James E. Gadek

Antielastases of the human alveolar structures. Implications for the protease-antiprotease theory of emphysema.

The current concepts of the pathogenesis of emphysema hold that progressive, chronic destruction of the alveolar structures occurs because there was in imbalance between the proteases and antiproteases in the lower respiratory tract. In this context, proteases, particularly neutrophil elastase, work unimpeded to destroy the alveolar structures. This concept has evolved from consideration of patients with alpha 1-antitrypsin deficiency, who have decreased levels of serum alpha 1-antitrypsin and who have progressive panacinar emphysema. To directly assess the antiprotease side of this equation, the lower respiratory tract of non-smoking individuals with normal serum antiproteases and individuals with PiZ homozygous alpha 1-antitrypsin deficiency underwent bronchoalveolar lavage to evaluate the antiprotease screen of their lower respiratory tract. These studies demonstrated that: (a) alpha 1-antitrypsin is the major antielastase of the normal human lower respiratory tract; (b) alpha 2-macroglobulin, a large serum antielastase, and the bronchial mucous inhibitor, an antielastase of the central airways, do not contribute to the antielastase protection of the human alveolar structures; (c) individuals with PiZ alpha 1-antitrypsin deficiency have little or no alpha 1-antitrypsin in their lower respiratory tract and have no alternative antiprotease protection against neutrophil elastase; and (d) the lack of antiprotease protection of the lower respiratory tract of PiZ individuals is a chronic process, suggesting their vulnerability to neutrophil elastase is always present.

Replacement Therapy in Hereditary Angioedema: Successful Treatment of Acute Episodes of Angioedema with Partly Purified C1 Inhibitor

Although considerable progress has been made during the past two decades in the use of androgens to prevent attacks of hereditary angioedema, replacement of the deficient C1-inhibitor protein would provide a useful menas of treatment once an attack has begun. We studied the clinical use of C1 inhibitor that was partly purified on a large scale from pooled plasma. The in vivo efficacy and safety of this protein concentrate were evaluated during 11 intravenous infusions in eight patients with hereditary angioedema. Three patients received the C1-inhibitor preparation during an asymptomatic period. Increases in serum C4 activity provided evidence of the biologic activity of the infused inhibitor. Intravenous administration of the concentrate during acute abdominal or laryngeal attacks of hereditary angioedema in five patients resulted in abatement of symptoms in addition to increased serum C4 activity. No untoward effects of the intravenous administration of the C1 inhibitor were observed in these eight patients. Thus, this C1-inhibitor preparation seems to offer the potential for safe, effective replacement therapy and may provide a means of controlling an attack of hereditary angioedema that is in progress.

Human Alveolar Macrophage-derived Chemotactic Factor for Neutrophils: STIMULI AND PARTIAL CHARACTERIZATION

The presence of neutrophils within the lung is a characteristic feature of a variety of lung diseases. To evaluate the potential role of alveolar macrophages in modulating the migration of neutrophils to the lung, normal human alveolar macrophages obtained from volunteers by bronchopulmonary lavage, were exposed for various periods of time in vitro to heat-killed microorganisms, and noninfectious particulates, immune complexes, and the macrophage supernates were evaluated for chemotactic activity. The microorganisms, noninfectious particulates, and immune complexes were chosen as stimuli for alveolar macrophages because these stimuli are representative of a spectrum of pathogenic agents that cause neutrophil accumulation in the lower respiratory tract. After incubation with each of these stimuli, alveolar macrophages released low molecular weight (400-600) chemotactic factor(s) (alveolar macrophage-derived chemotactic factor[s] [AMCF]) with relatively more activity for neutrophils than monocytes or eosinophils. Checker-board analysis of the AMCF revealed that the factor was primarily chemotactic and not chemokinetic for neutrophils. The selectivity for neutrophils vs. monocytes could not be explained by a selective deactivation of monocytes, because the AMCF was more potent in deactivating neutrophils than monocytes. Partial characterization of AMCF demonstrated it was heterogeneous with the following features: (a) stable to heating at 56 and 100 degrees C for 30 min; (b) stable over a pH range of 1.0 to 12.0 for 60 min; (c) stable after exposure to trypsin, papain, chymotrypsin, collagenase, and elastase; (d) partially inhibited by serum chemotactic factor inhibitor(s); (e) two major isoelectric points (pI 7.6 and 5.2); and (f) partially extractable into ethyl acetate, ether, and hexane. Although AMCF was, at least, partially lipid in nature, it did not appear to be similar to previously described lipid chemotactic factors (e.g., hydroxy-derivatives of 5,8,10,14-eicosatetraenoic acid); analysis by gas chromatography-mass spectrophotometry of AMCF extracted into ethyl acetate did not reveal the presence of 5,8,10,14-eicosatetraenoic acid. The macrophage supernates containing the AMCF also stimulated normal human neutrophils to release lysozyme and lactoferrin but not lactate dehydrogenase. These studies suggest that a wide variety of potentially pathogenic stimuli induce normal alveolar macrophages to generate a low molecular weight chemotactic factor(s) that preferentially attracts neutrophils. Because alveolar macrophages are normal residents of alveoli, it is likely that by releasing this factor(s) macrophages play a significant role in amplifying the inflammatory processes seen in many acute and chronic lung diseases.

Protein permeability in the adult respiratory distress syndrome. Loss of size selectivity of the alveolar epithelium.

Small amounts of plasma protein normally reach the alveolar epithelial surface by a size-selective process that restricts the passage of very large molecules. Size selectivity may be compromised in the lungs of patients with the adult respiratory distress syndrome (ARDS). To assess this question, bronchoalveolar lavage fluid (BALF) from normal volunteers (n = 11), cardiac edema patients (n = 3), and ARDS patients (n = 8) was compared. Mean total protein in ARDS BALF was greater than 12 times the levels in normals or cardiac edema patients. BALF/plasma total protein ratios and measurements of epithelial lining fluid protein also separated the patients groups. The large proteins IgM and alpha 2-macroglobulin were found in ARDS BALF at greater than 90 times the concentrations of normal or cardiac edema fluid. The relationship of distribution coefficient vs. log molecular weight for seven proteins (54,000-900,000 mol wt) hyperbolically increased in normals but was flat in ARDS patients. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a paucity of high molecular weight proteins in normal and cardiac edema BALF, but demonstrated the full spectrum of plasma proteins in ARDS BALF. We conclude that normal size selectivity is preserved in cardiac edema but is destroyed by the alveolar-capillary injury of ARDS.

Replacement therapy of alpha 1-antitrypsin deficiency. Reversal of protease-antiprotease imbalance within the alveolar structures of PiZ subjects.

The emphysema associated with the inherited serum deficiency of alpha 1-antitrypsin appears to result from an imbalance between neutrophil elastase and its major inhibitor within the alveolar structures. In the present study we assessed the feasibility of reversing this biochemical defect within the lung via parenteral replacement therapy with an alpha 1-antitrypsin concentrate of normal plasma. A 20--40% polyethylene glycol precipitate of pooled human donor plasma was used to obtain an enriched alpha 1-antitrypsin concentrate devoid of hepatitis B antigen and immunoglobulins. Using this material, five individuals with severe serum alpha 1-antitrypsin deficiency (PiZ phenotype) and advanced emphysema received 4 g of alpha 1-antitrypsin intravenously at weekly intervals for four doses. During this period of weekly replacement therapy alpha 1-antitrypsin serum levels were maintained at greater than or equal to 70 mg/dl, the level likely required for effective antielastase protection of the lung. In addition, assessment of lower respiratory tract antielastase activity by bronchoalveolar lavage demonstrated that parenteral replacement of alpha 1-antitrypsin resulted in establishment of effective antielastase activity within the alveolar structures. There were no untoward side effects consequent to this approach to the replacement therapy of alpha 1-antitrypsin. These results demonstrate that the parenteral replacement of alpha 1-antitrypsin provides a means of obtaining elastase-antielastase balance within the lung of individuals with this serum protease inhibitor deficiency.

Mechanisms of neutrophil accumulation in the lungs of patients with idiopathic pulmonary fibrosis.

Neutrophils are a characteristic feature of the alveolitis of idiopathic pulmonary fibrosis (IPF). a chronic disorder limited to lung. One mechanism by which neutrophils may be selectively attracted to lung and not other tissues is via the secretion of the neutrophil-specific chemotactic factor by alveolar macrophages. To evaluate the role of alveolar macrophages in modulating the migration of neutrophils to he lung in IPF, alveolar macrophages, obtained by bronchoalveolar lavage of patients with IPF, were evaluated for their ability to release a chemotactic factor for neutrophils. Unstimulated alveolar macrophages from normal individuals did not release the factor. In patients with IPF, there was a significant correlation between the proportions of neutrophils in lavage fluid and the release of a chemotactic factor for neutrophils by alveolar macrophages (p less than 0.001). The chemotactic factor released by IPF alveolar macrophages was of low molecular weight (400-600), at least partially lipid in nature, and preferentially attracted neutrophils compared with monocytes. Several lines of evidence suggested that immune complexes in the lung stimulated alveolar macrophages of patients with IPF to release the chemotactic factor. First, immune complexes stimulated normal macrophages to release the factor.Second, there was a significant correlation between the release of the chemotactic factor by IPF alveolar macrophages and the levels of immune complexes in bronchoalveolar lavage fluid. Third, bronchoalveolar lavage fluid containing immune complexes stimulated normal macrophages to release the factor. Fourth, IPF alveolar macrophages that released large amounts of the chemotactic factor had an apparent suppression of their immunoglobulin (Ig)G Fc receptor function, suggesting that immune complexes were bound to their surface. In contrast, the IgG Fc receptor function of IPF alveolar macrophages that released only small amounts of the factor was similar to that of normal macrophages. These studies suggest that neutrophils are attracted to the lung in patients with IPF by a potent chemotactic factor released by alveolar macrophages that have been stimulated, in vivo, via their IgG Fc receptor by immune complexes.

Maintenance of Granuloma Formation in Pulmonary Sarcoidosis by T Lymphocytes within the Lung

Pulmonary granulomata of sarcoidosis are composed primarily of mononuclear phagocytic cells that are probably derived from blood monocytes. To evaluate the concept that recruitment of blood monocytes to the sarcoid lung is mediated by chemoattractants produced by immune effector cells within the lung, we obtained mononuclear cells from lung and blood of six patients with active pulmonary sarcoidosis, six normal subjects, and six patients with active idiopathic pulmonary fibrosis and studied their ability to secrete a chemotactic factor for monocytes. Lung T lymphocytes from all sarcoidosis patients, but not from normal subjects or patients with idiopathic pulmonary fibrosis, spontaneously secreted such a mediator. Lung T lymphocytes from patients with sarcoidosis secreted more monocyte chemotactic factor than did blood T lymphocytes from the same patients. The accumulation of monocytes in the lung in patients with pulmonary sarcoidosis may be mediated by local production of monocyte chemotactic factor by lung T lymphocytes.

Collagenase in the lower respiratory tract of patients with idiopathic pulmonary fibrosis.

To test the hypothesis that idiopathic pulmonary fibrosis (IPF) is mediated through collagenase present in the lower respiratory tract, we used the fiberoptic bronchoscope to obtain fluid from the lower respiratory tract of 24 patients with IPF, 18 controls and nine patients with sarcoidosis. The fluid was analyzed for a variety of enzymes, including collagenase. Fifteen of 21 patients with IPF showed collagenase activity, whereas normal controls and patients with sarcoidosis showed none (P greater than 0.001, for all comparisons). In two patients with IPF who were re-evaluated after eight to 24 months, the collagenase activity was persistent. Fluid from patients with IPF also contained elevated levels of a non-specific neutral protease (P greater than 0.01 compared with controls), but there was no elastase activity in fluid from patients with IPF or from controls. The collagenase found in lavage fluid in IPF cleaved lung collagen into collagenase-specific TCA and TCB fragments. We conclude that in IPF the collagen of the lung is subjected to sustained lysis, followed by disordered resynthesis, and that the presence of active collagenase in the lower respiratory tract is a specific feature of the alveolitis associated with this disease.

Enteral nutrition with eicosapentaenoic acid, γ-linolenic acid, and antioxidants reduces alveolar inflammatory mediators and protein influx in patients with acute respiratory distress syndrome

ObjectivePreviously, we showed that acute respiratory distress syndrome patients fed an enteral diet containing eicosapentaenoic acid and &ggr;-linolenic acid and elevated antioxidants (EPA+GLA; Oxepa) had significantly reduced pulmonary inflammation, increased oxygenation, and improved clinical outcomes. In a subset of acute respiratory distress syndrome patients from this trial, we performed a preliminary examination of the potential mechanisms underlying these clinical improvements by retrospectively testing the hypothesis that enteral feeding with EPA+GLA could reduce alveolar-capillary membrane protein permeability and the production of interleukin (IL)-8, IL-6, tumor necrosis factor-&agr;, and leukotriene B4 that are responsible, in part, for pulmonary inflammation. DesignProspective, randomized, double-blind, controlled clinical trial. SettingIntensive Care Unit of the Ohio State University Medical Center. PatientsA total of 67 patients were enrolled who met defined criteria for acute lung injury/acute respiratory distress syndrome. InterventionsA total of 43 of 67 evaluable patients randomly received either EPA+GLA or an isonitrogenous, isocaloric standard diet that was tube fed at a minimum caloric delivery of 75% of basal energy expenditure times 1.33 for at least 4 to 7 days. Measurements and Main ResultsBronchoalveolar lavage (BAL) was performed at baseline and study days 4 and 7 to obtain BAL fluid (BALF) for measurement of total protein, ceruloplasmin, and transferrin, total neutrophil count, IL-8, IL-6, tumor necrosis factor-&agr;, and leukotriene B4. Oxygenation, measured as Pao2/Fio2, was assessed before BAL. Patients fed EPA+GLA had a significant reduction in BALF ceruloplasmin and IL-8 during the study as compared with patients fed the control diet. BALF levels of total protein, neutrophils, and leukotriene B4 tended to decrease in EPA+GLA patients over the course of the study as compared with control patients. BALF levels of IL-6 declined similarly during the study in both groups. A trend toward a reduction in BALF tumor necrosis factor-&agr; was observed on study day 7 in the EPA+GLA group as compared with control patients. Significant improvements in oxygenation (Pao2/Fio2) occurred in EPA+GLA patients on study day 4 as compared with controls. Correlation analysis revealed significant relationships between BALF neutrophil counts and indices of alveolar-capillary membrane protein permeability, IL-8, and leukotriene B4. ConclusionsThis preliminary investigation showing a decrease in BALF levels of IL-8 and leukotriene B4 and the associated reduction of BALF neutrophils and alveolar membrane protein permeability in acute respiratory distress syndrome patients fed EPA+GLA support, in part, the potential mechanisms underlying the previously described clinical improvements with this diet. Additional controlled studies are needed to confirm these findings.

Eosinophil-mediated injury to lung parenchymal cells and interstitial matrix. A possible role for eosinophils in chronic inflammatory disorders of the lower respiratory tract.

Eosinophils are a common component of the inflammation of the lower respiratory tract that characterizes the interstitial lung disorders. Bronchoalveolar lavage analyses (n = 680) of 251 patients with interstitial lung disease demonstrated that eosinophils represented greater than 5% of the effector cells comprising the alveolitis in 20% of all lavages. In contrast, lavage of normal individuals (n = 117) showed that eosinophils were never greater than 5% of the total effector cells recovered. To evaluate a possible role for eosinophils in mediating some of the cellular and connective tissue matrix derangements of the lung parenchyma found in interstitial disease, eosinophils were evaluated for the presence of proteases capable of cleaving connective tissue proteins found in the lung and for the ability to mediate cytotoxicity to lung parenchymal cells. Evaluation of guinea pig and human eosinophils demonstrated that eosinophil granules contained a collagenase that specifically cleaved human collagen types I and III, the two major connective tissue components of the human lung parenchyma. In contrast, the eosinophil did not contain an elastase or a nonspecific neutral protease. The eosinophil collagenase appeared to be a metalloprotease, as it was inhibited by ethylenediaminetetraacetate but not by phenylmethanesulfonyl-fluoride or alpha 1-antitrypsin. The eosinophil also has the capacity to injure lung parenchymal cells. Without further stimulation, eosinophils purified from peritoneal exudates of guinea pigs demonstrated spontaneous cytotoxicity for human lung fibroblasts (HFL-1), cat lung epithelial cells (AK-D) and rat lung mesothelial cells (I6B). Under identical conditions, the epithelial cells were more sensitive to eosinophil-mediated cytotoxicity than the fibroblasts or mesothelial cells (P less than 0.01), consistent with the clinical observation that in the interstitial disorders, the alveolar epithelial cells are damaged more commonly than fibroblasts or pleural cells. The eosinophil-mediated cytotoxicity could be partially inhibited by the antioxidants catalase and dimethylsulfoxide suggesting that toxic oxygen radicals play a role in mediating the cellular damage. Importantly, eosinophils purified from bronchoalveolar lavage of human interstitial lung disease also demonstrated spontaneous cytotoxicity for lung epithelial cells. These observations demonstrate that eosinophils are frequent participants of the alveolitis of the interstitial lung disorders and suggest that these cells have the potential to damage the parenchymal cells and collagen matrix of the lower respiratory tract.