Richard A. Robbins

Increased nitric oxide in exhaled air of asthmatic patients

Nitric oxide (NO) gas is produced by various cells within the lower respiratory tract, including inflammatory and epithelial cells, and is detectable in the exhaled air of normal human subjects. We have measured exhaled NO in patients with asthma, since several cell types that are activated in asthma can produce NO after induction. NO was measured reproducibly by a slow vital capacity manoeuvre and an adapted chemiluminescence analyser. NO was detectable in exhaled air of 67 control subjects (mean peak concentration 80.2 [SE 4.1] ppb) and was significantly reduced by inhalation of the specific NO synthase inhibitor NG-monomethyl-L-arginine. 61 non-steroid-treated asthmatic subjects had significantly higher peak expired NO concentrations than controls (283 [16] ppb, p < 0.001) but 52 asthmatic patients receiving inhaled corticosteroids had levels similar to controls (101 [7] ppb). High exhaled NO concentrations in asthmatic patients may reflect induction of NO synthase, which is known to be inhibited by steroids. Measurement of exhaled NO concentrations may be clinically useful in detection and management of cytokine-mediated inflammatory lung disorders.

Induction of cyclo‐oxygenase‐2 by cytokines in human pulmonary epithelial cells: regulation by dexamethasone

1 Cyclo‐oxygenase metabolizes arachidonic acid to prostaglandin H2 (PGH2) and exists in at least two isoforms. Cyclo‐oxygenase‐1 (COX‐1) is expressed constitutively whereas COX‐2 is induced by lipopolysaccharide (LPS) and some cytokines in vitro and at the site of inflammation in vivo. Epithelial cells may be an important source of prostaglandins in the airways and we have, therefore, investigated the expression of COX‐1 or COX‐2 isoforms in primary cultures of human airway epithelial cells or in a human pulmonary epithelial cell line (A549). 2 COX‐1 or COX‐2 protein was measured by western blot analysis using specific antibodies to COX‐2 and selective antibodies to COX‐1. The activity of COX was assessed by the conversion of either endogenous or exogenous arachidonic acid to four metabolites, PGE2, PGF2α, thromboxane B2 or 6‐oxo PGF1α measured by radioimmunoassay. Thus, COX‐1 or COX‐2 activity was measured under two conditions; initially the accumulation of the COX metabolites formed from endogenous arachidonic acid was measured after 24 h. In other experiments designed to measure COX activity directly, cells were treated with cytokines for 12 h before fresh culture medium was added containing exogenous arachidonic acid (30 μm) for 15min after which COX metabolites were measured. 3 Untreated primary cells or A549 cells contained low amounts of COX‐1 or COX‐2 protein. Bacterial LPS (1 μg ml−1 for 24 h) induced COX‐2 protein in the primary cells, a process which was enhanced by interferon‐γ, with no further increase in the presence of a mixture of cytokines (interleukin‐1β, tumour necrosis factor‐α and interferon‐γ, 10 ng ml−1 for all). In contrast, A549 cells contained only low levels of COX‐2 protein after exposure to LPS or LPS plus interferon‐γ, but contained large amounts of COX‐2 protein after exposure to the mixture of cytokines. 4 Untreated human pulmonary primary cells or A549 cells released low levels of all COX metabolites measured over a 24 h incubation period. This release was enhanced by treatment of either cell type with the mixture of cytokines (interleukin‐10, tumour necrosis factor‐α and interferon‐γ, 10ng ml−1 for all). PGE2 was the principal COX metabolite released by cytokine‐activated epithelial cells. The release of PGE2 induced by cytokines occurred after a lag period of more than 6 h. 5 The glucocorticosteroid, dexamethasone (1 μm; 30 min prior to cytokines) completely suppressed the cytokine‐induced expression of COX‐2 protein and activity in both primary cells and A549 cells. 6 In experiments where COX‐2 activity was supported by endogenous stores of arachidonic acid, treatment of A549 cells with interleukin‐10 but not tumour necrosis factor‐α or interferon‐γ alone caused a similar release of PGE2 to that seen when the cytokines were given in combination. However, both interleukin‐10 and necrosis factor‐α alone produced similar increases in COX‐2 activity (measured in the presence of exogenous arachidonic acid) as seen when the mixture of interleukin‐1β, tumour necrosis factor‐α and interferon‐γ were used to stimulate the cells. 7 These findings show that COX‐2 expression correlates with the exaggerated release of prostaglandins from cytokine‐activated human pulmonary epithelial cells and that the induction of the enzyme is suppressed by a glucocorticosteroid. These findings may be relevant to inflammatory diseases of the lung, such as asthma.

Aprotinin and methylprednisolone equally blunt cardiopulmonary bypass–induced inflammation in humans ☆ ☆☆ ★ ★★

Cardiopulmonary bypass induces an inflammatory state characterized by tumor necrosis factor-alpha release. Integrin CD11b is a neutrophil surface adhesive glycoprotein integrin that is rapidly and permanently unregulated by tumor necrosis factor-alpha exposure. The CD11b integrin is known to be the primary neutrophil integrin responsible for neutrophil lung and myocardial entrapment after cardiopulmonary bypass and subsequent reperfusion injury. Twenty-four adults admitted to the hospital for myocardial revascularization were equally randomized to one of three groups: group A (control), group B (methylprednisolone before cardiopulmonary bypass), and group C (low-dose aprotinin protocol). Blood was collected at three times: (1) baseline, (2) 50 minutes of cardiopulmonary bypass duration, and (3) 30 minutes after cardiopulmonary bypass termination. Neutrophil CD11b integrin expression was measured by fluorescence-activated cell sorter analysis and plasma tumor necrosis factor-alpha levels measured by enzyme-linked immunosorbent assay. Group A demonstrated significant (p < 0.05) increases in CD11b expression at times 2 and 3 when results were compared with those of the same group baseline and with those of groups B and C at similar times. No significant changes were noted between groups B and C at any time. Group A demonstrated a significant (p < 0.05) increase in levels of tumor necrosis factor-alpha at time 3 when results were compared with those of the same group baseline and of groups B and C at the same time. No significant changes were noted between B and C at any time. These results demonstrate low-dose aprotinin has a similar antiinflammatory effect to that of methylprednisolone in blunting cardiopulmonary bypass-induced systemic tumor necrosis factor-alpha release and neutrophil integrin CD11b upregulation.

Diffuse alveolar hemorrhage in autologous bone marrow transplant recipients.

PURPOSE The purpose of our work was to evaluate pulmonary complications in autologous bone marrow transplant recipients. PATIENTS AND METHODS A total of 141 consecutive autologous bone marrow transplant recipients were evaluated. In 29 patients, a clinical syndrome characterized by progressive dyspnea, hypoxia, cough, diffuse consolidation on chest roentgenography, and characteristic bronchoalveolar lavage findings developed over one to seven days. RESULTS In 29 patients, bronchoalveolar lavage performed by sequential instillation and aspiration of 20-ml aliquots of normal saline resulted in recovered lavage fluid that became progressively bloodier with each recovered aliquot. Autopsy and bronchoalveolar lavage in these patients revealed no pathogens that accounted for the clinical findings. Since the later aliquots sample predominantly alveolar material, this syndrome was termed diffuse alveolar hemorrhage (DAH). DAH was associated with a high inpatient mortality rate (23 of 29 died versus 14 of 112 without DAH, p less than 0.001) and was associated with age over 40 years, solid malignancies, high fevers, severe mucositis, white blood cell recovery, and renal insufficiency (p less than 0.05, compared with patients without DAH). However, DAH was not associated with prolonged prothrombin or partial thromboplastin times or decreased platelet counts compared with patients without DAH. CONCLUSION DAH is a frequent cause of respiratory compromise and a major cause of mortality in autologous bone marrow transplant recipients.

Immunological functions of the pulmonary epithelium

The mature pulmonary epithelium forms a continuous lining to the airspace. Recent data suggest that this specialized epithelium may also contribute to host defence via interactions with inflammatory cells. Pulmonary epithelial cells can serve as part of the local immune system, providing structures and functions crucial for the maintenance of normal pulmonary function. This article will briefly review the morphology and development of the pulmonary epithelial cells, their function with regard to host defence, alterations of the pulmonary epithelium associated with airway diseases, and potential therapeutic implications for the treatment of respiratory diseases.

Corticosteroids as adjunctive therapy for diffuse alveolar hemorrhage associated with bone marrow transplantation

BACKGROUND Diffuse alveolar hemorrhage is a frequent complication of treating malignancies with high-dose chemotherapy and bone marrow transplantation and is associated with very high mortality. This disorders association with pulmonary inflammation, its coincidence with marrow recovery, and the usefulness of corticosteroids for treating other pulmonary hemorrhage syndromes provided the rationale for this study. METHODS We retrospectively studied 65 episodes of diffuse alveolar hemorrhage that has occurred in 63 of 603 consecutively treated patients who had undergone high-dose chemotherapy with bone marrow transplantation. Patients were divided into three groups according to the therapy they had received for diffuse alveolar hemorrhage: supportive therapy alone (n = 12); low-dose corticosteroids (30 mg or less of methylprednisolone or its equivalent; n = 10); and high-dose corticosteroids (more than 30 mg methylprednisolone or its equivalent; n = 43). The primary outcome measures were overall survival and survival to hospital discharge, occurrence of respiratory failure requiring intubation, and development of infections subsequent to the diagnosis of diffuse alveolar hemorrhage. RESULTS Overall survival at the end of the follow-up period was significantly higher for the high-dose corticosteroid group compared with the supportive therapy group (P = 0.005); however, treatment with low-dose steroids did not increase survival over supportive therapy alone (P = 0.198). In addition, survival to discharge was significantly increased for the high-dose group compared with the other two groups combined (33% versus 9.1%, P = 0.038). Respiratory failure after the diagnosis of diffuse alveolar hemorrhage developed in only 12 of the 22 unintubated patients in the high-dose group compared with 9 of the 10 initially unintubated patients in the other two groups (P = 0.056). Although the incidence of infections was high (40%) subsequent to diffuse alveolar hemorrhage, neither high-dose nor low-dose corticosteroid treatment significantly increased the risk of infections (P > 0.4, all comparisons). CONCLUSIONS In this study, high-dose corticosteroid therapy for diffuse alveolar hemorrhage related to bone marrow transplantation was associated with improved total survival and survival to hospital discharge, and decreased development of respiratory failure in these patients. These results suggest the therapy is beneficial, and further prospective studies are warranted to verify the effectiveness of the treatment.

Aprotinin reduces interleukin-8 production and lung neutrophil accumulation after cardiopulmonary bypass.

Pulmonary neutrophil entrapment and resultant oxidative injury is thought to be the primary mechanism of cardiopulmonary bypass (CPB) induced lung injury.Interleukin-8 (IL-8), a potent neutrophil chemoattractant induced by cytokines, including tumor necrosis factor-alpha (TNF), is found in increased concentrations in bronchial alveolar lavage fluid (BALF) in lung inflammation. Since aprotinin reduces TNF release during CPB, the effects of aprotinin on BALF IL-8 concentrations and neutrophil levels were determined after CPB in adult humans. Study patients were equally divided into a control group (n = 8, Group 1) and an aprotinin treated group (n = 8, Group 2). In vitro neutrophil chemotaxis was done with volunteer neutrophils using three different chemoattractants: 1) N-formyl-1-methionyl-1-leucyl-1-phenylalanine (FMLP); 2) the supernatant of a human bronchial epithelial cell culture line, A549, after 24 h of TNF stimulation with or without aprotinin or N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK) (a potent protease inhibitor), and 3) BALF. Aprotinin treatment significantly (P < 0.05) reduced post-CPB BALF IL-8 concentrations and percentage of neutrophils. In vitro, BALF from Group 1 had significantly greater chemotactic ability when compared with Group 2. The TNF stimulated A549 cell culture supernatant had significantly (P < 0.05) greater chemotactic ability than control supernatant, while aprotinin and TLCK significantly (P < 0.05) reduced this chemotactic ability. These results demonstrate that aprotinin blunts IL-8 production and reduces neutrophil lung accumulation post-CPB. (Anesth Analg 1996;83:696-700)

Glucocorticoids Blunt Neutrophil Cd11b Surface Glycoprotein Upregulation During Cardiopulmonary Bypass in Humans

Neutrophil-endothelial adhesion is the initiating event in neutrophil migration to areas of infection or injury. The binding of neutrophils to endothelium depends upon adhesive glycoproteins, of which the CD11/CD18 glycoproteins are the most important. Because of known upregulation of one of these adhesive glycoproteins (CD11b) during cardiopulmonary bypass (CPB) in humans, we evaluated CD11a, CD11b, and CD11c surface expression before, during, and after CPB in humans, with or without pre-CPB administration of a glucocorticoid (methylprednisolone). Fourteen patients were randomized into two groups: Group S received methylprednisolone (1 g intravenously) 5 min prior to CPB; Group N received no steroid. CD11b was significantly upregulated (P < 0.01) during, and 24 h after, CPB in Group N when compared with controls and Group S at similar time intervals, while in Group S no significant changes were found. Since interleukin-1, tumor necrosis factor, and endotoxin are known to upregulate neutrophil CD11b surface expression and are released during CPB in humans, while steroids are known to suppress the release of these cytokines, the authors conclude that the blunting effect by steroids on CD11b surface expression upregulation during and after CPB in humans is attributed to suppressed cytokine release.

Nitric oxide synthase inhibitors attenuate human monocyte chemotaxis in vitro

Nitric oxide synthase (NOS) inhibitors have been shown to modulate neutrophil migration. We hypothesized that the NOS inhibitors NG‐monomethyl‐L‐ arginine (L‐NMMA), NG‐nitro‐L‐arginine methyl ester (L‐NAME), and L‐canavanine (L‐CAN) also modulate human peripheral blood monocyte chemotaxis. To test this hypothesis, monocyte chemotaxis toward formylmethionyl‐ leucyl‐phenylalanine (fMLP) was assessed using a modified blindwell chemotaxis chamber technique. L‐ NMMA and L‐NAME, but not D‐NMMA or L‐CAN, significantly attenuated fMLP‐induced monocyte chemotaxis (P < .05). L‐Arginine and sodium nitroprusside, but not D‐arginine, reversed NOS inhibitor‐induced responses. Dibutryl cyclic guanyl monophosphate (cGMP) attenuated the inhibitory effects of L‐NMMA on monocyte chemotaxis (P < .05). Finally, fMLP increased cGMP generation by monocytes, which was significantly attenuated by L‐NMMA (P < .05). These data indicate that the L‐arginine/NO biosynthetic pathway regulates human monocyte chemotaxis in vitro.

Cigarette smoke decreases inducible nitric oxide synthase in lung epithelial cells.

Cigarette smoking has been associated with decreased exhaled nitric oxide (NO). To investigate the mechanism of this decrease, the effects of a cigarette smoke extract were evaluated a murine lung epithelial cell line (LA-4), a human lung epithelial cell line (A549), and primary cultures of human lung epithelial cells induced to produce NO by cytokines. NO production was evaluated by measuring nitrite, a stable end product of NO, in cell culture supernatant fluids. Cigarette smoke extract caused a reduction in the cytokine-induced nitrite concentrations in the culture supernatant fluids from all 3 cell types (P<.01, all comparisons). To further investigate these observations, immunohistochemistry demonstrated a decrease in cytokine-induced inducible NO synthase (iNOS) protein expression and iNOS mRNA after cigarette smoke extract exposure in LA-4 cells. However, iNOS mRNA half-life was not altered by the smoke extract, suggesting that the smoke extract decreased NO by decreasing iNOS mRNA transcription. These findings demonstrate that cigarette smoke extract decreases iNOS expression and NO production from lung epithelial cells.