applied intranasally. Low concentrations (20 ng) induced eosinophilic airway inflammation as well as eosinophil degranulation, whereas intranasal exposure to higher concentrations (2,000 ng) resulted in neutrophilic airway inflammation, permanent airway destruction, toxic shock, and mortality. SEB-induced eosinophilic inflammatory response was enhanced in signal transducer and activator of transcription (STAT)-4-deficient HLA-DQ8 transgenic mice with defective interleukin-12 signaling. Intranasal administration of SPEA induced airway inflammation and systemic immune activation in HLA-DQ8 transgenic mice. In conclusion, repeated chronic intranasal exposure to bacterial superantigens causes airway inflammation and systemic immune activation.

Summary A patient with Waldenstrom’s macroglobulinaemia expresses a high titre IgM antibody in serum that binds both mouse and human dendritic cells (DC) in a B7-DC (PD-L2)-dependent manner. We have reported previously that purified antibody from patient serum activates immature and mature DC in vitro, enhancing the ability of these professional antigen-presenting cells to activate naive T cells, take up antigen, resist a cytokine-depleted environment and secrete immunomodulatory cytokines, such as interkeukin (IL)-6 and tumour necrosis factor (TNF)-α. Systemic treatment of experimental animals with this antibody induces potent anti-melanoma immunity and modulates protectively the recall response against antigen challenge through the airway in an experimental model of inflammatory airway disease. Here we describe a monoclonal IgM antibody derived from this serum immunoglobulin that recapitulates each of these earlier observations, providing direct evidence that M protein from the Waldenstrom’s patient mediates these potent immunomodulatory effects. Furthermore, cell lines expressing this recombinant form of the human antibody provide the basis for developing this reagent for clinical application.

The effects of platelet-activating factor (PAF) and IL-5 on intracellular pH were investigated in human eosinophils. Purified peripheral blood eosinophils were loaded with the ratiometric fluorescent pH indicator BCECF-AM ester. Stimulation of eosinophils with PAF produced time-dependent alkalinization of the cytoplasm from an initial pH of 7.1±0.04 to 7.5±0.05. A similar alkalinization response was produced by the calcium ionophore, ionomycin and by the calcium ATPase inhibitor, thapsigargin. These compounds as well as PAF produce significant increases in cytoplasmic calcium ([Ca2+]i). In contrast, IL-5 and the protein kinase C (PKC) activator phorbol myristate acetate (PMA) did not produce cytoplasmic alkalinization and had no effect on [Ca2+]i in eosinophils. PAF-stimulated alkalinization was not inhibited under conditions that blocked plasma membrane Na+-H+ exchange, proton channel or plasma membrane H+-ATPase activities. Measurements of intragranule pH with a cell permeant pH indicator (LysoSensor Yellow/Blue DND-160), which partitions into intracellular acidic compartments, revealed that PAF-stimulated cytosolic alkalinization correlated with intragranule acidification. These results suggest that the increase in [Ca2+]i after PAF stimulation activates a H+-ATPase present in the granule membranes, leading to enhanced granule acidification and cytoplasmic alkalinization. We propose that granule acidification is an important step in solubilization of major basic protein crystals, which are stored within the granule core, in preparation for degranulation and release of these proteins.

Objective.Eosinophil-associated proteins, especially eosinophil-derived neurotoxin, may be important contributors to the neurologic pathology and symptoms caused byBaylisascaris procyonis infection.Methods.Two cases of severe B procyonisencephalitis with evidence of marked eosinophil degranulation in the central nervous system are presented. Serial cerebrospinal fluid (CSF) specimens were collected from each patient during the course of their illness. Antibodies against B procyonis were measured in the patients' serum and CSF. Levels of the eosinophilopoietin interleukin-5 (IL-5) and 2 important eosinophil proteins, eosinophil-derived neurotoxin and major basic protein, were assayed in the CSF.Results.Both patients had rapidly progressive central nervous system disease with evidence of eosinophilic meningoencephalitis. Both tested positive for antibodies to B procyonis in serum and CSF and had progressively worsening deep white matter changes on magnetic resonance images of the brain. CSF levels of IL-5, eosinophil-derived neurotoxin, and major basic protein were markedly elevated over controls.Conclusions.This is the first report of the measurement of IL-5, eosinophil-derived neurotoxin, and major basic protein in human CSF. In addition to traumatic damage and necrosis caused by migrating larvae, eosinophil-derived neurotoxin from associated eosinophilic inflammation may be an important contributory factor in the pathogenesis of B procyonis encephalitis. parasite, eosinophil-derived-neurotoxin, major basic protein, eosinophilia, hypereosinophilia, interleukin-5, encephalitis, child.

Human peripheral blood eosinophils released eosinophil survival-enhancing activity when stimulated with the calcium ionophore, ionomycin. The release of activity was detected as early as 3 h after stimulation and was inhibited by an immunomodulating agent, cyclosporin A. The survival-enhancing activity was completely abolished by treatment with anti-interleukin 3 (IL-3) and anti-granulocyte/macrophage colony-stimulating factor (GM-CSF) monoclonal antibodies. Moreover, IL-3 and GM-CSF were measurable in ionomycin-stimulated eosinophil supernatants by immunoassay. Eosinophils produced approximately one-half as much IL-3 and one-fifth as much GM-CSF as ionomycin-stimulated mononuclear cells. Neutrophils also produced IL-3 and GM-CSF, but the amounts were less than those produced by eosinophils. These observations suggest a novel role for eosinophils in pathophysiology of allergic inflammation and host defense mechanisms.

SUMMARY Eosinophils and eosinophil granule proteins may play an important role in the pathogenesis of asthma. BALF from 40 patients with symptomatic asthma were analysed for cytokine activity by the eosinophil survival assay. BALF from 15 patients showed increased survival activity. Survival activities in BALF from four of these patients were almost completely blocked by anti-IL-5 MoAb, and the remaining activities were blocked by anti-granulocyte-macrophage colony-stimulating factor (GM-CSF), anti-IL-3 antibody, or both. Surprisingly, BALF samples from the other 25 patients decreased eosinophil viabilities below the levels of medium control. The inhibitory factor in these BALF was of low molecular weight, was heat-stable, was largely overcome by excess exogenously added cytokines, and was positively correlated with the concentrations of lidocaine in the BALF. Lidocaine itself inhibited eosinophil survival at concentrations less than those present in the BALF. These findings indicate that lidocaine is an inhibitor of cytokines in the eosinophil survival assay, and they suggest the need for caution in analyses of BALF containing lidocaine or other local anaesthetics.

Protein kinase C (PKC) activation in human eosinophils increases NADPH oxidase activity, which is associated with plasma membrane depolarization. In this study, membrane potential measurements of eosinophils stimulated with phorbol ester (phorbol 12-myristate 13-acetate; PMA) were made using a cell-permeable oxonol membrane potential indicator, diBAC4(3). Within 10 minutes after PMA stimulation, eosinophils depolarized from–32.9±5.7 mV to +17.3±1.8 mV. The time courses of depolarization and proton channel activation were virtually identical. Blocking the proton conductance with 250 μM ZnCl2(+43.0±4.2 mV) or increasing the proton channel activation threshold by reducing the extracellular pH to 6.5 (+44.4±1.4 mV) increased depolarization compared with PMA alone. Additionally, the protein kinase C(PKC) δ-selective blocker, rottlerin, inhibited PMA-stimulated depolarization, indicating that PKCδ was involved in regulating depolarization associated with eosinophil NADPH oxidase activity. Thus, the membrane depolarization that is associated with NADPH oxidase activation in eosinophils is sufficient to produce marked proton channel activation under physiological conditions.

Abstract Oxidized low-density lipoprotein (oxLDL) has been reported as a major participant in the pathogenesis of atherosclerosis. We hypothesized that oxLDL can also interact with granulocytes during inflammatory airway diseases, such as asthma. To test the chemotactic effect of oxLDL, isolated human peripheral granulocytes were added to the upper chambers of Transwell filters and migration in response to oxLDL was determined. Cu+2-oxidized LDL stimulated neutrophil (23.4 ± 3.2% for 100 μg/ml oxLDL versus 2.9 ± 1.1% for buffer, P < 0.05) and eosinophil (19.3 ± 3.5% versus 0.6 ± 0.02% for buffer, P < 0.05) chemotaxis in a concentration-dependent manner. The magnitude of chemotaxis was dependent on the degree of LDL oxidation. Granulocyte transmigration across IL-1β–activated human pulmonary microvascular endothelial cell monolayers was similarly stimulated by oxLDL. OxLDL activated significant degranulation of both neutrophils (100.9 ± 9.8 versus 49.6 ± 8.4 ng lactoferrin released/5 × 105 neutrophils for buffer, P < 0.05) and eosinophils (342 ± 115.4 versus 85.8 ± 30.4 ng eosinophil-derived neurotoxin/1 × 106 eosinophils for buffer, P < 0.05). Therefore, in vivo influx and oxidation of LDL may be an important mediator for the initiation of bronchial inflammation where granulocytes are recruited to the lung.