Neutral metalloproteinases produced by human mononuclear phagocytes. Enzyme profile, regulation, and expression during cellular development.

Mononuclear phagocytes are developmentally and functionally complex cells that play critical roles in extracellular matrix remodeling. We hypothesized that differentiated mononuclear phagocytes, typified by alveolar macrophages, use a spectrum of metalloproteinases to degrade various matrix macromolecules. To test this hypothesis, we have evaluated synthesis and secretion of four metalloproteinases (interstitial collagenase, stromelysin, 72-kD type IV collagenase, and 92-kD type IV collagenase) by human mononuclear phagocytes with regard to (a) the effect of cellular differentiation, (b) regulation of secretion, and (c) comparisons/contrasts with a prototype metalloproteinase-secretory cell, the human fibroblast. We found that regulated secretion of greater quantities and a wider spectrum of metalloenzymes correlated with a more differentiated cellular phenotype. As extreme examples, the 92-kD type IV collagenase was released by peripheral blood monocytes and uninduced U937 monocyte-like cells, whereas stromelysin was secreted only by lipopolysaccharide-stimulated alveolar macrophages. Macrophage production of interstitial collagenase, stromelysin, and 72-kD type IV collagenase was approximately 20%, 10%, and 1-2%, respectively, of that from equal numbers of fibroblasts; secretion of the 92-kD type IV collagenase was not shared by fibroblasts. This work confirms the potential of macrophages to directly degrade extracellular matrix via secreted metalloproteinases in a manner that differs both qualitatively and quantitatively from that of fibroblasts. Moreover, varying regulation of metalloenzyme synthesis, evidenced by distinct patterns of basal and stimulated secretion during differentiation, can be studied at a molecular level in this model system.

Clinical course and outcome of patients admitted to an ICU for status asthmaticus.

STUDY OBJECTIVES: To describe the prognostic factors, clinical course, and outcome of patients with status asthmaticus treated in a medical ICU (MICU). DESIGN: Analysis of prospective data. SETTING: A multidisciplinary MICU of an inner-city university hospital. PATIENTS: We collected data on 132 hospital admissions of 89 patients with status asthmaticus treated in our MICU from August 1995 through July 1998. MEASUREMENTS: APACHE (acute physiology and chronic health evaluation) II scores were among the parameters measured. RESULTS: Seventy-nine percent of the patients were female, and 67% were African American (mean +/- SD age, 42.4 +/- 15.1 years). Patients in 48 of the 132 hospital admissions (36%) required invasive mechanical ventilation; sepsis developed in patients during 17 hospital admissions (13%), nonpulmonary organ failure developed during 16 hospital admissions (12%), and ARDS developed during 2 hospital admissions (2%). Pneumothorax developed in four patients and required tube thoracostomy in all four patients. The median APACHE II score was 11. Predicted mortality and actual mortality were 6.7% and 8.3%, respectively. The two most common immediate causes of death were pneumothorax (n = 3) and nosocomial infection (n = 3). All the deaths occurred in female patients. Compared with survivors, nonsurvivors had higher APACHE II scores (median, 26 vs 15; p < 0.0001), PaCO(2) (63.8 +/- 21.3 mm Hg vs 47.8 +/- 19.1 mm Hg, p = 0.0101), and lower arterial pH (7.09 +/- 0.12 vs 7.27 +/- 0.12, p < 0.0001), respectively. Patients in 10 of 48 hospital admissions (21%) who required mechanical ventilation died. CONCLUSIONS: The hospital mortality of patients admitted to an MICU for status asthmaticus is higher than expected. Higher APACHE II score and PaCO(2) and lower arterial pH within 24 h of hospital admission are associated with increased mortality. Sepsis and nonpulmonary organ failure are more likely to develop in nonsurvivors than survivors.

Successful medical therapy of Rhodococcus equi pneumonia in a patient with HIV infection.

A 34-year-old HIV-infected man was successfully treated with antimicrobial therapy alone for Rhodococcus equi pneumonia and has survived longer than six months. In the current literature, only two of seven HIV-infected patients so treated have survived as long as six months. Based on our experience and the available literature, it seems reasonable to treat HIV-infected patients with R equi pneumonia who do not require surgical intervention with prolonged intravenous therapy followed by long-term oral therapy with at least two effective antibiotics. The optimal choice and duration of antibiotic therapy need to be determined.

Protons safely allow coverage of high-risk nodes for patients with regionally advanced non-small-cell lung cancer.

Our objective was to determine if protons allow for the expansion of treatment volumes to cover high-risk nodes in patients with regionally advanced non-small-cell lung cancer. In this study, 5 consecutive patients underwent external-beam radiotherapy treatment planning. Four treatment plans were generated for each patient: 1) photons (x-rays) to treat positron emission tomography (PET)-positive gross disease only to 74 Gy (XG); 2) photons (x-rays) to treat high-risk nodes to 44 Gy and PET-positive gross disease to 74 Gy (XNG); 3) protons to treat PET-positive gross disease only to 74 cobalt gray equivalent (PG); and 4) protons to treat high-risk nodes to 44 CGE and PET-positive gross disease to 74 CGE (PNG). We defined high-risk nodes as mediastinal, hilar, and supraclavicular lymph nodal stations anatomically adjacent to the foci of PET-positive gross disease. Four-dimensional computed tomography was utilized for all patients to account for tumor motion. Standard normal-tissue constraints were utilized. Our results showed that proton plans for all patients were isoeffective with the corresponding photon (x-ray) plans in that they achieved the desired target doses while respecting normal-tissue constraints. In spite of the larger volumes covered, median volume of normal lung receiving 10 CGE or greater (V10Gy/CGE), median V20Gy/CGE, and mean lung dose were lower in the proton plans (PNG) targeting gross disease and nodes when compared with the photon (x-ray) plans (XG) treating gross disease alone. In conclusion, proton plans demonstrated the potential to safely include high-risk nodes without increasing the volume of normal lung irradiated when compared to photon (x-ray) plans, which only targeted gross disease.

Massive theophylline dosing in a heavy smoker receiving both phenytoin and phenobarbital.

OBJECTIVE: To report unusually high theophylline dosing requirements in a smoker receiving concomitant therapy with phenytoin and phenobarbital. DESIGN: Single case report. SETTING: 517-bed, university teaching hospital. PATIENT: 29-year-old woman with newly diagnosed asthma, heavy smoking history, and a seizure disorder. RESULTS: The additive influence of smoking, phenytoin, and phenobarbital greatly increased the theophylline dosing requirements. Doses of up to 4 g/d (59 mg/kg/d) were required to achieve adequate symptomatic relief of her asthma as well as to provide therapeutic serum theophylline concentrations. CONCLUSIONS: Multiple polymorphisms may additively influence theophylline metabolism and exceptionally large theophylline doses may be required in some patients who smoke and are comedicated with phenytoin and phenobarbital.

Pharmacist-managed, physician-directed asthma management program reduces emergency department visits.

OBJECTIVE: To determine whether the number of emergency department (ED) visits for acute asthma exacerbations could be decreased by providing patients with a comprehensive program of asthma management delivered by a pharmacist and a physician. DESIGN: Patients were selected from the ED and asked to attend a special asthma clinic that provided education about asthma and proper use of asthma medications, regular telephone contact between the pharmacist and patient, and an open-door clinic policy. SETTING: A university-affiliated urban teaching hospital. PARTICIPANTS: The study population consisted of 25 asthmatic patients who were at least 18 years of age and who were seen in the ED a minimum of 3 times in a 12-month period. MAIN OUTCOME MEASURES: The number of visits to the ED for acute exacerbations of asthma was measured. Patients served as their own controls. The number of ED visits for asthma during the 6-month study period was compared with two 6-month periods prior to the study period for each patient. RESULTS: The total number of ED visits for the 25 enrolled patients six months prior to their enrollment into the study was 92; the number of ED visits during the same months of the study in the prior year was 47. During the study period, there were only 6 ED visits for asthma exacerbations. CONCLUSIONS: The comprehensive asthma management program reduced the number of ED visits for acute exacerbations of asthma.

The assessment of alpha 1 proteinase inhibitor form and function in lung lavage fluid from healthy subjects.

The form and function of alpha 1 proteinase inhibitor in lung lavage fluid from healthy smoking and non smoking individuals has been accurately assessed using critically appraised techniques. The present study demonstrated that it is possible to accurately assess alpha 1 PI function in unconcentrated lavage fluid but that sample collection, storage and subsequent processing may all affect the results. Absolute levels of alpha 1 PI were elevated in subjects who smoke and a substantial quantity of inactive protein was found in both smokers and non smokers. The proportion of inactive alpha 1 PI was similar for both groups, which by inference implies that normal smoking subjects do not have decreased protection by this inhibitor at the bronchoalveolar level. Physicochemical analysis of the alpha 1 PI in these normal subjects showed that it was different from alpha 1 PI previously reported from patients with established disease and this may have important implications regarding the pathogenesis of their condition. Western immunoblotting of bronchoalveolar lavage fluid (BALF) showed that all of the alpha 1 PI was present in the native molecular mass form (54,000 Da). Pre-incubation of samples with methionine sulphoxide peptide reductase restored alpha 1 PI function only by approximately 10% suggesting the presence of little reversibly oxidised alpha 1 PI in either group. Anion exchange HPLC of BALF revealed the presence of two alpha 1 PI species, one of which co-eluted with native, oxidised or proteolyzed forms and the other which was more cationic and did not inhibit porcine pancreatic elastase. Finally, thirteen out of sixteen BALF samples inhibited more neutrophil elastase than could be accounted for by the amounts of functional alpha 1 PI present, suggesting that the presence of other inhibitors is a feature of normal lavage fluids.

Monocyte procollagenase and tissue inhibitor of metalloproteinases. Identification, characterization, and regulation of secretion.

Alveolar macrophages have been shown to secrete a procollagenase and the tissue inhibitor of metalloproteinases (TIMP), which are similar or identical to the corresponding proteins of human skin fibroblasts. Little is known, however, about the collagenolytic activity of normal human monocytes. We have applied immunologic, biochemical, and molecular biologic tools to examine the collagenolytic profile of freshly isolated peripheral blood monocytes. Our studies indicate that: 1) monocytes are capable of producing both procollagenase and TIMP that are identical to the corresponding products of skin fibroblasts, alveolar macrophages, and U-937 cells; 2) unstimulated monocytes in vitro secrete high levels of TIMP, but little or no procollagenase; 3) an as yet unidentified component(s) of serum are required for in vitro production of TIMP (but not procollagenase) by monocytes; 4) even when stimulated, monocytes secrete much smaller quantities of procollagenase in comparison with macrophages; and 5) regulation of the secretion of procollagenase and TIMP by monocytes exhibits a high degree of individual variability, but is nevertheless subject to clearly different control mechanisms than our previous findings would indicate for alveolar macrophages. Monocytes thus express a macrophage-like, rather than a neutrophil-like, profile of proteins capable of mediating collagen turnover, the regulation of which is distinct from that of more differentiated alveolar macrophages. Further study of monocyte and macrophage collagenolytic activities may provide insights into both the cell biology of mononuclear phagocyte maturation and the mechanisms by which such cells mediate the turnover of interstitial collagens.

Selective up-regulation of human alveolar macrophage collagenase production by lipopolysaccharide and comparison to collagenase production by fibroblasts.

Collagenase catalyzes the initial and rate-limiting step in interstitial collagen degradation. Human alveolar macrophages produce both a fibroblast-like procollagenase and tissue inhibitor of metalloproteinases (TIMP). To define the potential of macrophages to express collagenase and TIMP, we have studied the effects of certain cell culture variables and LPS on in vitro production of these proteins. Our data indicate: 1) human macrophages cultured in a 1/1 (v/v) mixture of HAM F-12:DME produce two- to three-fold greater quantities of procollagenase (but not TIMP) as compared to HAM F-12, DME, or alpha-MEM alone; 2) maximal collagenase expression requires the further addition of LPS, whereas TIMP production is optimized by 5% fetal bovine serum alone; 3) the up-regulation of macrophage procollagenase by LPS represents a highly selective biologic response when compared to changes induced in other secreted and intracellular proteins; 4) measurements of steady state procollagenase mRNA by Northern blot analysis suggest that the LPS effect is mediated at a pre-translational level; and finally 5) on a per cell basis, human alveolar macrophages cultured under optional conditions secrete approximately 20% of the collagenase and approximately 10% of the TIMP elaborated by stimulated human fibroblasts. We conclude that procollagenase and TIMP secretion by human alveolar macrophages in vitro is strikingly responsive to variations in cell culture conditions and that an especially noteworthy selective upregulation of procollagenase secretion by LPS is probably modulated by a transcriptional mechanism. The macrophage synthetic potential for procollagenase suggests a potentially important role for these cells in directly mediating collagen turnover.

Neutral proteinases of human mononuclear phagocytes. Cellular differentiation markedly alters cell phenotype for serine proteinases, metalloproteinases, and tissue inhibitor of metalloproteinases.

Mononuclear phagocytes have the capacity to directly participate in extracellular matrix turnover via secretion of neutral proteinases. We have studied the effects of in vivo and in vitro differentiation upon cellular content or secretion of a spectrum of neutral proteinases, along with a counter-regulatory metalloproteinase inhibitor (TIMP). We found 1) matrix-degradative serine proteinases (leukocyte elastase and cathepsin G) were lost during cellular maturation and/or differentiation; 2) the 92-kDa type IV/type V collagenase and TIMP were secreted earliest in mononuclear phagocyte differentiation, whereas stromelysin secretion was observed only by LPS-stimulated alveolar macrophages; 3) exposure of alveolar macrophages, but not monocytes, to phorbol esters and LPS resulted in markedly augmented secretion of all studied metalloproteinases and TIMP; 4) monocyte-derived macrophages partially (but not completely) mimicked the metalloproteinase secretory phenotype of alveolar macrophages; and 5) the secretory phenotype of alveolar macrophages for interstitial collagenase (but not TIMP) was largely lost during in vitro culture. These results underscore the complexity of the process of differentiation in human mononuclear phagocytes, and provide insights into the variable capacity of mononuclear phagocytes to degrade extracellular matrix components. Moreover, we anticipate that human mononuclear phagocytes at various stages of differentiation will provide a useful model system for study of the variable regulation of secretion of human matrix-degrading metalloproteinases.

Elastin degradation by human alveolar macrophages. A prominent role of metalloproteinase activity.

Macrophages are thought to play an important role in the turnover of extracellular matrix, but the capacity of human macrophages to degrade elastin, and the elastolytic mechanisms of these cells, have been controversial. Particular difficulty has been encountered in efforts to establish whether human macrophages secrete a metalloelastase that is analogous to the enzyme secreted by rodent macrophages. We studied elastin degradation by human alveolar macrophages cultured directly in contact with radiolabeled elastin using media containing 10% fetal bovine serum, and for comparison performed parallel studies of P388D1 murine macrophagelike cells that are known to secrete metalloelastase. With both cell types, we observed elastin degradation and the following: (1) direct contact between the cells and elastin substrate was required for elastin degradation; (2) elastin degradation was inhibited by the tissue inhibitor of metalloproteinases, but minimally or not at all by inhibitors of cysteine proteinases (E-64, CBZ-phe-phe-CHN2, CBZ-phe-ala-CHN2, and cystatin C), or by the serine proteinase inhibitor eglin-c; (3) elastin degradation increased sharply after the cells were in contact with elastin for 24 h, and required new protein synthesis as indicated by sensitivity to cycloheximide; (4) inclusion of dexamethasone (10(-6) to 10(-8) M) in the cultures led to decreased elastin degradation. Also, with both cell types, elastin degradation occurred despite the finding that cell-conditioned media did not contain elastase activity and could inhibit P388D1-derived metalloproteinase elastase. These results indicate a prominent role for metalloproteinase activity in elastin degradation by both human and murine macrophages and support the concept that events at the cell-substrate interface are critically important to macrophage-mediated elastin degradation.