Long-term overexpression of human granulocyte colony-stimulating factor in transgenic mice: persistent neutrophilia with no increased mortality for more than one year

To investigate possible adverse consequences of persistent neutrophil overproduction, mice transgenic for human granulocyte colony-stimulating factor (hG-CSF) were studied for more than 1 year. They showed marked granulocytopoiesis and neutrophilia. Continuous medullary and extramedullary granulocytopoiesis resulted in marked changes in bone and liver. In the liver, haemorrhage and focal necrosis and a few haemangiosarcomas were present, presumably caused by the destructive granulocytopoiesis. Despite the high incidence of lung infiltration by mature neutrophils, lung lesions rarely appeared. Although there was a persistent increase in neutrophils, mortality of the mice did not differ from that of non-transgenic littermates at least within 1 year after birth. Factors other than overproduction of G-CSF and extensive neutrophilia could be required for the development of neutrophil-mediated acute and chronic tissue damage.     

Stress-induced effects, which inhibit host defenses, alter leukocyte trafficking

Acute cold restraint stress (ACRS) has been reported to suppress host defenses against Listeria monocytogenes, and this suppression was mediated by beta1-adrenoceptors (β1-ARs). Although ACRS appears to inhibit mainly early innate immune defenses, interference with leukocyte chemotaxis and the involvement of β1-AR (or β2-AR) signaling had not been assessed. Thus, the link between sympathetic nerve stimulation, release of neurotransmitters, and changes in blood leukocyte profiles, including oxidative changes, following ACRS was evaluated. The numbers of leukocyte subsets in the blood were differentially affected by β1-ARs and β2-ARs following ACRS; CD3⁺ (CD4 and CD8) T-cells were shown to be decreased following ACRS, and the T cell lymphopenia was mediated mainly through a β2-AR mechanism, while the decrease in CD19⁺ B-cells was influenced through both β1- and β2-ARs, as assessed by pharmacological and genetic manipulations. In contrast to the ACRS-induced loss of circulating lymphocytes, the number of circulating neutrophils was increased (i.e., neutrophilia), and this neutrophilia was mediated through β1-ARs. The increase in circulating neutrophils was not due to an increase in serum chemokines promoting neutrophil emigration from the bone marrow; rather it was due to neutrophil release from the bone marrow through activation of a β1-AR pathway. There was no loss of glutathione in any of the leukocyte subsets suggesting that there was minimal oxidative stress; however, there was early production of nitric oxide and generation of some protein radicals. Premature egress of neutrophils from bone marrow is suggested to be due to norepinephrine induction of nitric oxide, which affects the early release of neutrophils from bone marrow and lessens host defenses.     

Kinetics and mechanisms of recombinant human interleukin 1 and tumor necrosis factor-alpha-induced changes in circulating numbers of neutrophils and lymphocytes

Human recombinant interleukins 1 alpha and 1 beta (rIL-1 alpha and -1 beta) both induced monophasic peripheral neutrophilia and lymphopenia in Lewis rats 1.5 hr after i.v. injection. The kinetics of rIL-1 alpha- and -1 beta-induced neutrophilia were similar to those induced by human monocyte-derived IL-1, IL-1 alpha, and IL-1 beta, and the peripheral neutrophilia was accompanied by a marked decrease in marrow neutrophils. Arachidonic acid metabolites are implicated as biochemical intermediates in the production of the neutrophilia but not lymphopenia, since indomethacin and dexamethasone both completely abrogated IL-1-induced neutrophilia but did not affect the IL-1-induced lymphopenia. Acetylsalicylic acid, a cyclooxygenase inhibitor, did not inhibit IL-1-induced neutrophilia, suggesting that products of the lipoxygenase rather than the cyclooxygenase pathway of arachidonate metabolism may contribute to the neutrophilia. Human recombinant tumor necrosis factor-alpha (rTNF) administered i.v. to Lewis rats induced peripheral neutropenia, two peaks of neutrophilia, and lymphopenia. A wide range of doses of rTNF resulted in an initial neutropenia at 0.5 hr after injection followed by a first peak of neutrophilia at 1.5 hr and a second peak of neutrophilia at 6 hr. The initial neutropenia and the first peak of neutrophilia were not inhibited by pretreatment of rats with dexamethasone, indomethacin, or aspirin. The second peak of neutrophilia was inhibited by both dexamethasone and indomethacin, but was not at all inhibited by aspirin, suggesting that the second peak of neutrophilia is mediated by the release of endogenous cytokines, especially by IL-1, since exogenous IL-1-induced neutrophilia is also completely inhibited by dexamethasone and indomethacin but not by aspirin. The TNF-induced peripheral neutrophilia is also accompanied by a significant depletion of bone marrow neutrophils, indicating that the source of increased circulating neutrophils is, at least in part, via recruitment of marrow neutrophils. Systemic blood pressure was not affected by IL-1 or rTNF at the dosages employed, showing that the changes in circulating leukocyte subsets were not attributable to hemodynamic changes nor to the hemodynamic change-related release of adrenal hormones. Adrenalectomy did not alter the IL-1- or rTNF-induced neutrophilia or lymphopenia, also demonstrating that neither monokine mediates its hematologic effects on peripheral blood leukocytes via the release of adrenal hormones.(ABSTRACT TRUNCATED AT 400 WORDS)     

Functional expression of transient receptor potential melastatin- and vanilloid-related channels in pulmonary arterial and aortic smooth muscle

Although the accumulation of neutrophils in the lungs and airways is common to many inflammatory lung diseases, including acute lung injury, the alterations that neutrophils undergo as they leave the peripheral circulation and migrate into the lungs have not been well characterized. Human volunteers were exposed to endotoxin by bronchoscopic instillation. The resulting air space neutrophil accumulation and peripheral blood neutrophils were isolated 16 h later, compared with circulating neutrophils isolated before or after to the pulmonary endotoxin exposure, and compared with circulating neutrophils exposed to endotoxin in vitro. Microarray analysis was performed on air space, circulatory, and in vitro endotoxin-stimulated neutrophils. Functional analysis included the determination of neutrophil apoptosis, chemotaxis, release of cytokines and growth factors, and superoxide anion release. Dramatic gene expression differences were apparent between air space and circulating neutrophils: approximately 15% of expressed genes have altered expression levels, including broad increases in inflammatory- and chemotaxis-related genes, as well as antiapoptotic and IKK-activating pathways. Functional analysis of air space compared with circulating neutrophils showed increased superoxide release, diminished apoptosis, decreased IL-8-induced chemotaxis, and a pattern of IL-8, macrophage inflammatory protein-1beta, monocyte chemoattractant protein-1, and tumor necrosis factor-alpha release different from either unstimulated or LPS-stimulated circulating neutrophils. Many of these changes are not elicited by in vitro treatment with endotoxin. Limited differences were detected between circulating neutrophils isolated before and 16 h after pulmonary endotoxin instillation. These results suggest that neutrophils sequestered in the lung become fundamentally different from those resident in the circulation, and this difference is distinct from in vitro activation with endotoxin.     

In vivo radioprotection by 5-androstenediol: stimulation of the innate immune system

We showed previously that 5-androstenediol stimulates myelopoiesis, increases the numbers of circulating neutrophils and platelets, and enhances resistance to infection in gamma-irradiated mice. We have extended those studies to include monocytes, natural killer (NK) cells, eosinophils and basophils, and we have measured the activation marker CD11b using flow cytometry. Androstenediol (160 mg/kg) was administered subcutaneously to female B6D2F1 mice 24 h before whole-body gamma irradiation. Androstenediol treatments increased the blood levels of neutrophils, monocytes and NK cells in unirradiated animals; decreased the numbers of circulating eosinophils; and ameliorated radiation-induced decreases in neutrophils, monocytes, NK cells, erythrocytes and platelets. The androstenediol treatments had no significant effect on the numbers of circulating B cells or T cells. CD11b labeling intensity on monocytes was decreased slightly after androstenediol treatment. In contrast, radiation or androstenediol alone caused increases in CD11b labeling intensity on NK cells. Androstenediol and radiation combined caused a marked increase in NK cell CD11b. The results indicate that androstenediol increases the numbers of the three major cell types of the innate immune system (neutrophils, monocytes and NK cells), that androstenediol-induced changes in blood elements in irradiated animals persist for at least several weeks, and that there is a significant positive interaction between radiation and administration of androstenediol in the activation of NK cells.     

Major Neutrophilia Observed in Acute Phase of Human Leptospirosis Is Not Associated with Increased Expression of Granulocyte Cell Activation Markers

It has long been known that pathogenic Leptospira can mobilize the immune system but the specific contribution of neutrophils to control the infectious challenge remains to be clarified. We herein analyzed the phenotype of circulating neutrophils of patients with leptospirosis and healthy controls for the expression of toll-like receptor (TLR) type 2 (TLR2, to sense the leptospiral LPS) and several activation markers: interleukin 8 chemokine receptor CD182 (CXCR2), CD11b of the integrin/opsonin complement receptor type 3 (CR3) and CD15 (ligand of the selectin). The plasmatic level of the main CD182 ligand, interleukin 8 (CXCL8), was measured by ELISA. Hospitalized leptospirosis cases showed marked neutrophilia, particularly in the most severe cases. Interestingly, TLR2 was significantly increased in leptospirosis but identical levels of CD182 and CD11b were detected when compared to controls. CD15 was significantly decreased on neutrophils in leptospirosis but returned to normal within 1 month. Basal levels of IL-8 were measured in control subjects and were not increased in leptospirosis cases at the initial stage of the disease. In conclusion, we observed that neutrophils failed to regulate the expression of several of the receptors involved in cell activation and recruitment. This study further emphasizes the paradigm that neutrophils may be impaired in their overall capacity to thwart bacterial infection in leptospirosis patients.     

Characterization of f-Met-Leu-Phe-stimulated fluid pinocytosis in human polymorphonuclear leukocytes by flow cytometry

N-formylated chemotactic peptide stimulation of human neutrophils initiates a number of cellular processes, such as lysosomal enzyme release and superoxide anion production, that are indicative of the events of neutrophil activation during the acute inflammatory response in disease. This study characterizes a newly recognized neutrophil activation event, N-formylated chemotactic peptide-stimulated fluid pinocytosis in human neutrophils, using a novel flow cytometric assay for this activity. Fluid pinocytosis was found to be inhibited by acidic pH and low temperature but could be enhanced by cytochalasin B treatment or surface adherence by neutrophils. The activity measured by this new assay of fluid pinocytosis appears to be separate and distinct from lysosomal enzyme release and receptor-mediated adsorptive endocytosis in neutrophils. The physiologic significance of N-formylated chemotactic peptide-stimulated fluid pinocytosis is not known, but a possible relationship to neutrophil locomotion is discussed.     

Neutrophil myeloperoxidase deficiency associated with canine hepatozoonosis

Hepatozoonosis is a very important disease in dogs in Nigeria. Hepatozoonosis was reported in Nigeria in 18 dogs. The clinical signs included fever, anorexia, loss of weight, lameness, oculonasal discharge and conjunctivitis. Hematologic findings included leukocytosis due to neutrophilia and eosinophilia. Parasitemia varied from 1 to 9% of the circulating neutrophils in the peripheral blood smears of the dogs examined. Hepatozoon canis gametocytes were identified in circulating neutrophils of dogs. Peripheral blood smears from dogs confirmed to have natural H. canis infection were cytochemically stained for myeloperoxidase. Parasitized neutrophils were myeloperoxidase deficient while non-parasitized neutrophils were myeloperoxidase positive. This is considered important, because deficiency of the enzyme may be responsible for poor response of H. canis to chemotherapeutic agents.     

Activation of the aryl hydrocarbon receptor increases pulmonary neutrophilia and diminishes host resistance to influenza A virus

Unlike their role in bacterial infection, less is known about the role of neutrophils during pulmonary viral infection. Exposure to pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) results in excess neutrophils in the lungs of mice infected with influenza A virus. TCDD is the most potent agonist for the aryl hydrocarbon receptor (AhR), and exposure to AhR ligands has been correlated with exacerbated inflammatory lung diseases. However, knowledge of the effects of AhR agonists on neutrophils is limited. Likewise, the factors regulating neutrophil responses during respiratory viral infections are not well characterized. To address these knowledge gaps, we determined the in vivo levels of KC, MIP-1alpha, MIP-2, LIX, IL-6, and C5a in infected mouse lungs. Our data show that these neutrophil chemoattractants are generally produced transiently in the lung within 12-24 h of infection. We also report that expression of CD11a, CD11b, CD49d, CD31, and CD38 is increased on pulmonary neutrophils in response to influenza virus. Using AhR-deficient mice, we demonstrate that excess neutrophilia in the lung is mediated by activation of the AhR and that this enhanced neutrophilia correlates directly with decreased survival in TCDD-exposed mice. Although AhR activation results in more neutrophils in the lungs, we show that this is not mediated by deregulation in levels of common neutrophil chemoattractants, expression of adhesion molecules on pulmonary neutrophils, or delayed death of neutrophils. Likewise, exposure to TCDD did not enhance pulmonary neutrophil function. This study provides an important first step in elucidating the mechanisms by which AhR agonists exacerbate pulmonary inflammatory responses.     

IL-6 plays an essential role in neutrophilia under inflammation

In the present study, we explored the involvement of interleukin-6 (IL-6) in neutrophilia under inflammatory conditions. The neutrophil count in the peripheral blood was high in arthritic monkeys, and anti-IL-6 receptor antibody reduced neutrophil counts to normal levels. IL-6 injection into normal monkeys significantly increased neutrophil counts in the blood 3h after injection. The expression of cluster of differentiation (CD) 162 on circulating neutrophils was reduced by IL-6 injection. IL-6 treatment in vitro did not affect CD162 expression on neutrophils from human blood. In IL-6-treated monkeys, IL-8 and granulocyte-macrophage colony-stimulating factor (GM-CSF) levels in plasma were clearly elevated. IL-8 and GM-CSF treatment in vitro reduced cell-surface CD162 expression on human neutrophils, and moreover, increased soluble CD162 expression in the cell supernatant. The addition of IL-6 into human whole peripheral blood induced IL-8 production and reduced CD162 expression on neutrophils. Furthermore, IL-8 and GM-CSF augmented mRNA expression of a disintegrin and metalloprotease like domain 10 (ADAM10) in neutrophils. Knock-down of ADAM10 by siRNA in neutrophil-like HL-60 cells partially reversed the expression of CD162 reduced by GM-CSF and IL-8 on HL-60 cells. In conclusion, IL-6 induced neutrophilia and reduced CD162 expression on neutrophils in inflammation.     

Neutrophil Activation in Morbid Obesity,Chronic Activation of Acute Inflammation

Recent studies show that morbid obesity is associated with activation of the innate immune response. Neutrophil activation is a fundamental process in the innate immune response. Therefore, the activation state of neutrophils in severely obese subjects and the effect of bariatric surgery on neutrophil activation was evaluated. Neutrophil activation was assessed by measuring circulating concentrations of myeloperoxidase (MPO) and calprotectin in 37 severely obese and 9 control subjects (enzyme‐linked immunosorbent assay). Moreover, membrane expression of CD66b on circulating neutrophils was measured using flow cytometry in a group of seven severely obese and six control subjects. Immunohistochemical detection of MPO was performed in adipose and muscle tissue. Plasma MPO and calprotectin levels were significantly increased in severely obese subjects as compared to healthy controls, 27.1 ± 10.8 vs. 17.3 ± 5.5 ng/ml (P < 0.001) and 115.5 ± 43.5 vs. 65.1 ± 23.1 ng/ml (P < 0.001) for MPO and calprotectin, respectively. In line, CD66b expression was significantly increased in severely obese individuals, 177.3 ± 43.7 vs. 129.7 ± 9.2 (mean fluorescence intensity) (P < 0.01). Bariatric surgery resulted in decreased calprotectin, but MPO plasma levels remained elevated. Adipose and muscle tissue did not contain increased numbers of MPO expressing cells in severely obese individuals. These results point out that circulating neutrophils are activated to a greater extent in severely obese subjects. Our data support the finding that the innate immune system is activated in severely obese individuals. Moreover, because neutrophils have a short life span, this indicates that the chronic inflammatory condition associated with morbid obesity is characterized by a continuous activation of the innate immune system.     

Partial recovery of neutrophil functions during prolonged hypothermia in pigs

The changes in circulation and migration of mature and immature neutrophils during 12 h of hypothermia have been studied using an experimental pig model. At 29 degrees C the number of circulating neutrophils fell from 5 +/- 1.1 at 37 degrees C to 3.5 +/- 0.6 X 10(9)/l and then remained unchanged while hypothermia was maintained. The number of circulating immature neutrophils did not fall during hypothermia. During hypothermia, hydrocortisone failed to stimulate the release of mature and immature neutrophils from the bone marrow. In contrast, endotoxin caused a profound neutropenia followed by a gradual increase in the number of circulating mature neutrophils, which by 6 h, was similar to the number circulating before endotoxin administration. At 29 degrees C the number of circulating immature neutrophils also fell following endotoxin but then increased over the number circulating before endotoxin administration by approximately 10-fold. Compared with neutrophil migration at 37 degrees C, very few mature or immature neutrophils migrated to an inflammatory site during the 12 h of hypothermia (29 degrees C). Unlike hypothermia in vitro, where neutrophil function may improve with time in vivo, neutrophil function remains compromised.     

Neutrophil chemotactic factors promote leukocytosis. A common mechanism for cellular recruitment from bone marrow.

We investigated cellular responses in a rabbit to i.v. administration of five established chemotactic factors (leukotriene B4 (LTB4), platelet-activating factor (PAF), C5a, N-Formyl-Met-Leu-Phe (F-MLF), and IL-8), and each exerted a characteristic effect on circulating white blood cell levels. All five factors induced a rapid and transient leukopenia. The blood was nearly devoid of circulating neutrophils 5 min after administration of each chemotactic factor. Other leukocytes were also variably depleted during the leukopenic phase, including eosinophils, basophils, monocytes, and lymphocytes. The lymphocyte numbers remained significantly depressed (approximately 30%) for as long as 3 h after administration of PAF or f-MLF. Each chemotactic factor produced a marked neutrophilia (i.e., 250-400% of baseline levels) after the initial leukopenia. Eosinophil numbers were elevated along with the neutrophil response in the C5a- and LTB4-treated animals. Basophil levels were significantly elevated only in LTB4-treated animals. The cellular response to PAF, f-MLF, and IL-8 appeared to be specific for the neutrophils. The kinetic profiles of the neutrophilia induced by PAF (10 micrograms/kg) or f-MLF (2.5 micrograms/kg) were similar, with maximal responses occurring 3 to 4 h after administration. In contrast, LTB4 (10 micrograms/kg), IL-8 (2.5 micrograms/kg), and C5a (5 micrograms/kg) induced a more rapid neutrophilia, with peak responses occurring 1 to 1.5 h after injection, and remaining elevated for 3 to 4 h. In all animals the neutrophilia was accompanied by a relative increase in the number of nonsegmented neutrophils (bands), suggesting that a major component of leukocytosis is caused by the release of bone marrow reserves. Phenidone (10 mg/kg), a dual cyclooxygenase/5-lipoxygenase inhibitor, affected neither the neutropenia nor the neutrophilia induced by C5a, f-MLF, or PAF. The protein synthesis inhibitor actinomycin D also failed to suppress neutrophil responses induced by either C5a or PAF. These results suggest that leukocytosis is a common response induced by all neutrophil chemotactic factors. Leukocytosis appears to be a direct result of the dynamic adaptive response of neutrophils to chemotactic factor stimulation without involvement of a secondary mediator system.     

N-Glycans differentially regulate eosinophil and neutrophil recruitment during allergic airway inflammation

Allergic airway inflammation, including asthma, is usually characterized by the predominant recruitment of eosinophils. However, neutrophilia is also prominent during severe exacerbations. Cell surface-expressed glycans play a role in leukocyte trafficking and recruitment during inflammation. Here, the involvement of UDP-N-acetylglucosamine:α-6-D-mannoside β1,6-N-acetylglucosaminyltransferase V (MGAT5)-modified N-glycans in eosinophil and neutrophil recruitment during allergic airway inflammation was investigated. Allergen-challenged Mgat5-deficient (Mgat5(-/-)) mice exhibited significantly attenuated airway eosinophilia and inflammation (decreased Th2 cytokines, mucus production) compared with WT counterparts, attributable to decreased rolling, adhesion, and survival of Mgat5(-/-) eosinophils. Interestingly, allergen-challenged Mgat5(-/-) mice developed airway neutrophilia and increased airway reactivity with persistent elevated levels of proinflammatory cytokines (IL-17A, TNFα, IFNγ)). This increased neutrophil recruitment was also observed in LPS- and thioglycollate (TG)-induced inflammation in Mgat5(-/-) mice. Furthermore, there was significantly increased recruitment of infused Mgat5(-/-) neutrophils compared with WT neutrophils in the peritoneal cavity of TG-exposed WT mice. Mgat5(-/-) neutrophils demonstrated enhanced adhesion to P-selectin as well as increased migration toward keratinocyte-derived chemokine compared with WT neutrophils in vitro along with increased calcium mobilization upon activation and expression of elevated levels of CXCR2, which may contribute to the increased neutrophil recruitment. These data indicate an important role for MGAT5-modified N-glycans in differential regulation of eosinophil and neutrophil recruitment during allergic airway inflammation.     

Effect of inhaled platelet-activating factor on circulating neutrophils and platelets in vivo and ex vivo in man

We studied the effect of five successive inhalations of platelet-activating factor (PAF) on airway calibre, circulating neutrophil and platelet counts and the activation of these cells ex vivo in normal subjects. PAF (24 ug) caused a mean maximal fall of the expiratory flow rate at 70% vital capacity from a partial manoeuvre (Vp30) of 46.4 +/- 6.2% (p less than 0.001); there was a tachyphylactic response to further doses of PAF. Circulating neutrophil counts fell by 54.3 +/- 10.6% (p less than 0.005) with immediate recovery and with a rebound neutrophilia by the fifth inhalation. Platelet counts showed no significant changes. Aggregation of platelets in platelet-rich plasma to PAF and ADP in vitro at 15 min after the first, second and fifth PAF inhalations was not significantly altered. Chemiluminescence responses of neutrophils to PAF (0.01, 0.1, 1 and 10 microM) in vitro were reduced at 15 min after the fifth PAF inhalation, but this was only significant at 1 microM PAF. Methacholine inhalation did not cause any changes in responsiveness of neutrophils to PAF ex vivo. We conclude that ex vivo platelet desensitisation cannot be used as an index of endogenous PAF release, but reduced responsiveness of neutrophils ex vivo is not a sensitive indicator.     

Porcine granulocyte-colony stimulating factor (G-CSF) delivered via replication-defective adenovirus induces a sustained increase in circulating peripheral blood neutrophils

The use of immunomodulators is a promising area for biotherapeutic, prophylactic, and metaphylactic use to prevent and combat infectious disease. Cytokines, including granulocyte-colony stimulating factor (G-CSF), have been investigated for potential value as biotherapeutic proteins. G-CSF enhances the production and release of neutrophils from bone marrow and is already licensed for use in humans. A limitation of cytokines as immunomodulators is their short half-life which may limit their usefulness as a one-time injectable in production-animal medicine. Here we report that administration of recombinant G-CSF induced a transient neutrophilia in pigs; however, delivery of porcine G-CSF encoded in a replication-defective adenovirus (Ad5) vector significantly increased the neutrophilia pharmacodynamics effect. Pigs given one injection of the Ad5-G-CSF had a neutrophilia that peaked between days 3–11 post-treatment and neutrophil counts remained elevated for more than 2 weeks. Neutrophils from Ad5-G-CSF treated pigs were fully functional based on their ability to release neutrophil extracellular traps and oxidative metabolism after in vitro stimulation. Since acceptable alternatives to the use of antibiotics in food-animal production need to be explored, we provide evidence for G-CSF as a possible candidate for agents in which neutrophils can provide protection.     

Effect of inhaled platelet-activating factor on circulating neutrophils and platelets in vivo and ex vivo in man

We studied the effect of five successive inhalations of platelet-activating factor (PAF) on airway calibre, circulating neutrophil and platelet counts and the activation of these cells in normal subjects. PAF (24 ug) caused a mean maximal fall of the expiratory flow rate at 70% vital capacity from a partial manoeuvre (Vp30) of 46.4 ± 6.2% (p < 0.001); there was a tachyphylactic response to further doses of PAF. Circulating neutrophil counts fell by 54.3 ± 10.6% (p < 0.005) with immediate recovery and with a rebound neutrophilia by the fifth inhalation. Platelet counts showed no significant changes. Aggregation of platelets in platelet-rich plasma to PAF and ADP at 15 min after the first, second and fifth PAF inhalations was not significantly altered. Chemiluminescence responses of neutrophils to PAF (0.01, 0.1, 1 and 10 μM) were reduced at 15 min after the fifth PAF inhalation, but this was only significant at 1 μM PAF. Methacholine inhalation did not cause any changes in responsiveness of neutrophils to PAF . We conclude that platelet desensitisation cannot be used as an index of endogenous PAF release, but reduced responsiveness of neutrophils is not a sensitive indicator.     

Metabolic requirements for maintenance of the chlortetracycline-labeled pool of membrane-bound calcium in human neutrophils

Human neutrophils labeled with chlortetracycline (CTC), commonly used as a probe of membrane-bound calcium, release lysosomal enzymes and exhibit a rapid decrease in fluorescence when exposed to the chemotactic peptide fMet-Leu-Phe or the lectin Con A. This decrease has been attributed to the release of calcium from a membrane-associated "trigger pool." The nature of this putative pool has been further characterized by examining the effects of various inhibitors on the CTC fluorescence response and lysosomal enzyme release from stimulated neutrophils. These agents included inhibitors of glycolysis (2-deoxyglucose and iodoacetate), an uncoupler of oxidative- phosphorylation (KCN), and a sulfhydryl inhibitor (N-ethylmaleimide). Resting neutrophils labelled with CTC demonstrated an enhanced decay of baseline fluorescence when exposed to 2-deoxyglucose or iodoacetate. This suggested that the pool of membrane-bound calcium labelled by this probe was maintained by glycolytic metabolism. Furthermore, 2-deoxyglucose and iodoacetate inhibited both the stimulated decrease in CTC fluorescence and lysosomal enzyme release induced by fMet-Leu-Phe and Con A in a time-dependent manner. KCN did not inhibit either response to stimulation, but did retard the recovery of CTC fluorescence observed when fMet-Leu-Phe was used as the stimulus. High concentrations of N-ethylmaleimide (100 microM) completely inhibited both the CTC fluorescence response and lysosomal enzyme release almost immediately; low concentrations of N-ethylmaleimide (30 microM) inhibited lysosomal enzyme release in a time-dependent manner without significantly affecting changes in CTC fluorescence. These results are consistent with the hypothesis that CTC serves as a probe of membrane-bound "trigger" calcium, the release of which is dependent upon intact glycolysis and is a requirement for lysosomal enzyme release.     

Lysosome proteinases and protein turnover in the liver, spleen and thymus of rats undergoing antigenic stimulation and fasting

Antigenic stimulation (AS) inhibited cathepsin A and D activation in the liver induced by fasting. AS after short-term fasting (4 days) induced the activation increase in the most of lysosomal proteinases by 20-80% in the spleen and thymus, while AS of animals, upon 6- and 8-day fasting depressed the activation of all the investigated lysosomal hydrolases (cathepsin A, B, C, D and arylsulphatases A and B) and decreased the total protein half-life. The revealed peculiarities in the lysosomal proteolytic system function and intensified protein turnover in lymphoid organs may reflect the general reaction of the organism, directed towards the maintenance of immunological homeostasis and, in particular, towards antibody production.     

Histamine release and circulating cells after antigen inhalation in allergic dogs

Histamine can be recovered from the blood of ragweed-sensitized dogs after aerosol antigen challenge, although its source is unknown. Neutrophils and eosinophils have been recovered from bronchoalveolar lavage fluid (BALF) obtained under identical conditions. We investigated the time course of changes in histamine levels in plasma and BALF taken from ragweed-sensitized dogs after aerosol challenge. Changes in the numbers of circulating neutrophils, eosinophils, lymphocytes, monocytes, and platelets were also studied. After 3 min, total pulmonary resistance (RL) was maximally increased and systolic blood pressure was maximally decreased. Histamine levels in plasma and BALF were increased and circulating eosinophils and neutrophils were decreased. After 15 min, platelet numbers were reduced. By 90 min, changes in RL, blood pressure, plasma and BALF histamine concentrations, and circulating neutrophils and eosinophils had returned to base-line values, but platelet numbers remained significantly decreased. Sham challenge caused no significant changes in any of these variables. Intravenous administration of histamine in doses large enough to attain plasma levels comparable with those achieved after aerosol antigen challenge resulted in no concomitant rise in BALF histamine levels. We conclude that antigen challenge in sensitized dogs causes increases in BALF and plasma histamine levels and is associated with a reduction in circulating neutrophils, eosinophils, and platelets. It is likely that antigen causes airway mast cells to release mediators that move down a concentration gradient from the airways to the pulmonary circulation.