F. J. van Overveld

Methods of sputum processing for cell counts, immunocytochemistry and in situ hybridisation

Since the first attempts to use standardised methods for sampling induced airways sputum, two methods for processing the expectorate have evolved. The first involves selecting all viscid or denser portions from the expectorated sample with the aid of an inverted microscope 1, 2. This method has been extensively evaluated and reported in detail 2–4. The second approach involves processing the entire expectorate, comprising sputum plus variable amounts of saliva 5. Recent modifications to this method include collecting saliva and sputum separately in order to reduce salivary contamination 6–8. Both methods have advantages and disadvantages. The advantages of using selected sputum are: squamous cell contamination is 20% of all recovered cells 4. There is conflicting data as to whether or not differential cell counts (DCCs) differ between the two methods. One study reported a higher percentage of eosinophils in sputum processed by the selection method compared to the entire expectorate 9 but this has not been confirmed in other studies 2, 6, 10. Although, both the selected …

Potential role of Clara cell protein, an endogenous phospholipase A2 inhibitor, in acute lung injury

It is now recognized that epithelial cells lining airways and alveoli are capable of releasing various mediators, which have the potential to modulate local inflammatory reactions. The amount of the 16 kDa Clara cell protein (CC16), an inhibitor of phospholipase A2 activity produced by pulmonary epithelial cells, was measured by means of a sensitive immunoassay in the unconcentrated bronchoalveolar lavage fluid (BALF) of 13 control subjects, and in patients with acute lung injury (14 with the full-blown adult respiratory distress syndrome (ARDS); 21 after standard cardiopulmonary bypass surgery, a known risk factor for ARDS). The level of CC16 was compared with other markers of inflammation with a wide range of molecular weights: albumin (nephelometry); total protein (spectrophotometry); beta 2-microglobulin (latex immunoassay); cystatin C (latex immunoassay); alpha 1-antitrypsin (immunoradiometry), and lipocortin-1 (enzyme-linked immunosorbent assay (ELISA)). The Clara cell protein (CC16) was detectable in all BALF, and significantly higher levels of this protein were observed in BALF from patients with acute lung injury. Changes in BALF Clara cell protein levels differed from those of alpha 2-macroglobulin and the natural phospholipase inhibitor lipocortin-1. Alpha 2-macroglobulin levels were not significantly enhanced in patients at risk for ARDS, but were increased in patients with ARDS; whereas, lipocortin 1 levels were not elevated in either group. Pretreatment of patients at risk for ARDS with high dose methylprednisolone did not alter the amount of Clara cell protein recovered in BALF. The mean CC16 level in BALF from patients with ARDS who died was significantly lower than from those who survived. The data presented in this study suggest that pulmonary epithelial cells secrete a natural anti-inflammatory protein during acute lung injury, which might have a protective and immunosuppressive role.

N-acetylcysteine pretreatment of cardiac surgery patients influences plasma neutrophil elastase and neutrophil influx in bronchoalveolar lavage fluid

ObjectiveStudy of leukocyte activation and release of toxic mediators during extracorporeal circulation (ECC). ECC can be used to study the potential protective effect of a pharmacon against neutrophil-mediated lung injury. Clinical studies have indicated that N-acetylcysteine (NAC) may improve systemic oxygenation and reduce the need for ventilatory support when given to patients with acute lung injury.DesignCardiac surgery patients were pretreated with high-dose NAC in order to assess the potential role of NAC to interfere with neutrophil-mediated inflammation and lung injury.Patients18 patients who underwent ECC: group 1 (n=8) no premedication (only placebo); group 2 (n=10) NAC (72 mg/kg i.v. as a bolus, later 72 mg/kg over 12 h).Measurements and resultsIn group 2, the partial pressure of oxygen in arterial blood/fractional inspired oxygen 4 h after surgery was significantly higher than in group 1 (213±31 vs 123±22;p=0.044). NAC pretreatment prevented an increase in plasma neutrophil elastase activity (18.9±6.9 vs 49.9±5.6 ng/ml in group 1 at the end of ECC;p=0.027). Release of myeloperoxidase (MPO) was not affected (group 1: 1105±225 ng/ml vs group 2: 1127±81 at the end of ECC;p=0.63). At the end of ECC, total antigenic human neutrophil elastase (group 1: 671±72 ng/ml vs group 2: 579±134;p=0.37) and complex formation between elastase and α1-proteinase inhibitor were no different in the two groups. There were no significant differences in cellular composition and mediators in the lavage fluid, although values for total number of neutrophils, elastase, MPO and interleukin-8 were lower in group 2.ConclusionPretreatment with NAC may prevent lung injury by diminishing elastase activity. Since the release of mediators, especially MPO, is not affected, this diminished activity of elastase may be achieved by enhanced inactivation by antiproteases after initial treatment.

Role of elastases in the pathogenesis of chronic obstructive pulmonary disease: Implications for treatment

Neutrophil elastase, metalloproteinases, and their inhibitors play an important role in the development of chronic obstructive pulmonary disease (COPD), resulting in extensive tissue damage and malfunctioning of the airways. Nearly fifty years after the protease-antiprotease imbalance hypothesis has been suggested for the cause of emphysema, it is still appealing, but it does not explain the considerable variation in the clinical expressions of emphysema. However, there are many recent research findings to support the imbalance hypothesis as will be shown in this review. Although limited, there might be openings for the treatment of the disease.

Induced sputum of patients with chronic obstructive pulmonary disease (COPD) contains adhesion-promoting, therapy-sensitive factors

Abstract.Objective: The aim of this study was to investigate whether sputum of COPD patients before and after treatment with inhaled corticosteroids (IHC) or N-acetylcysteine (NAC) exerts any effect on the adhesion of isolated polymorphonuclear cells (PMNs) to cultured endothelial cells.¶Methods: A human endothelial cell line was grown to confluence before use in adhesion experiments. PMNs were obtained from normal, non-smoking volunteers and preincubated (30 min, 37°C) with diluted sputum sol obtained from COPD patients before the cells were put on the endothelial cells.¶Results: Basal adhesion of unstimulated PMNs after 30 min at 37°C in 5% CO2 was 15.9 ± 1.1% (mean ± SEM, n = 9). A significant enhancement of the adhesion to 33.0 ± 1.4% (n = 11, P < 0.0001) was observed with sputum obtained from COPD patients before treatment with IHC, and 34.6 ± 1.5% (n = 10, P<0.0001) before treatment with NAC. Administration of IHC for 8 weeks resulted in an adhesion of 27.7 ± 2.4%, which is an inhibition of 31% (n = 11, P<0.05). However, treatment for 8 weeks with NAC showed no change in the adhesion of stimulated PMNs. Long-term treatment with NAC showed a gradual decrease of adhesion (n = 9, P<0.05), whereas long-term treatment with IHC lead to an increase in adhesion (n = 10, P<0.02).¶Conclusions: These results indicate that factors locally produced in the airways of COPD patients may pro-mote adhesion of neutrophils to endothelium. They further suggest that glucocorticoids may only have a short-term transient effect on adhesion, whereas NAC showed effects on the adhesion after administration for longer periods.¶

Nitroprusside, a nitrogen oxide generating drug, inhibits release of histamine and tryptase from human skin mast cells

We have investigated the effects of the nitric oxide donor sodium nitroprusside on the immunologic release of histamine and tryptase from human mast cells. Isolated human skin mast cells were purified using Percoll gradient centrifugation. Mast cells with a purity of 70–90% were preincubated for 30 minutes with sodium nitroprusside and subsequently stimulated with anti-IgE. Upon challenge with anti-IgE, a release of 6.37±0.46 nmol histamine and 612±49 ng tryptase per 106 cells was observed. In presence of sodium nitroprusside a dose-dependent decrease of the release occurred. The results suggest that nitric oxide is effective in reducing the release of histamine and tryptase from human skin mast cells.

Pretreatment with methylprednisolone in coronary artery bypass grafting influences the levels of histamine and tryptase in serum but not in bronchoalveolar lavage fluid.

1. The presence of histamine and tryptase in serum during and after coronary artery bypass grafting may be an indication of the induction of inflammation. 2. One group of patients received no glucocorticoids and a second group received methylprednisolone before extracorporeal circulation. In the steroid group no effects were seen on the basal levels of histamine (2.84 +/- 0.12 ng/ml) and tryptase (0.50 +/- 0.05 ng/ml) during and after surgery. In the other group two peak levels of histamine were observed: one at 10 min after starting extracorporeal circulation (4.19 +/- 1.79 ng/ml) and another at 4 h after surgery (8.26 +/- 4.85 ng/ml). In this group tryptase was only elevated during the period of extracorporeal circulation (1.54 +/- 0.16 ng/ml). 3. There were no differences between the two groups in complement activation. C3a levels rose to 170 +/- 8% and 180 +/- 10% of the initial value in the steroid and non-steroid group, respectively. 4. It was concluded that during surgery mast cells were activated, but since tryptase levels decreased in the post-operative period, the second increase in the histamine level can be explained by activation of basophils or by an unknown mechanism for the release of histamine but not tryptase by mast cells. 5. In the bronchoalveolar lavage fluid the levels of histamine and tryptase showed no differences between the two groups of patients, but histamine was enhanced compared with normal levels.

Release of arachidonic acid metabolites from isolated human alveolar type II cells

Human alveolar type II cells are thought to play a role in the pathogenesis of lung injury. Patterns of mediator release of arachidonic acid metabolism by type II cells were therefore studied after challenge with calcium ionophore A23187, opsonized zymosan and hydrogen peroxide. A time- and concentration dependent release of cyclooxygenase products was observed, with release of PGE2 greater than 6-keto-PGF1 alpha greater than TxB2. Addition of glutathione or bicarbonate further increased the production of PGE2. N-ethylmaleimide, a sulfhydryl (SH) reactant, induced a dose-dependent increase in the release of TxB2 and 6-keto-PGF1 alpha, but not of PGE2. This relates most likely to the SH-dependency and glutathione requirement of the PGE2 isomerase and SH-independence of thromboxane and prostacyclin isomerase.

Use of ICAM-1 antibodies and antisense oligonucleotides to inhibit transmigration of neutrophils

AbstractObjective: The increase in adhesion molecule expression during the initial phase of inflammation leads to transmigration of neutrophils. Inhibition of this transmigration could decrease inflammatory response and tissue damage. Materials and methods: Uptake of fluorescein-labelled oligonucleotides, expression of ICAM-1 and neutrophil migration were studied using the bilayer of epithelial (H292) and endothelial (ECV-304) cell lines and neutrophils from peripheral blood of healthy volunteers. Results: The inhibition of ICAM-1 expression was time dependent for both cell lines, with inhibition of more than 50% after 48 h. Antisense oligos inhibited transmigration already after 4 h of incubation (6.6 ± 2.5% versus 18.6 ± 2.5% inhibition, p < 0.01). Antisense was more effective in preventing fMLP-induced neutrophil migration than ICAM-1 antibodies (88 ± 3.8% versus 67 ± 7% inhibition, p = 0.02). Conclusions: Antisense oligos cause a decrease in ICAM-1 expression and inhibit transmigration of neutrophils. However the effectiveness of antisense is higher than monoclonal antibodies, neither of them is able to block the migration completely. This suggests the existence of ICAM-1 independent mechanisms that are responsible for migration.

Mast cell subtypes from human lung tissue: their identification, separation, and functional characteristics

The contribution of mast cell subtypes and their different mediators to the pathogenesis of chronic obstructive lung diseases (COLD) has not yet been established. In the present study, enzymatic digestion, centrifugal elutriation and Percoll gradient centrifugation were used to obtain two populations of mast cell subtypes from human lung tissue. Mast cell subtypes were challenged with anti-human IgE, propranolol, compound 48/80, or opsonized zymosan. Both subtypes were able to release histamine, but differed in the amount of the amine released. Only the formalin-sensitive and alcian blue-positive type (FS-AB) released histamine on challenge with opsonized zymosan. The same subtype was able to release leukotriene C4 (LTC4) after challenge with anti-human IgE. The other subtype, the formalin-insensitive and alcian blue-positive type (FI-AB), did not respond to opsonized zymosan and did not release LTC4 after challenge with anti-human IgE. Stimulation with propranolol or compound 48/80 did not release histamine from the FS-AB mast cells while the FI-AB mast cells released only about 10% of their histamine content upon challenge with these secretagogues.