Pro-inflammatory interactions of streptolysin O toxin with human neutrophils in vitro.

The recent global resurgence of severe infections caused by the Group A streptococcus (GAS) pathogen, Streptococcus pyogenes, has focused attention on this microbial pathogen, which produces an array of virulence factors, such as the pore-forming toxin, streptolysin O (SOT). Importantly, the interactions of SOT with human neutrophils (PMN), are not well understood. The current study was designed to investigate the effects of pretreatment of isolated human PMN with purified SOT on several pro-inflammatory activities, including generation of reactive oxygen species (ROS), degranulation (elastase release), influx of extracellular calcium (Ca2+) and release of extracellular DNA (NETosis), using chemiluminescence, spectrophotometric and fluorimetric procedures, respectively. Exposure of PMN to SOT alone caused modest production of ROS and elastase release, while pretreatment with the toxin caused significant augmentation of chemoattractant (fMLP)-activated ROS generation and release of elastase by activated PMN. These effects of treatment of PMN with SOT were associated with both a marked and sustained elevation of cytosolic Ca2+concentrations and significant increases in the concentrations of extracellular DNA, indicative of NETosis. The current study has identified a potential role for SOT in augmenting the Ca2+-dependent pro-inflammatory interactions of PMN, which, if operative in a clinical setting, may contribute to hyper-activation of PMN and GAS-mediated tissue injury.

Decreased severity of disease during the first global omicron variant covid-19 outbreak in a large hospital in tshwane, south africa.

INTRODUCTION: The coronavirus disease 2019 (COVID-19) first reported in Wuhan, China in December 2019 is a global pandemic that is threatening the health and wellbeing of people worldwide. To date there have been more than 274 million reported cases and 5.3 million deaths. The Omicron variant first documented in the City of Tshwane, Gauteng Province, South Africa on 9 November 2021 led to exponential increases in cases and a sharp rise in hospital admissions. The clinical profile of patients admitted at a large hospital in Tshwane is compared with previous waves. METHODS: 466 hospital COVID-19 admissions since 14 November 2021 were compared to 3962 admissions since 4 May 2020, prior to the Omicron outbreak. Ninety-eight patient records at peak bed occupancy during the outbreak were reviewed for primary indication for admission, clinical severity, oxygen supplementation level, vaccination and prior COVID-19 infection. Provincial and city-wide daily cases and reported deaths, hospital admissions and excess deaths data were sourced from the National Institute for Communicable Diseases, the National Department of Health and the South African Medical Research Council. RESULTS: For the Omicron and previous waves, deaths and ICU admissions were 4.5% vs 21.3% (p<0.00001), and 1% vs 4.3% (p<0.00001) respectively; length of stay was 4.0 days vs 8.8 days; and mean age was 39 years vs 49,8 years. Admissions in the Omicron wave peaked and declined rapidly with peak bed occupancy at 51% of the highest previous peak during the Delta wave. Sixty two (63%) patients in COVID-19 wards had incidental COVID-19 following a positive SARS-CoV-2 PCR test . Only one third (36) had COVID-19 pneumonia, of which 72% had mild to moderate disease. The remaining 28% required high care or ICU admission. Fewer than half (45%) of patients in COVID-19 wards required oxygen supplementation compared to 99.5% in the first wave. The death rate in the face of an exponential increase in cases during the Omicron wave at the city and provincial levels shows a decoupling of cases and deaths compared to previous waves, corroborating the clinical findings of decreased severity of disease seen in patients admitted to the Steve Biko Academic Hospital. CONCLUSION: There was decreased severity of COVID-19 disease in the Omicron-driven fourth wave in the City of Tshwane, its first global epicentre.

Pulmonary nodules associated with JAK inhibitor therapy in rheumatoid arthritis: A case report.

Patients with rheumatoid arthritis (RA) may receive Janus kinase (JAK) inhibitors to achieve optimal control of their disease. We report a case of a patient who received a selective JAK1 inhibitor and subsequently developed multiple pulmonary nodules with cavitation. Biopsies confirmed the presence of cryptococcosis and the patient responded well to anti-fungal therapy.

Lymphopenia and IgG2 subclass deficiency in patients with severe COVID-19 pneumonia.

BACKGROUND: COVID-19 caused by the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) manifests with a range of disease severities. A small proportion of COVID-19 patients are severely ill; however, a significant proportion of these patients are critically ill, and require admission and mechanical ventilation, which is associated with a high mortality. OBJECTIVE: To identify factors that may predispose patients with COVID-19 to severe disease that requires mechanical ventilation (MV). METHODS: We performed a retrospective audit of patients admitted with COVID-19 pneumonia to the intensive care unit (ICU) and medical wards to evaluate the potential associations between comorbid conditions, lymphopenia and IgG subclass deficiency with a need for MV. RESULTS: A total of 51 patients were included in the study. Almost half of the patients (47%; n=24) were documented to have IgG2 deficiency, 43% (n=22) had lymphopenia and 37% (n=19) had combined lymphopenia and IgG2 subclass deficiency. Of the 24 patients who required MV, 75% had IgG2 subclass deficiency, 73% had lymphopenia and 50% had both. The relative risk for requiring MV was 2.64, 3.38 and 2.81 for lymphopenia, IgG2 subclass deficiency and both, respectively. CONCLUSION: These findings suggest that lymphopenia, low IgG2 concentrations or the combination of both may be used to identify patients with severe COVID-19 that are at increased risk for MV. This may facilitate earlier identification of patients at high risk, who may benefit from more intensive therapy.

Adverse effects of biologic anti-inflammatory agents on the respiratory system: A review.

The therapy of autoimmune rheumatological conditions has undergone significant changes with the introduction of biologic antiinflammatory agents including cytokine antagonists and agents that interfere with the function of T and B cells or those that inhibit intracellular enzymes such as Janus kinase (JAK). Although useful to control inflammation, these agents may be associated with druginduced lung disease, which may be difficult to differentiate from pulmonary disorders caused by the underlying autoimmune diseases. This review aims to provide a description of lung disease, both infectious and non-infectious, that may be induced by the administration of biologic anti-inflammatory agents with emphasis on inhibitors of tumour necrosis factor, interleukin-1, interleukin-6 and JAK.

Pneumolysin activates neutrophil extracellular trap formation.

The primary objective of the current study was to investigate the potential of the pneumococcal toxin, pneumolysin (Ply), to activate neutrophil extracellular trap (NET) formation in vitro. Isolated human blood neutrophils were exposed to recombinant Ply (5-20 ng ml(-1) ) for 30-90 min at 37°C and NET formation measured using the following procedures to detect extracellular DNA: (i) flow cytometry using Vybrant® DyeCycle™ Ruby; (ii) spectrofluorimetry using the fluorophore, Sytox(®) Orange (5 µM); and (iii) NanoDrop(®) technology. These procedures were complemented by fluorescence microscopy using 4', 6-diamino-2-phenylindole (DAPI) (nuclear stain) in combination with anti-citrullinated histone monoclonal antibodies to visualize nets. Exposure of neutrophils to Ply resulted in relatively rapid (detected within 30-60 min), statistically significant (P < 0·05) dose- and time-related increases in the release of cellular DNA impregnated with both citrullinated histone and myeloperoxidase. Microscopy revealed that NETosis appeared to be restricted to a subpopulation of neutrophils, the numbers of NET-forming cells in the control and Ply-treated systems (10 and 20 ng ml(-1) ) were 4·3 (4·2), 14.3 (9·9) and 16·5 (7·5), respectively (n = 4, P < 0·0001 for comparison of the control with both Ply-treated systems). Ply-induced NETosis occurred in the setting of retention of cell viability, and apparent lack of involvement of reactive oxygen species and Toll-like receptor 4. In conclusion, Ply induces vital NETosis in human neutrophils, a process which may either contribute to host defence or worsen disease severity, depending on the intensity of the inflammatory response during pneumococcal infection.

Cysteinyl leukotriene receptor-1 antagonists as modulators of innate immune cell function.

Cysteinyl leukotrienes (cysLTs) are produced predominantly by cells of the innate immune system, especially basophils, eosinophils, mast cells, and monocytes/macrophages. Notwithstanding potent bronchoconstrictor activity, cysLTs are also proinflammatory consequent to their autocrine and paracrine interactions with G-protein-coupled receptors expressed not only on the aforementioned cell types, but also on Th2 lymphocytes, as well as structural cells, and to a lesser extent neutrophils and CD8(+) cells. Recognition of the involvement of cysLTs in the immunopathogenesis of various types of acute and chronic inflammatory disorders, especially bronchial asthma, prompted the development of selective cysLT receptor-1 (cysLTR1) antagonists, specifically montelukast, pranlukast, and zafirlukast. More recently these agents have also been reported to possess secondary anti-inflammatory activities, distinct from cysLTR1 antagonism, which appear to be particularly effective in targeting neutrophils and monocytes/macrophages. Underlying mechanisms include interference with cyclic nucleotide phosphodiesterases, 5'-lipoxygenase, and the proinflammatory transcription factor, nuclear factor kappa B. These and other secondary anti-inflammatory mechanisms of the commonly used cysLTR1 antagonists are the major focus of the current review, which also includes a comparison of the anti-inflammatory effects of montelukast, pranlukast, and zafirlukast on human neutrophils in vitro, as well as an overview of both the current clinical applications of these agents and potential future applications based on preclinical and early clinical studies.

Pharmacological approaches to regulate neutrophil activity.

Although indispensable in host defense against microbial pathogens, misdirected hyperacute and chronic activation of neutrophils presents the potential hazard of tissue damage, organ dysfunction, and carcinogenesis. In many clinical settings, particularly inflammatory disorders of the airways, over-reactivity of neutrophils is exacerbated by their relative resistance to conventional, pharmacological anti-inflammatory therapies, including, but not limited to, corticosteroids. Notwithstanding their sheer numbers, which can increase rapidly and dramatically during inflammatory responses, these cells are not only pre-programmed to release reactive oxygen species, proteinases, and eicosanoids/prostanoids immediately on exposure to pro-inflammatory stimuli but may also subsequently undergo the process of netosis, thereby enhancing and protracting their inflammatory potential. All of these mechanisms are likely to underpin the resistance of neutrophils to pharmacological control and have triggered the search for alternatives to corticosteroids. In addition to macrolides and adenosine 3',5'-cyclic adenosine monophospate-elevating agents, more recent innovations in the control of neutrophilic inflammation include activators of histone deacetylases and antagonists of chemokine receptors, as well as monoclonal antibodies which target neutrophil-activating cytokines and their receptors. These and other neutrophil-targeted strategies represent the focus of the current review.

Manganese promotes increased formation of hydrogen peroxide by activated human macrophages and neutrophils in vitro.

Although pro-inflammatory mechanisms have been implicated in the pathogenesis of manganese (Mn²âº)-related neurological and respiratory disorders, relatively little is known about the potential of this metal to interact pro-oxidatively with human phagocytes. The primary objective of the current study was to investigate the effects of Mn²âº as MnCl2 (0.5-100 µM) on the generation of the reactive oxygen species (ROS), superoxide, hydrogen peroxide (H2O2), and hypohalous acids by isolated human blood neutrophils and monocyte-derived macrophages following activation of these cells with the chemotactic tripeptide, FMLP (1 µM), or the phorbol ester, PMA (25 ng/mL). Generation of ROS was measured using the combination of oxygen consumption, lucigenin/luminol-enhanced chemiluminescence, spectrofluorimetric detection of oxidation of 2,7-dichlorodihydrofluorescein, radiometric assessment of myeloperoxidase (MPO)-mediated protein iodination, release of MPO by ELISA, and spectrophotometric measurement of nitrite formation. Treatment of activated neutrophils with either FMLP or PMA resulted in significantly decreased reactivity of superoxide in the setting of increased formation of H2O2 and MPO-mediated iodination, with no detectable effects on either oxygen consumption or MPO release. Similar effects of the metal with respect to superoxide reactivity and H2O2 formation were observed with activated macrophages, while generation of NO was unaffected. Taken together with the findings of experiments using cell-free ROS-generating systems, these observations are compatible with a mechanism whereby Mn²âº, by acting as a superoxide dismutase mimetic, increases the formation of H2O2 by activated phagocytes. If operative in vivo, this mechanism may contribute to the toxicity of Mn²âº.

Evaluation of circulating soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) to predict risk profile, response to antimicrobial therapy, and development of complications in patients with chemotherapy-associated febrile neutropenia: a pilot study.

The soluble triggering receptor expressed on myeloid cells 1 (sTREM-1) is a useful marker of infection in patients with sepsis, but has not been adequately evaluated in patients with chemotherapy-associated febrile neutropenia (FN). The value of sTREM-1 in this setting has been tested in a retrospective, pilot study using stored serum from 48 cancer patients with documented FN. On presentation, patients were categorized according to the Talcott risk-index clinical score. Circulating soluble sTREM-1 was measured using an ELISA procedure, while procalcitonin (PCT) or interleukins 6 (IL-6) and 8 (IL-8), included for comparison, were measured using an immunoluminescence-based assay and Bio-Plex® suspension bead array system, respectively. Circulating concentrations of both sTREM-1 and PCT were significantly (P < 0.05) elevated in patients at high risk for complications or death, as predicted by the Talcott score and were significantly lower in patients who responded to empiric antimicrobial agents. Neither IL-6 nor IL-8 accurately predicted serious complications in patients with FN. These observations, albeit from a pilot study, demonstrate that sTREM-1 is indeed elevated in high-risk patients with FN and is potentially useful to predict their clinical course, either together with, or as an alternative to PCT.

Soluble triggering receptor expressed on myeloid cells in sputum of patients with community-acquired pneumonia or pulmonary tuberculosis: a pilot study.

Soluble triggering receptor expressed on myeloid cells (s-TREM-1) is upregulated on the surface of inflammatory cells in the presence of bacterial infections, apparently excluding those due to Mycobacterium tuberculosis. Therefore, sputum concentrations of s-TREM-1 may be of value in distinguishing bacterial pneumonia from pulmonary tuberculosis (PTB) in patients with respiratory infections. The current pilot study was designed to evaluate whether s-TREM-1 concentrations measured in the sputum of patients with suspected community-acquired pneumonia (CAP) allowed differentiation of those patients with PTB from other causes of pneumonia and to correlate s-TREM-1 with CURB-65, a marker of disease severity. Soluble s-TREM-1 concentrations were measured in sputum samples from patients admitted to a tertiary hospital with CAP or PTB by means of an ELISA procedure. Soluble-TREM-1 was readily detectable and quantifiable in sputum samples from patients with both CAP and PTB, with concentrations of 234±47 and 178±36 pg/ml respectively, but did not differ significantly between the two groups. However, patients with PTB had significantly lower leukocyte counts, 9±1.3 vs 15±1.4 × 10(9)/l compared with those without PTB. Interestingly, sputum s-TREM-1 concentrations correlated significantly with the CURB-65 pneumonia severity score calculated at the time of admission. Soluble-TREM-1 expression is upregulated in patients with both CAP and PTB, but does not differentiate between these two conditions. Sputum concentrations of s-TREM-1 may predict the severity of disease in patients with CAP.

Interactive inhibitory effects of formoterol and montelukast on activated human neutrophils.

The research question addressed in the current study was: do formoterol (1 and 10 nM) and montelukast (2 µM) possess interactive inhibitory effects on activated human neutrophils, particularly in relation to alterations in cyclic AMP and cytosolic Ca²(+) fluxes? Isolated human blood neutrophils were activated with the chemoattractant N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) (1 µM) in combination with cytochalasin B (CB; 3 µM). Fura-2-acetoxymethyl ester-based spectrofluorimetry, lucigenin-enhanced chemiluminescence, colorimetric and flow cytometric procedures were used to measure cytosolic Ca²(+) fluxes, production of superoxide, elastase release and beta-2 integrin (CR3) expression, respectively, while cyclic AMP and leukotriene (LT)B4 were assayed using competitive binding ELISA procedures. Activation of the cells with fMLP/CB resulted in abrupt and sustained increases in cytosolic Ca²(+), as well as release of elastase and production of superoxide and LTB4, and expression of CR3, all of which were attenuated by formoterol and montelukast individually, and especially by the combination of these agents. These anti-inflammatory effects of each agent, as well as the combination, were associated with significant increases in cyclic AMP. The findings of the current study may explain the efficacy of montelukast and formoterol when used in combination with inhaled corticosteroids in the treatment of severe asthma, possibly by controlling neutrophil-driven inflammation of the airways.

Posaconazole attenuates leukotriene B4 release and uptake of calcium by chemoattractant-activated human neutrophils: a potential strategy to control neutrophil-mediated inflammation.

OBJECTIVES: This study was designed to investigate the neutrophil-targeted anti-inflammatory potential of posaconazole (0.1-5 microM, equivalent to 0.7-3.9 mg/L) by measuring the effects of this agent on the release of leukotriene B(4) (LTB(4)) and store-operated uptake of Ca(2+) following stimulation of human neutrophils with platelet-activating factor (200 nM). METHODS: LTB(4) release and uptake of Ca(2+) by the cells were measured using an enzyme immunoassay and fura-2/AM-based spectrofluorimetric procedures, respectively. RESULTS: Treatment of neutrophils with posaconazole resulted in dose-related attenuation of PAF-activated release of LTB(4) and influx of Ca(2+), which attained statistical significance at 1 microM of the antimycotic. CONCLUSIONS: Although primarily an antimycotic, posaconazole possesses secondary anti-inflammatory activities, which may contribute to the therapeutic efficacy of this agent in patients with sepsis.

Leukotrienes C4 and D4 sensitize human neutrophils for hyperreactivity to chemoattractants.

OBJECTIVE AND DESIGN: To investigate the sensitizing effects of the cysteinyl leukotrienes (CysLTs) C(4) and D(4) on the proinflammatory responses of chemoattractant-activated human neutrophils in vitro. MATERIALS: Neutrophils were isolated from venous blood taken from healthy, adult, human volunteers. TREATMENT: Cells were exposed to LTC(4) and LTD(4) (50-300 nM) prior to activation with 1 microM of N-formyl-L-methionyl- L-leucyl-L-phenylalanine (fMLF). METHODS: A fura-2/AM-based spectrofluorimetric procedure, lucigenin-enhanced chemiluminescence (LECL), a colourimetric method and an ELISA procedure, were used to measure Ca(2+) mobilization, superoxide production, elastase and MMP-8 release respectively following activation of LTC(4)/ D(4)-primed neutrophils with fMLF. Superoxide generation was also measured in the presence and absence of the CysLT receptor 1 antagonist, montelukast (100 nM). RESULTS: Exposure of neutrophils to either LTC(4) or LTD(4) alone had modest effects on Ca(2+) mobilization, while superoxide generation and elastase release were unaffected. However, relative to the responses of neutrophils activated with fMLF in the absence of the CysLTs, pre-treatment of the cells with either LTC(4)or LTD(4) resulted in significant, augmentation of fMLF-activated elastase and MMP-8 release and superoxide generation, which was attenuated by montelukast. CONCLUSION: These previously undocumented sensitizing interactions of LTs C(4) and D(4) with neutrophils may contribute to the activation of these cells in acute and chronic inflammation of both atopic and non-atopic aetiology, while identifying a role for montelukast in regulating neutrophil reactivity.

Itraconazole-mediated inhibition of calcium entry into platelet-activating factor-stimulated human neutrophils is due to interference with production of leukotriene B4.

The primary objective of this study was to probe the involvement of leukotriene B(4) (LTB(4)) in itraconazole (0.1-5 microM)-mediated inhibition of Ca(2+) uptake by chemoattractant-activated human neutrophils. Following exposure of the cells to platelet-activating factor (PAF, 200 nM), LTB(4) was measured by immunoassay, while neutrophil cytosolic Ca(2+) concentrations were determined by a fura-2/AM-based spectrofluorimetric procedure. Activation of neutrophils was accompanied by an abrupt and sustained (for about 1 min) elevation in cytosolic Ca(2+) which was associated with increased generation of LTB(4), both of which were attenuated significantly by itraconazole at 0.5 microM and higher. The inhibitory effect of the anti-mycotic on Ca(2+) uptake by PAF-activated cells was mimicked by an LTB(4) antibody, as well as by LY255283 (1 microM) and MK886 (0.5 microM), an antagonist of LTB(4) receptors and an inhibitor of 5'-lipoxygenase-activating protein, respectively, while addition of itraconazole to purified 5'-lipoxygenase resulted in inhibition of enzyme activity. A mechanistic relationship between itraconazole-mediated inhibition of LTB(4) production and Ca(2+) influx was also supported by the observation that pulsed addition of purified LTB(4) to PAF-activated neutrophils caused substantial restoration of Ca(2+) uptake by cells treated with the anti-mycotic. Taken together, these observations suggest that the potentially beneficial anti-inflammatory interactions of itraconazole with activated neutrophils result from interference with production of LTB(4), with consequent attenuation of a secondary LTB(4)-mediated wave of Ca(2+) uptake by the cells.

Taming the neutrophil: calcium clearance and influx mechanisms as novel targets for pharmacological control.

Neutrophils are relatively insensitive to the anti-inflammatory actions of conventional chemotherapeutic agents, including corticosteroids, emphasizing the requirement for novel pharmacological strategies to control the potentially harmful proinflammatory activities of these cells. In the case of commonly-occurring inflammatory diseases of the airways, the neutrophil is the primary mediator of inflammation in conditions such as chronic obstructive pulmonary disease, cystic fibrosis, acute respiratory distress syndrome, bronchiectasis and non-eosinophilic bronchial asthma. Recent insights into the mechanisms utilized by neutrophils to restore Ca(2+) homeostasis following activation with Ca(2+)-mobilizing, proinflammatory stimuli have facilitated the identification of novel targets for anti-inflammatory chemotherapy in these cells. The most amenable of these from a chemotherapeutic perspective, is the cyclic AMP-dependent protein kinase-modulated endomembrane Ca(2+)-ATPase which promotes clearance of the cation from the cytosol of activated neutrophils. Second generation type 4 phosphodiesterase inhibitors and adenosine receptor agonists operative at the level of subtype A2A adenosine receptors, which are currently undergoing clinical and preclinical assessment respectively, hold promise as pharmacologic modulators during the restoration of Ca(2+) homeostasis. If this promise is realized, it may result in novel chemotherapeutic strategies for the control of hyperacute and chronic inflammatory conditions in which neutrophils are primary offenders. Alternative, potential future targets include the Na(+), Ca(2+)-exchanger and store-operated Ca(2+) channels, which cooperate in the refilling of intracellular Ca(2+) stores.

Endogenous adenosine regulates neutrophil pro-inflammatory activities by cyclic AMP-dependent accelerated clearance of cytosolic calcium.

OBJECTIVE AND DESIGN: To identify the involvement of adenosine in restoration of Ca2+ homeostasis to activated human neutrophils. MATERIALS: Neutrophils were isolated from venous blood taken from healthy, adult, human volunteers. TREATMENT: The cells were exposed to adenosine deaminase (ADA, 0.1-2 units/ml) for 10 min at 37 degrees C prior to activation with N-formyl-L-methionyl-L-leucyl-L-phenylala-nine (FMLP, 1 microM). METHODS: Cytosolic Ca2+ concentrations and transmembrane fluxes of the cation in FMLP-activated neutrophils +/- ADA were measured using spectrofluorimetric and radiometric procedures respectively, while intracellular cAMP and inositol triphosphate were measured by radioassay, and superoxide production and elastase release by, chemiluminescence and colourimetric methods respectively. Levels of statistical significance were calculated using the Mann-Whitney U-test and ANOVA. RESULTS: Although FMLP-activated generation of inositol triphosphate and mobilisation of Ca2+ from neutrophil internal stores, as well as the magnitude of the subsequent efflux and store-operated influx of the cation were unaffected by ADA, there was a prolonged elevation in cytosolic Ca2+ in the presence of the enzyme, which was associated with failure to activate adenylate cyclase and with increased production of superoxide and release of elastase. These effects of ADA were attenuated by dibutyryl cAMP (4 mM), CGS 21680 (1 microM) and rolipram (0.5 microM), as well as by EGTA (10 mM). CONCLUSIONS: These results are compatible with a physiological role for adenosine in promoting deactivation of neutrophils, possibly by promoting cAMP-dependent clearance of Ca2+ from the cytosol of the cells by the endo-membrane Ca2+-ATPase.

Regulation of calcium homeostasis in activated human neutrophils--potential targets for anti-inflammatory therapeutic strategies.

OBJECTIVES: The objectives of the current study were to: (i) present an integrated model for the restoration of calcium homeostasis in activated human neutrophils based on current knowledge and recent research; and (ii) identify potential targets for the modulation of calcium fluxes in activated neutrophils based on this model and to investigate the effects of intracellular probes which target key processes involved in calcium homeostasis and pro-inflammatory activity in these cells. DESIGN AND SETTING: Laboratory-based experimental research using purified human neutrophils from healthy, adult human volunteers. OUTCOME MEASURES: Calcium metabolism and pro-inflammatory activity of neutrophils. RESULTS: Modulation of calcium fluxes in activated human neutrophils can be achieved by cAMP-dependent upregulation of the activity of the endomembrane Ca(2+)-ATPase which resequesters cytosolic Ca2+. Formoterol, a long-acting beta 2-agonist, elevates intracellular cAMP levels, accelerates Ca2+ restoration in activated neutrophils and downregulates the pro-inflammatory responses of these cells. Alterations in the membrane potential of activated neutrophils may play a role in regulating calcium reuptake into the cells as attenuation of the membrane depolarisation response is associated with accelerated calcium influx. CONCLUSIONS: Modulation of the activity of the endomembrane Ca(2+)-ATPase in human neutrophils represents an important target for anti-inflammatory

The anti-inflammatory interactions of epinephrine with human neutrophils in vitro are achieved by cyclic AMP-mediated accelerated resequestration of cytosolic calcium.

This study was designed to evaluate the effects of epinephrine (0.01-1 microM) on superoxide production by, and release of elastase from human neutrophils activated with the chemotactic tripeptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) (1 microM) in vitro, and to relate alterations in these responses to changes in adenosine 3,5' cyclic monophosphate (cAMP) and cytosolic free Ca(2+). Cyclic AMP, superoxide production and elastase release were measured by radioimmunoassay, lucigenin-enhanced chemiluminescence, and a colorimetric procedure respectively. Cytosolic Ca(2+) fluxes were measured by fura-2 spectrofluorimetry in combination with radiometric procedures that enable distinction between net efflux and influx of the cation. Epinephrine treatment of neutrophils resulted in increased cAMP and dose-related inhibition of both superoxide production and elastase release, which was potentiated by the type 4 phosphodiesterase inhibitor, rolipram, and attenuated by propranolol, but not by selective beta(1)-, alpha(1)- or alpha(2)-adrenoreceptor antagonists. Although epinephrine did not affect the FMLP-activated abruptly-occurring increase in fura-2 fluorescence intensity, indicating no effects on the release of Ca(2+) from neutrophil intracellular stores, this agent accelerated the rate of decline in fluorescence in the setting of decreased efflux and a reduction in store-operated influx of Ca(2+). These effects of epinephrine on the clearance of Ca(2+) from the cytosol of FMLP-activated neutrophils were attenuated by propranolol, and are compatible with enhancement of the activity of the cAMP-dependent Ca(2+) sequestering/resequestering endo-membrane Ca(2+)-ATPase. We conclude that epinephrine down-regulates the pro-inflammatory activities of neutrophils by cAMP-mediated enhancement of the clearance of cytosolic Ca(2+).

Accelerated calcium influx and hyperactivation of neutrophils in chronic granulomatous disease.

The relationship between activation of NADPH-oxidase, alterations in membrane potential and triggering of Ca2+ fluxes in human phagocytes has been investigated using neutrophils from four subjects with chronic granulomatous disease (CGD). Cytosolic Ca2+ and membrane potential were measured by spectrofluorimetry, and net efflux and influx of Ca2+ by radiometric procedures. Exposure of normal neutrophils to the chemotactic tripeptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP; 1 microM) was accompanied by an abrupt increase in cytosolic Ca2+ coincident with membrane depolarization and efflux of the cation. These events terminated at around 30 s after the addition of FMLP and were followed by membrane repolarization and store-operated influx of Ca2+, both of which were superimposable and complete after about 5 min. Activation of CGD neutrophils was also accompanied by an increase in cytosolic Ca2+, which, in spite of an efficient efflux response, was prolonged in relation to that observed in normal cells. This prolonged increase in cytosolic Ca2+ in activated CGD neutrophils occurred in the setting of trivial membrane depolarization and accelerated influx of Ca2+, and was associated with hyperactivity of the cells according to excessive release of elastase and increased activity of phospholipase A2. Treatment of CGD neutrophils with the type 4 phosphodiesterase inhibitor, rolipram (1 microM) restored Ca2+ homeostasis and attenuated the increase in elastase release. These findings support the involvement of NADPH-oxidase in regulating membrane potential and Ca2+ influx in activated neutrophils, and may explain the disordered inflammatory responses and granuloma formation which are characteristic of CGD.