Human neutrophil elastase abolishes interleukin-8 chemotactic activity.

A large body of literature supports the role of interleukin-8 (IL-8) in inflammatory lung disease. Numerous factors induce the local synthesis and secretion of this potent chemokine leading to the recruitment and activation of polymorphonuclear leukocytes. However, little is currently known about the fate of IL-8 secreted at sites of inflammatory injury. We have found that incubation of recombinant human IL-8 with purified human neutrophil elastase (HNE) results in the loss of IL-8 chemotactic activity in a dose- and time-dependent fashion. This loss in bioactivity is accompanied by a similar loss of IL-8 immunoreactivity. Western blot analysis revealed that IL-8 chemotactic activity is lost by proteolysis of the parent molecule into undetectable small fragments. The terminal digestion of IL-8 was specific to HNE as no loss of bioactivity was observed with equimolar concentrations of the serine proteases urokinase, plasmin, thrombin, or cathepsin G. This effect on chemotactic activity is not limited to recombinant IL-8 because HNE also digested IL-8 secreted by human monocytes. HNE-mediated proteolysis offers a novel mechanism for down-regulating the inflammatory cascade initiated by IL-8.

A cytosolic inhibitor of human neutrophil elastase and cathepsin G.

The neutrophil serine proteinases elastase and cathepsin G produce connective tissue injury, the extent of which depends on the balance between these enzymes and their inhibitors. The most important of these inhibitors is alpha 1-proteinase inhibitor, a member of a superfamily of homologous proteins known as serpins. Neutrophil cytosol inhibited the activities of human neutrophil elastase and cathepsin G in a dose-dependent fashion. To demonstrate formation of an enzyme-inhibitor complex, we combined 125I-elastase or 125I-cathepsin G with neutrophil cytosol or alpha 1-proteinase inhibitor and analyzed the products by polyacrylamide gel electrophoresis. Unbound elastase and cathepsin G each migrated to an apparent molecular weight of 25 kDa. In the presence of cytosol from neutrophils both radiolabeled enzymes migrated with a relative size of 68 kDa, whereas in the presence of alpha 1-proteinase inhibitor the relative size was 85 kDa. Enzyme-inhibitor complexes were stable in sodium dodecyl sulfate at 100 degrees C but were dissociated by hydrolysis in ammonium hydroxide (1.5 mol/L) at 37 degrees C. Formation of each complex was prevented by pretreatment of elastase or cathepsin G with diisopropylfluorophosphate, indicating that the inhibitor binds to the active site of the enzyme. Exposure of either alpha 1-proteinase inhibitor or neutrophil cytosol to the myeloperoxidase-H2O2-halide system prevented complex formation, suggesting the presence of an oxidizable amino acid at the binding site of the inhibitor. By electrophoretic analysis, the molecular weight of the cytosolic inhibitor was 43 kDa and neutrophils contained approximately 1 attomol of inhibitor per cell. The isoelectric points of the elastase and cathepsin G inhibitor were 5.5-5.9 and inhibitors of the two proteinases coeluted using size exclusion chromatography. These data demonstrate that human neutrophil cytosol contains a single serpinlike protein that inhibits elastase and cathepsin G. The inhibitor may be important in protecting the intracellular environment from proteolytic injury during degranulation.

CD11b/CD18 mediates the neutrophil chemotactic activity of fibrin degradation product D domain.

Coagulation and fibrinolysis universally accompany tissue injury and repair. The accumulation of regionally generated fibrin degradation products (FDP) may modify the local inflammatory response. We have found FDP to be potent neutrophil chemotaxins. We separated plasmin FDP by chromatofocusing and found chemotactic activity limited to fractions containing the fibrinogen D domain (D-D dimer and D monomer). The bioactivity of the D-D dimer did not require an intact cross link site as removal of this sequence with puff adder venom or hypocalcemic plasmic digestion did not decrease chemotaxis. Peptide inhibition studies confirmed that the chemotactic region did not involve terminal gamma chain sequences or alpha chain RGD motifs. The internal gamma chain peptide KYGWTVFQKRLDGSV (P1), known to bind CD11b/CD18, exhibited concentration dependent chemotactic activity. Similarly, monoclonal antibodies directed against CD11b/CD18 blocked PMN migration to FDP without similar inhibition of chemotaxis to IL-8 or LTB4. Thus, neutrophil chemotaxis to FDP is mediated by interactions between the fibrinogen D domain and CD11b/CD18.

Prognostic role of eosinophils in pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) and pulmonary fibrosis associated with a collagen vascular disorder (PF-CVD) are chronic inflammatory lung disorders which may be characterized in various subgroups of patients by increased numbers of macrophages, neutrophils, lymphocytes, and/or eosinophils. Previous studies have suggested that the cell populations recovered with bronchoalveolar lavage (BAL) may be important in predicting disease progression and response to therapy. We evaluated this hypothesis in 27 patients by determining if the cell populations recovered with BAL differed between patients who improved, remained stable, or worsened in their pulmonary functions (as defined by at least a 15 percent change in forced vital capacity) over a six-month observation period. The findings suggested that BAL eosinophilia may be a marker of progressive lung disease in patients with IPF and PF-CVD.

Nodular pulmonary opacities in patients with rheumatoid arthritis. A diagnostic dilemma.

Nodular opacities are a well-known pulmonary manifestation of rheumatoid arthritis (RA), occurring most often in seropositive men who smoke and have subcutaneous nodules. In the past 15 years two cases of lung carcinoma presenting as pulmonary nodules have been reported in patients with rheumatoid disease. We present seven patients with seropositive RA and subcutaneous nodules who had new pulmonary nodule(s) noted on chest roentgenograms. All but one were current smokers. Carcinoma was found in all patients at bronchoscopy or thoracotomy. Four patients had solitary nodules (one was cavitary); the remaining three patients had multiple bilateral nodules that cavitated in one case. All patients had interstitial abnormality (peribronchial/vascular thickening) with basal predominance in three, and there was evidence of pleural thickening/fluid in three patients. These results strongly suggest that histologic proof of presumed rheumatoid pulmonary nodules be obtained.

Outcome after cardiopulmonary resuscitation in a medical intensive care unit.

Cardiopulmonary resuscitation (CPR) is often performed in modern critical care units, but its efficacy has not been evaluated in this setting. It is important to evaluate CPR in critical care units because these patients often have multisystem disorders and suffer from diseases reported to carry a poor outcome after CPR. Inappropriate resuscitation of patients in this setting results in increased cost of care (both financial and emotional), with little tangible benefit. To address the question of successful resuscitation in the medical intensive care unit (MICU), we retrospectively reviewed the records of 114 patients who underwent CPR in our MICU over a three-year period. Eighty patients (70 percent) were not successfully resuscitated, 21 patients (18 percent) were successfully resuscitated but died before discharge, and 13 patients (11 percent) survived to leave the hospital. We evaluated a number of prearrest conditions (diagnoses, age, sex, duration of hospitalization, length of ICU stay, and severity of illness as measured by APACHE 2 scores) and arrest conditions (the initial cardiac rhythm and duration of CPR) to determine if the outcome after CPR was influenced by any of these parameters. Among the prearrest conditions, only a diagnosis of hypotension or sepsis and an elevated APACHE 2 acute physiology score were independently associated with a poor outcome after CPR. The only arrest condition found to be independently associated with outcome following CPR was the duration of resuscitative effort (p less than 0.01). The patients who were successfully resuscitated but died before discharge were not different from the patients who were not successfully resuscitated in any parameter that we evaluated. These results demonstrate that CPR can be successful in the MICU and that there are prearrest and arrest parameters which are useful in identifying those patients most likely to benefit from CPR in the critical care setting.

Predicting postoperative pulmonary function in patients undergoing lung resection.

OBJECTIVE: Our aim was to determine the effect of lung resection on spirometric lung function and to evaluate the accuracy of simple calculation in predicting postoperative pulmonary function in patients undergoing lung resection. DESIGN: We reviewed preoperative and postoperative pulmonary function test results on patients who were followed in the multidisciplinary lung cancer clinic between July 1991 and March 1994 and who underwent lung resection. The predicted postoperative FEV1 and FVC were calculated based on the number of segments resected and were compared with the actual postoperative FEV1 and FVC. SETTING: This study was conducted at a university, tertiary referral hospital. PATIENTS: All patients were evaluated at a multidisciplinary lung cancer clinic and underwent lung resection by one surgeon (L.A.L.). MEASUREMENTS AND MAIN RESULTS: Sixty patients undergoing 62 pulmonary resections were reviewed. The predicted postoperative FEV1 and FVC were calculated using the following formula: predicted postoperative FEV1 (or FVC) = preoperative FEV1 (or FVC) x (1-(S x 0.0526)); where S = number of segments resected. The actual postoperative FEV1 and FVC correlated well with the predicted postoperative FEV1 and FVC for patients undergoing lobectomy (r = 0.867 and r = 0.832, respectively); however, the predicted postoperative FEV1 consistently underestimated the actual postoperative FEV1 by approximately 250 mL. For patients undergoing pneumonectomy, the actual postoperative FEV1 and FVC did not correlate as well with the predicted postoperative FEV1 and FVC (r = 0.677 and r = 0.741, respectively). Although there was considerable variability, the predicted postoperative FEV1 consistently underestimated the actual postoperative FEV1 by nearly 500 mL. Of the patients undergoing lobectomy, eight also received postoperative radiation therapy. When analyzed separately, patients receiving combined therapy lost an average of 5.47% of FEV1 per segment resected. This contrasts with a 2.84% per segment reduction in FEV1 for patients who did not receive radiation therapy. CONCLUSIONS: This simple calculation of predicted postoperative FEV1 and FVC correlates well with the actual postoperative FEV1 and FVC in patients undergoing lobectomy. The predicted postoperative FEV1 consistently underestimated the actual postoperative FEV1 by approximately 250 mL. The postoperative FEV1 and FVC for patients undergoing pneumonectomy is not accurately predicted using this equation. The predicted postoperative FEV1 for patients undergoing pneumonectomy was underestimated by an average of 500 mL and by greater than 250 mL in 12 of our 13 patients. Thus, by adding 250 mL to the above calculation of predicted postoperative FEV1, we improve our ability to we identify a minimal postoperative FEV1 for patients undergoing pneumonectomy. Finally, combined modality treatment with surgery followed by radiation therapy may result in additive lung function loss.

Correlation of chest roentgenograms with pulmonary function and bronchoalveolar lavage in interstitial lung disease.

We used the ILO classification for occupational lung disease to determine whether there was any correlation between the type and/or severity of pulmonary infiltration on chest roentgenograms and either pulmonary function tests or the types of inflammatory cells present in BAL fluid in patients with interstitial lung disease. Of the 62 patients evaluated (27 with sarcoidosis, 18 with IPF, and 17 with a CV disease and lung involvement), 49 had irregular linear opacities and 13 had normal chest x-rays. There were no significant correlations between the types of cells present in BAL fluid and the various categories of infiltrate or profusion of the infiltrates within each disease group. In patients with sarcoidosis, more extensive infiltration (profusion) was associated with lower FEV, (p less than 0.01). In patients with IPE, linear opacity type, profusion, and the presence or absence of honeycombing were not related to the severity of pulmonary function abnormalities. We conclude that the ILO classification for analysis of chest roentgenograms can be applied to patients with interstitial lung disease not associated with an occupational exposure and that this approach is useful, especially for communication. However, these data provide no information regarding the inflammatory process in the lung and limited information regarding abnormalities in pulmonary function.

Realizing the promise: delivering pulmonary continuing medical education over the Internet.

STUDY OBJECTIVES: Continuing medical education (CME) is meant to bridge the gap between new scientific observations and clinical practice. However, traditional CME has not been effective at altering the behaviors of physicians. One reason for this failure of traditional CME programs may be their inflexibility. In traditional CME, the clinician does not choose the topic, the pace of the program, or the place of learning, and the CME material cannot be easily delivered to the point of care where the clinician needs the information. Computers and computer networks have the potential to accomplish these goals. CME has begun to appear on the Internet; however, there have been few evaluations of its usefulness, acceptance, and effectiveness. Over the last 18 months, we have developed three on-line pulmonary CME programs, and we have delivered them on the Virtual Hospital, the University of Iowa's digital health sciences library on the Internet. We report our initial experience with this CME material. DESIGN: We measured the frequency with which the Internet-delivered CME is accessed by monitoring page accessions and by using a log file analysis program (Analog 1.2.3; University of Cambridge Statistical Laboratory; Cambridge, UK). In addition, we collected all completed CME examinations and evaluation forms submitted by registered users. MEASUREMENTS AND RESULTS: We have found that the frequency with which the Internet-delivered CME is accessed has continued to increase with time (2.3-fold increase over 18 months), that evaluations of technical and content issues are strongly favorable, and that some clinicians have been willing to pay to receive CME through the medium of the Internet. CONCLUSIONS: We feel that with adequate peer review and quality control, physicians will use the Internet-delivered CME. However, several obstacles to wide use remain. These obstacles include issues regarding training in using the Internet for physicians, reluctance of physicians to participate in on-line commerce, and the current unavailability of CME to be delivered in small-grained quantities to the point of care. As these issues are addressed, we feel that on-line CME will represent an increasingly important CME medium for clinicians.

Protamine interaction with the epithelial cell surface.

We have previously reported that exposing cultured Madin Darby canine kidney (MDCK) cells to the polycation protamine (PRO) results in increased short-circuit current and decreased barrier integrity as measured by mannitol permeability and transepithelial electrical resistance. To further investigate the interaction of PRO with the surface of epithelial cells, we labeled PRO with [14C] with use of reductive alkylation. [14C]PRO bound to the cells in a biphasic pattern. Approximately 10% of the [14C]PRO was bound to the cells in the first 5 min, followed by an additional 10% that was bound over the next 25 min. No additional [14C]PRO bound to the cells after the initial 30 min. Binding of [14C]PRO was inhibited by "cold" PRO, which suggested specificity. Binding was also inhibited by polyanions, serum, and albumin, agents previously found to protect MDCK cells from PRO-induced injury. The binding of PRO to MDCK cells was not inhibited by incubation of the MDCK cells with neuraminidase, to remove surface sialic acid residues, or with heparinase, to remove surface heparan sulfate, even though metabolic labeling experiments demonstrated that neuraminidase decreased cell sialic acid and heparinase decreased cell heparan sulfate. Neuraminidase and heparinase offered no protection from PRO injury and had no effect themselves on mannitol permeability. Incubation of the cells with trypsin, however, blunted both the binding of PRO to the cells and the increase in mannitol permeability after exposure of the cells to PRO.(ABSTRACT TRUNCATED AT 250 WORDS)

Protamine increases the permeability of cultured epithelial monolayers.

Polycations, including protamine, have been reported to decrease the barrier integrity of cultured rat pulmonary type II epithelial monolayers. In contrast, protamine has been reported to increase the transepithelial electrical resistance of gallbladder epithelium. The present study was done using Madin Darby canine kidney epithelial cells (MDCK) to determine whether the effect of protamine on type II epithelial monolayers was species or organ specific or was dependent on the presence of nonepithelial cells and to investigate the effect of protamine on the actin cytoskeleton. Exposure of MDCK monolayers to protamine resulted in decreased transepithelial electrical resistance (Rt), increased short-circuit current (Isc) across the monolayers, and increased mannitol permeability (Pmann) of the monolayers. The decrease in Rt and increase in Isc was seen only after the addition of protamine to the apical surface of the cells. The importance of charge in this action was supported by the fact that exposure of the monolayer to the polycation poly-L-lysine also resulted in increased Pmann, and both the decreased Rt and increased Pmann seen after the addition of protamine were prevented if the monolayers were exposed in the presence of the polyanions heparin or sulfated dextran. The increase in Pmann appeared to be the result of increased permeability in the paracellular pathway, because increased mannitol uptake by the cells represented only a fraction of the increase in Pmann. Subtle changes in the actin cytoskeleton were seen after exposure of the monolayers to protamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Cationic neutrophil proteins increase transendothelial albumin movement.

Neutrophils play a role in the development of pulmonary edema in many models of the adult respiratory distress syndrome, but the mechanism of their action is not completely understood. We asked whether two neutrophil secretory products, human neutrophil cationic protein (NCP) and human neutrophil elastase (HNE), would nonenzymatically alter the movement of albumin across a cultured endothelial monolayer. Both enzymes were inactivated by heating before use. HNE was additionally enzymatically inactivated with a chloromethylketone oligopeptide (CMK) inhibitor and with alpha 1-proteinase inhibitor (alpha 1-PI). Heated NCP, heated HNE, and CMK-complexed HNE all increased transendothelial albumin transfer. The cation protamine also increased albumin transfer across the endothelium and this increase was blocked by heparin. Alpha 1-PI and fetal bovine serum also prevented the cationic proteins from increasing albumin transfer. Using the release of lactate dehydrogenase as a marker of cytotoxicity, heated HNE was toxic to endothelial cells, heated NCP had only minimal toxicity, and protamine had no toxicity. Changes in endothelial cell shape with gap formation was seen after exposure to both heated HNE and heated NCP. Both the cytotoxicity associated with heated HNE and the cell shape changes associated with heated NCP and heated HNE could be blocked by heparin. These results suggest that in addition to neutrophil proteases and reactive O2 molecules, neutrophil-derived cationic proteins can directly and nonenzymatically contribute to edema formation during acute inflammation.

Silica directly increases permeability of alveolar epithelial cells.

Alveolar epithelial cell injury and increased alveolar-capillary membrane permeability are important features of acute silicosis. To determine whether silica particles contribute directly to this increased permeability, we measured paracellular permeability of rat alveolar epithelium after exposure to silica, in vitro, using markers of the extracellular space. Silica (Minusil) markedly increased permeability in a dose- and time-dependent manner. This was not the result of cytolytic injury, because lactate dehydrogenase release from monolayers exposed to silica was not increased. Pretreatment of the silica with serum, charged dextrans, or aluminum sulfate blocked the increase in permeability. Scanning electron microscopy demonstrated adherence of the silica to the surface of the alveolar epithelial cells. Thus silica can directly increase permeability of alveolar epithelium.

Evaluation of an Internet-based decision-support system for applying the ATS/CDC guidelines for tuberculosis preventive therapy.

Preventive therapy for patients infected with tuberculosis (TB) remains an important component of TB control. To guide physicians in applying preventive therapy, the American Thoracic Society and Centers for Disease Control (ATS/CDC) developed guidelines based on PPD reactivity and on pretest probability of infection. The guidelines have become complex, and many clinicians find them challenging to apply. The authors developed a computerized decision-support system to assist clinicians in applying the ATS/CDC guidelines. This tool, published on the World Wide Web using hypertext markup language, delivers patient-specific recommendations based on physician-delivered patient-specific information. Four local TB experts derived eight TB infection scenarios and validated the web-based tool, which was tested for effectiveness using general internal medicine residents, randomly divided into two groups. Group A (n = 12) used the web-based tool and group B (n = 17) used pre-existing understanding of the guidelines and/or written resources to determine the need for preventive therapy in the case scenarios. Group A correctly used therapy in 92/96 possible cases (95.8%), group B in only 77/136 (56.6%) (p < 0.001). Group A required a mean of three mouse-clicks and 1.5 minutes per scenario to reach their choices, and they rated the web-based tool both intuitive and effective. These data demonstrate that a computer-based decision-support system for applying TB treatment guidelines can be delivered over the Internet and provide an efficient and effective resource for clinicians.

Evaluation of internet-based clinical decision support systems.

BACKGROUND: Scientifically based clinical guidelines have become increasingly used to educate physicians and improve quality of care. While individual guidelines are potentially useful, repeated studies have shown that guidelines are ineffective in changing physician behavior. The Internet has evolved as a potentially useful tool for guideline education, dissemination, and implementation because of its open standards and its ability to provide concise, relevant clinical information at the location and time of need. OBJECTIVE: Our objective was to develop and test decision support systems (DSS) based on clinical guidelines which could be delivered over the Internet for two disease models: asthma and tuberculosis (TB) preventive therapy. METHODS: Using open standards of HTML and CGI, we developed an acute asthma severity assessment DSS and a preventative tuberculosis treatment DSS based on content from national guidelines that are recognized as standards of care. Both DSS's are published on the Internet and operate through a decision algorithm developed from the parent guidelines with clinical information provided by the user at the point of clinical care. We tested the effectiveness of each DSS in influencing physician decisions using clinical scenario testing. RESULTS: We first validated the asthma algorithm by comparing asthma experts' decisions with the decisions reached by nonpulmonary nurses using the computerized DSS. Using the DSS, nurses scored the same as experts (89% vs. 88%; p = NS). Using the same scenario test instrument, we next compared internal medicine residents using the DSS with residents using a printed version of the National Asthma Education Program-2 guidelines. Residents using the computerized DSS scored significantly better than residents using the paper-based guidelines (92% vs. 84%; p <0.002). We similarly compared residents using the computerized TB DSS to residents using a printed reference card; the residents using the computerized DSS scored significantly better (95.8% vs. 56.6% correct; p<0.001). CONCLUSIONS: Previous work has shown that guidelines disseminated through traditional educational interventions have minimal impact on physician behavior. Although computerized DSS have been effective in altering physician behavior, many of these systems are not widely available. We have developed two clinical DSS's based on national guidelines and published them on the Internet. Both systems improved physician compliance with national guidelines when tested in clinical scenarios. By providing information that is coupled to relevant activity, we expect that these widely available DSS's will serve as effective educational tools to positively impact physician behavior.

Neutrophil cathepsin G increases transendothelial albumin flux.

Neutrophils are involved in the development of inflammatory edema in some animal models, but their mechanisms of action are not completely understood. Among injurious agents available for neutrophil-mediated injury are cationic proteins such as cathepsin G. Previous articles have reported that cationic agents decrease the barrier properties of the endothelium both in vivo and in vitro. Using an ex vitro isolated, perfused rabbit lung and a cultured porcine pulmonary artery endothelial cell monolayer (ENDO) as models, we asked whether neutrophil cathepsin G could decrease the barrier properties of the intact vasculature and cultured endothelial monolayers. After the addition of cathepsin G (10 micrograms/ml) to the perfusate of the isolated, perfused lung, there was a fourfold increase in net weight gain after a venous pressure challenge (p less than 0.01). The addition of cathepsin G to a cultured ENDO directly increased the movement of albumin across the monolayers in a dose-dependent fashion (10 micrograms/ml led to a 59% +/- 5% increase, 15 micrograms/ml to a 135% +/- 55% increase, 20 micrograms/ml to a 247% +/- 78% increase, and 30 micrograms/ml to a 381% +/- 89% increase, p less than 0.05 at all concentrations). Heat-inactivation of the enzyme only partially protected the ENDO, but exposure in the presence of either the polyanion heparin or the polyanion dextran sulfate completely protected the ENDO. Normal human serum also protected the ENDO, but serum from two patients with alpha 1-proteinase inhibitor (alpha 1-PI) deficiency was only partially protective. The protection afforded by human serum was time dependent, because addition of the serum 5 or 15 minutes after the addition of cathepsin G offered no protection. Oxidation of alpha 1-PI, as may occur at sites of inflammation, also destroyed its protective effect. Cytotoxic injury of the porcine pulmonary artery endothelial cell monolayer by cathepsin G, which was also prevented by the polyanions, only partially explained these results, because cathepsin G increased albumin transfer across the ENDO at concentrations of 10 to 20 micrograms/ml, which were minimally cytolytic to the ENDO. Additionally, cathepsin G caused cell retraction, with the development of intercellular gaps visible on light microscopy of the ENDO. This effect was also prevented by the polyanions. These results demonstrate that cathepsin G has direct effects on a cultured ENDO that may be caused by the charge or charged site(s) on cathepsin G.