Clin. exp. Immunol. (1990) 80, 344-349
Increased elastase secretion by peripheral blood monocytes in cystic fibrosis patients M. M. JONES, D. K. SEILHEIMER*, G. B. PIERt & R. D. ROSSEN Immunology Research Laboratory, The Veterans Administration Medical Center, * The Departments of Microbiology and Immunology, Internal Medicine, and Pediatrics, Baylor College of Medicine, Houston, TX, and t The Channing Laboratory, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA
(Acceptedfor publication 15 January 1990)
SUMMARY Morbidity and mortality in cystic fibrosis (CF) is predominantly due to destruction of pulmonary tissue. The host immune response may, in part, play a pathogenic role in pulmonary destruction in these patients. To further understand host immune response in CF, we examined the state of activation of peripheral blood monocytes in CF. Baseline elastase activity was 2-2-fold greater in the CF monocytes than in controls. Pseudomonas aeruginosa mucoid exopolysaccharide (MEP) and high molecular weight polysaccharide (HMP) increased elastase activity in both control and CF monocytes, with a greater absolute increase in the CF monocytes. There was no difference in baseline or MEP-stimulated secretion of interleukin- I (IL-I) or interleukin-6 (IL-6) between CF and control monocytes. Ibuprofen enhanced both MEP and HMP-stimulated elastase activity, whereas dexamethasone suppressed both baseline and stimulated elastase activity > 20% in both CF and control monocytes. These results suggest that circulating monocytes in CF are stimulated in vivo, resulting in a remarkably elevated elastase activity in vitro. Elevated elastase release by peripheral blood monocytes as they enter the lung in response to chemotactic stimuli may contribute to lung destruction in CF. Keywords
monocytes cystic fibrosis
elastase interleukins
INTRODUCTION Cystic fibrosis (CF) is an autosomal recessive disorder characterized by abnormal exocrine gland secretion. Almost all patients develop chronic pulmonary infection and inflammation, resulting in respiratory failure and early death, despite intensive antibiotic therapy (David & di Sant' Agnese, 1984). Examination of lung tissue from CF patients shows evidence of extensive elastolysis and pulmonary secretions from CF patients have high levels of several proteolytic enzymes, including elastase and cathepsin G (Suter et al., 1984; Bruce et al., 1985). In CF, elastases in pulmonary secretions are derived from leucocytes, and also from Pseudomonas aeruginosa (PsA). In addition to destruction of lung extra-cellular matrix (Campbell, Senior & Welgus, 1987), these elastases interfere with important mechanisms for bacterial clearance: leucocyte elastase inactivates complement receptors on leucocytes (Tosi, Zakem & Berger, 1988) and PsA elastase degrades immunoglobulins, complement proteins, and human plasma al proteinase inhibitor
(Doring et al., 1985; Fick et al., 1985; Speert, 1985), thus potentiating elastolytic lung destruction. The human alveolar macrophage, derived from circulating blood monocytes, plays a fundamental role in virtually every aspect of the immune and inflammatory cell response and provides one of the first lines of host defense in the lung against invasion by pathogens (Bernaudin et al., 1988; Unanue & Allen, 1987). The role of the monocyte/macrophage system in CF lung damage has not been well characterized. Therefore, we measured the production of three monocyte products, elastase, IL- 1, and IL-6 in CF patients. MATERIALS AND METHODS Materials Dexamethasone, elastin, ibuprofen, and polymyxin B were purchased from Sigma Chemical Company (St Louis, MO), lipopolysaccharide B, E. coli 055.B5 (LPS-B) from Difco Laboratories (Detroit, MI), PsA refined endotoxin from Ribi Immunochemical Research, (Hamilton, MN), exotoxin A from List Biological Laboratories (Campbell, CA), and fetal calf serum (FCS) from Hyclone Laboratories (Logan, UT). Purified mucoid exopolysaccharide (MEP) and high molecular weight
Correspondence: Mary M. Jones, MD, Children's Hospital National Medical Center, Division of Rheumatology, 111 Michigan Avenue NW, Washington, DC 20010, USA.
344
Monocyte elastase and cystic fibrosis polysaccharide (HMP) were isolated as previously described (Pier et al., 1978; Pier, Matthews & Eardley, 1983). All media used contained less than 0-01 ng/ml of endotoxin, as demonstrated by the Oimulus amoebocyte lysate assay (Associates of Cape Cod, Woods Hole, MA). MEP contained 1 x 10-4 ng endotoxin/pg, and HMP had no detectable endotoxin ( < 4 x 10 ng/pg) by Limulus testing. Subjects Twelve Caucasian patients with CF (mean age 18 5 + 7 years), diagnosed on the basis of abnormal sweat chloride determination and characteristic clinical course, participated in the study. The patients were selected from the CF in-patient population followed at Texas Children's Hospital, on the basis of availability and willingness to submit to testing. At the time of study, 1 I children were receiving intravenous antibiotics, seven inhaled on oral bronchodilators, seven oral corticosteroids (range 20-50 mg prednisone/day), one child was enrolled in the randomized double-blinded study of oral corticosteroids, and was receiving either placebo or alternate day oral prednisone (30 or 60 mg every other day), and three-non-steroidal anti-inflammatory drugs. No CF patient had received anti-inflammatory drugs in the 12 h prior to sampling. Control subjects consisted of two groups: healthy young adults (mean age 28 + 4 years, n = 12) and young adults with symptomatic asthma on medication (mean age 35 years, n = 5). Each sample from a CF or asthma subject was paired with a sample from a healthy control assayed simultaneously. These studies were approved by the Institutional Review Board, Baylor College of Medicine, and written informed consent was obtained from all subjects.
Measurement of monocyte elastase The elastolytic activity of peripheral blood monocytes was determined by the release of [3H] from [3H]-elastin-coated plates. [3H]-elastin was prepared from bovine ligamentum elastin tritiated by reductive alkylation with [3H]-sodium borohydride (ICN, Irving, CA), diluted to 16 mg/ml with water, and kept at -70°C until assay (Banda, Dovey & Werb, 1981). The specific activity of the [3H]-elastin was 448 ct/min per pg of protein. An aliquot of [3H]-elastin was thawed, washed once in pyrogen-free water, resuspended to a final concentration of. 2 mg/ml, autoclaved for 15 min, sonicated for 45 sec using a Bronwill Biosonik III sonicator (Bronwill Scientific, Rochester, NY) on setting 50, at room temperature. [3H]-elastin (200 pg) was added to each 16-mm well of a 24-well polystyrene plate, and dried at 50°C. Plates were frozen at -70°C until assay (Chapman, Stone & Vaurin, 1984). The [3H]-elastin-coated wells were washed once with 0 5 ml HBSS before use. Elastolytic activity, expressed as pg [3H]-elastin solubilized/24 h/106 monocytes, was determined by counting the [3H] released into the monocyte supernatant. In this system, 10 pg of purified human leucocyte elastase derived from purulent sputum (Elastin products, Pacific, MO) solubilized 1155 + 2-6 pg of [3H]-elastin in 24 h. Preparation of monocytes Heparinized peripheral blood was layered over sodium diatrizoate/Ficoll (LSM, Organon Technika, Durham, NC) and centrifuged, and the interface was harvested and washed three times with HBSS without Ca2+ or Mg2+. The cells were incubated for 2 h on ice with 2-aminoethyl-isothiouronium bromide (AET)-modified sheep erythrocytes to rosette T cells
345
(Saxon, Feldhaus & Robins, 1976). The rosetting cells were separated from the non-rosetting cells by centrifugation over cold LSM. After washing twice, the monocytes suspended in culture medium (RPMI 1640, 10% heat-inactivated FCS, 100 U/ml penicillin, 100 pg/ml streptomycin, and 4 mM L-glutamine (PSG)). Monocytes (0 5 x 106/well) were added to 16-mm polystyrene wells coated with [3H]-elastin and allowed to adhere for 2 h at 37°C in a humidified 5% CO2 incubator. Non-adherent cells were aspirated, and the wells were washed three times. This procedure results in an adherent cell population of >85% monocytes, as demonstrated by non-specific esterase staining, and less than 1% T cells or PMNs (Rossen et al., 1985). The monocytes were then covered with 0 5 ml of a serum-free medium (RPMI 1640, 1% Nutridoma HU (Boehringer Mannheim, Indianapolis, IN), PSG, and 1 mm sodium pyruvate).
MEP, HMP, ibuprofen, dexamethasone, and/or appropriate
control medium were added to the cells at this time. After 24-h incubation in 5% CO2 at 37°C, the plates were centrifuged, and the supernatants removed. Two 100-pl aliquots were counted for 3H-elastin release. Dilutions of the remaining monocyte supernatants were made and the amounts of IL-6 and IL-1 determined.
Determination of IL-] and IL-6 The monocyte supernatants were assayed for their IL- 1 content by the murine thymocyte proliferation assay, as previously described (Mizel, Oppenheim & Rosenstreich, 1978). A standard dose-response curve of human rIL- I a (Genzyme, Boston, MA) was run with each assay and used to compute the units/ml of IL- 1 in each supernatant. The amount of IL-6 in the supernatant was measured by testing the ability to increase Immunoglobulin A (IgA) secretion by an Epstein-Barr virustransformed B cell- line, JB/FF, and the quantity of IgA determined by an enzyme-linked immunoassay as described (Rossen et al., 1985). A unit of activity was determined by the dilution of monocyte supernatant causing a doubling of the baseline IgA secretion. This cell line is sensitive to IL-6, but unresponsive to purified IL-1.
Quantifictation of DNA content of monocyte cultures After removal of the monocyte supernatant, 0 5 ml phosphatebuffered saline (PBS) was added to each well, and the plates were frozen at -90°C until time of assay. The cells were hydrolysed, the DNA precipitated with cold perchloric acid, then extracted with hot (70°C) perchloric acid, and measured by spectrophotometry after reaction with diphenylamine as described (Adams,
1981). Statistical analysis The null hypothesis to be tested was that there would be no difference in the quantity of monocyte secretory products produced by CF patients and controls. The statistical significance of the paired data (CF samples paired with controls assayed simultaneously) were tested using the Wilcoxon signed rank statistic (Glantz, 1987). For all statistical analyses, a probability of 0-05 was used to reject the null hypothesis. RESULTS Baseline elastase activity is increased in CF Monocytes from CF patients exhibited a 2-2-fold greater
M. M. Jones et al.
346 5
2 q
(NJ
4
-C
a) 0 A0
"I (h
40
3
0'
0
-o
a'
2
"I,
E.n
0) _0
LdJ
*I
C,
-A
A
CF
588688
CTL
-D _0
I
I0 CY%
rNX-.\.\
KL=
-
Asthma
Fig. 1. Baseline elastase activity is increased in CF but not in asthmatics. Adherent peripheral blood monocytes from the CF patients, healthy controls, and symptomatic adult asthmatics were cultured for 24 h on [3H]-elastin-coated plates. CF patients exhibited a 2 2-fold increase in elastase secretion (P 0 1). (Mean + s.e.m., CF & control n = 12; asthma n 5.) =
elastase activity compared with controls, with a mean activity of 3-62+0-9 jg/24 h/106 cells in the CF patients, compared with 1l66+0 25 Iug/24 h/106 cells for the control population (P< 0 01). In contrast to the results seen with the CF patients, there was no difference in baseline elastase activity between the asthmatic patients and the healthy controls (Fig. 1), suggesting that the increased activity of the CF monocytes cannot be explained on the basis of concurrent bronchodilator use or the simple presence of non-infectious lung disease. There was no correlation between age and the amount of elastolytic activity in either the CF patients or the healthy controls. (Spearman rank correlation coefficient for 12 CF=0-035, P>0 5, 12 controls 0 049, P > 0 5; and for CF and control combined (n = 24), coefficient = 0-26, P> 02) (Glantz, 1987). In order to demonstrate that the difference between CF and healthy controls is not simply due to a difference in the number of adherent monocytes (i.e. the CF monocytes are 'activated' by chronic stimulation, and thus more likely to adhere) with increased elastase activity resulting from increased numbers of CF monocytes adhering to plastic, we assayed the DNA content of the monocytes adherent to the plastic wells, and found no difference between the six pairs tested (P < 0-2, data not shown.) In our study, neither the plasma nor the wash fluids from CF or control monocytes contained detectable elastolytic activity against [3H]-elastin (not shown), indicating that the increased elastase activity of monocytes could not represent carryover of free elastase from the CF plasma. =
-
Regulation of elastase secretion with Pseudomonas aeruginosa products and anti-inflammatory drugs MEP 25 ug/ml significantly increased elastase activity in both
Cystic fibrosis
Control
Fig 2. Regulation of elastase secretion with products of Pseudomonas Aeruginosa and antiinflammatory drugs. Peripheral blood monocytes from CF patients and adult control were cultured as in Fig. 1 in the presence of mucoid exopolysaccharide 25 pg/ml (MEP), dexamethasone 10-7 M (DEX), and or ibuprofen 50 pg/ml (IBU). Both the baseline and stimulated elastase secretion was significantly higher in the CF patients compared with controls (CF versus CTL: Media P
Increased elastase secretion by peripheral blood monocytes in cystic fibrosis patients M. M. JONES, D. K. SEILHEIMER*, G. B. PIERt & R. D. ROSSEN Immunology Research Laboratory, The Veterans Administration Medical Center, * The Departments of Microbiology and Immunology, Internal Medicine, and Pediatrics, Baylor College of Medicine, Houston, TX, and t The Channing Laboratory, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA
(Acceptedfor publication 15 January 1990)
SUMMARY Morbidity and mortality in cystic fibrosis (CF) is predominantly due to destruction of pulmonary tissue. The host immune response may, in part, play a pathogenic role in pulmonary destruction in these patients. To further understand host immune response in CF, we examined the state of activation of peripheral blood monocytes in CF. Baseline elastase activity was 2-2-fold greater in the CF monocytes than in controls. Pseudomonas aeruginosa mucoid exopolysaccharide (MEP) and high molecular weight polysaccharide (HMP) increased elastase activity in both control and CF monocytes, with a greater absolute increase in the CF monocytes. There was no difference in baseline or MEP-stimulated secretion of interleukin- I (IL-I) or interleukin-6 (IL-6) between CF and control monocytes. Ibuprofen enhanced both MEP and HMP-stimulated elastase activity, whereas dexamethasone suppressed both baseline and stimulated elastase activity > 20% in both CF and control monocytes. These results suggest that circulating monocytes in CF are stimulated in vivo, resulting in a remarkably elevated elastase activity in vitro. Elevated elastase release by peripheral blood monocytes as they enter the lung in response to chemotactic stimuli may contribute to lung destruction in CF. Keywords
monocytes cystic fibrosis
elastase interleukins
INTRODUCTION Cystic fibrosis (CF) is an autosomal recessive disorder characterized by abnormal exocrine gland secretion. Almost all patients develop chronic pulmonary infection and inflammation, resulting in respiratory failure and early death, despite intensive antibiotic therapy (David & di Sant' Agnese, 1984). Examination of lung tissue from CF patients shows evidence of extensive elastolysis and pulmonary secretions from CF patients have high levels of several proteolytic enzymes, including elastase and cathepsin G (Suter et al., 1984; Bruce et al., 1985). In CF, elastases in pulmonary secretions are derived from leucocytes, and also from Pseudomonas aeruginosa (PsA). In addition to destruction of lung extra-cellular matrix (Campbell, Senior & Welgus, 1987), these elastases interfere with important mechanisms for bacterial clearance: leucocyte elastase inactivates complement receptors on leucocytes (Tosi, Zakem & Berger, 1988) and PsA elastase degrades immunoglobulins, complement proteins, and human plasma al proteinase inhibitor
(Doring et al., 1985; Fick et al., 1985; Speert, 1985), thus potentiating elastolytic lung destruction. The human alveolar macrophage, derived from circulating blood monocytes, plays a fundamental role in virtually every aspect of the immune and inflammatory cell response and provides one of the first lines of host defense in the lung against invasion by pathogens (Bernaudin et al., 1988; Unanue & Allen, 1987). The role of the monocyte/macrophage system in CF lung damage has not been well characterized. Therefore, we measured the production of three monocyte products, elastase, IL- 1, and IL-6 in CF patients. MATERIALS AND METHODS Materials Dexamethasone, elastin, ibuprofen, and polymyxin B were purchased from Sigma Chemical Company (St Louis, MO), lipopolysaccharide B, E. coli 055.B5 (LPS-B) from Difco Laboratories (Detroit, MI), PsA refined endotoxin from Ribi Immunochemical Research, (Hamilton, MN), exotoxin A from List Biological Laboratories (Campbell, CA), and fetal calf serum (FCS) from Hyclone Laboratories (Logan, UT). Purified mucoid exopolysaccharide (MEP) and high molecular weight
Correspondence: Mary M. Jones, MD, Children's Hospital National Medical Center, Division of Rheumatology, 111 Michigan Avenue NW, Washington, DC 20010, USA.
344
Monocyte elastase and cystic fibrosis polysaccharide (HMP) were isolated as previously described (Pier et al., 1978; Pier, Matthews & Eardley, 1983). All media used contained less than 0-01 ng/ml of endotoxin, as demonstrated by the Oimulus amoebocyte lysate assay (Associates of Cape Cod, Woods Hole, MA). MEP contained 1 x 10-4 ng endotoxin/pg, and HMP had no detectable endotoxin ( < 4 x 10 ng/pg) by Limulus testing. Subjects Twelve Caucasian patients with CF (mean age 18 5 + 7 years), diagnosed on the basis of abnormal sweat chloride determination and characteristic clinical course, participated in the study. The patients were selected from the CF in-patient population followed at Texas Children's Hospital, on the basis of availability and willingness to submit to testing. At the time of study, 1 I children were receiving intravenous antibiotics, seven inhaled on oral bronchodilators, seven oral corticosteroids (range 20-50 mg prednisone/day), one child was enrolled in the randomized double-blinded study of oral corticosteroids, and was receiving either placebo or alternate day oral prednisone (30 or 60 mg every other day), and three-non-steroidal anti-inflammatory drugs. No CF patient had received anti-inflammatory drugs in the 12 h prior to sampling. Control subjects consisted of two groups: healthy young adults (mean age 28 + 4 years, n = 12) and young adults with symptomatic asthma on medication (mean age 35 years, n = 5). Each sample from a CF or asthma subject was paired with a sample from a healthy control assayed simultaneously. These studies were approved by the Institutional Review Board, Baylor College of Medicine, and written informed consent was obtained from all subjects.
Measurement of monocyte elastase The elastolytic activity of peripheral blood monocytes was determined by the release of [3H] from [3H]-elastin-coated plates. [3H]-elastin was prepared from bovine ligamentum elastin tritiated by reductive alkylation with [3H]-sodium borohydride (ICN, Irving, CA), diluted to 16 mg/ml with water, and kept at -70°C until assay (Banda, Dovey & Werb, 1981). The specific activity of the [3H]-elastin was 448 ct/min per pg of protein. An aliquot of [3H]-elastin was thawed, washed once in pyrogen-free water, resuspended to a final concentration of. 2 mg/ml, autoclaved for 15 min, sonicated for 45 sec using a Bronwill Biosonik III sonicator (Bronwill Scientific, Rochester, NY) on setting 50, at room temperature. [3H]-elastin (200 pg) was added to each 16-mm well of a 24-well polystyrene plate, and dried at 50°C. Plates were frozen at -70°C until assay (Chapman, Stone & Vaurin, 1984). The [3H]-elastin-coated wells were washed once with 0 5 ml HBSS before use. Elastolytic activity, expressed as pg [3H]-elastin solubilized/24 h/106 monocytes, was determined by counting the [3H] released into the monocyte supernatant. In this system, 10 pg of purified human leucocyte elastase derived from purulent sputum (Elastin products, Pacific, MO) solubilized 1155 + 2-6 pg of [3H]-elastin in 24 h. Preparation of monocytes Heparinized peripheral blood was layered over sodium diatrizoate/Ficoll (LSM, Organon Technika, Durham, NC) and centrifuged, and the interface was harvested and washed three times with HBSS without Ca2+ or Mg2+. The cells were incubated for 2 h on ice with 2-aminoethyl-isothiouronium bromide (AET)-modified sheep erythrocytes to rosette T cells
345
(Saxon, Feldhaus & Robins, 1976). The rosetting cells were separated from the non-rosetting cells by centrifugation over cold LSM. After washing twice, the monocytes suspended in culture medium (RPMI 1640, 10% heat-inactivated FCS, 100 U/ml penicillin, 100 pg/ml streptomycin, and 4 mM L-glutamine (PSG)). Monocytes (0 5 x 106/well) were added to 16-mm polystyrene wells coated with [3H]-elastin and allowed to adhere for 2 h at 37°C in a humidified 5% CO2 incubator. Non-adherent cells were aspirated, and the wells were washed three times. This procedure results in an adherent cell population of >85% monocytes, as demonstrated by non-specific esterase staining, and less than 1% T cells or PMNs (Rossen et al., 1985). The monocytes were then covered with 0 5 ml of a serum-free medium (RPMI 1640, 1% Nutridoma HU (Boehringer Mannheim, Indianapolis, IN), PSG, and 1 mm sodium pyruvate).
MEP, HMP, ibuprofen, dexamethasone, and/or appropriate
control medium were added to the cells at this time. After 24-h incubation in 5% CO2 at 37°C, the plates were centrifuged, and the supernatants removed. Two 100-pl aliquots were counted for 3H-elastin release. Dilutions of the remaining monocyte supernatants were made and the amounts of IL-6 and IL-1 determined.
Determination of IL-] and IL-6 The monocyte supernatants were assayed for their IL- 1 content by the murine thymocyte proliferation assay, as previously described (Mizel, Oppenheim & Rosenstreich, 1978). A standard dose-response curve of human rIL- I a (Genzyme, Boston, MA) was run with each assay and used to compute the units/ml of IL- 1 in each supernatant. The amount of IL-6 in the supernatant was measured by testing the ability to increase Immunoglobulin A (IgA) secretion by an Epstein-Barr virustransformed B cell- line, JB/FF, and the quantity of IgA determined by an enzyme-linked immunoassay as described (Rossen et al., 1985). A unit of activity was determined by the dilution of monocyte supernatant causing a doubling of the baseline IgA secretion. This cell line is sensitive to IL-6, but unresponsive to purified IL-1.
Quantifictation of DNA content of monocyte cultures After removal of the monocyte supernatant, 0 5 ml phosphatebuffered saline (PBS) was added to each well, and the plates were frozen at -90°C until time of assay. The cells were hydrolysed, the DNA precipitated with cold perchloric acid, then extracted with hot (70°C) perchloric acid, and measured by spectrophotometry after reaction with diphenylamine as described (Adams,
1981). Statistical analysis The null hypothesis to be tested was that there would be no difference in the quantity of monocyte secretory products produced by CF patients and controls. The statistical significance of the paired data (CF samples paired with controls assayed simultaneously) were tested using the Wilcoxon signed rank statistic (Glantz, 1987). For all statistical analyses, a probability of 0-05 was used to reject the null hypothesis. RESULTS Baseline elastase activity is increased in CF Monocytes from CF patients exhibited a 2-2-fold greater
M. M. Jones et al.
346 5
2 q
(NJ
4
-C
a) 0 A0
"I (h
40
3
0'
0
-o
a'
2
"I,
E.n
0) _0
LdJ
*I
C,
-A
A
CF
588688
CTL
-D _0
I
I0 CY%
rNX-.\.\
KL=
-
Asthma
Fig. 1. Baseline elastase activity is increased in CF but not in asthmatics. Adherent peripheral blood monocytes from the CF patients, healthy controls, and symptomatic adult asthmatics were cultured for 24 h on [3H]-elastin-coated plates. CF patients exhibited a 2 2-fold increase in elastase secretion (P 0 1). (Mean + s.e.m., CF & control n = 12; asthma n 5.) =
elastase activity compared with controls, with a mean activity of 3-62+0-9 jg/24 h/106 cells in the CF patients, compared with 1l66+0 25 Iug/24 h/106 cells for the control population (P< 0 01). In contrast to the results seen with the CF patients, there was no difference in baseline elastase activity between the asthmatic patients and the healthy controls (Fig. 1), suggesting that the increased activity of the CF monocytes cannot be explained on the basis of concurrent bronchodilator use or the simple presence of non-infectious lung disease. There was no correlation between age and the amount of elastolytic activity in either the CF patients or the healthy controls. (Spearman rank correlation coefficient for 12 CF=0-035, P>0 5, 12 controls 0 049, P > 0 5; and for CF and control combined (n = 24), coefficient = 0-26, P> 02) (Glantz, 1987). In order to demonstrate that the difference between CF and healthy controls is not simply due to a difference in the number of adherent monocytes (i.e. the CF monocytes are 'activated' by chronic stimulation, and thus more likely to adhere) with increased elastase activity resulting from increased numbers of CF monocytes adhering to plastic, we assayed the DNA content of the monocytes adherent to the plastic wells, and found no difference between the six pairs tested (P < 0-2, data not shown.) In our study, neither the plasma nor the wash fluids from CF or control monocytes contained detectable elastolytic activity against [3H]-elastin (not shown), indicating that the increased elastase activity of monocytes could not represent carryover of free elastase from the CF plasma. =
-
Regulation of elastase secretion with Pseudomonas aeruginosa products and anti-inflammatory drugs MEP 25 ug/ml significantly increased elastase activity in both
Cystic fibrosis
Control
Fig 2. Regulation of elastase secretion with products of Pseudomonas Aeruginosa and antiinflammatory drugs. Peripheral blood monocytes from CF patients and adult control were cultured as in Fig. 1 in the presence of mucoid exopolysaccharide 25 pg/ml (MEP), dexamethasone 10-7 M (DEX), and or ibuprofen 50 pg/ml (IBU). Both the baseline and stimulated elastase secretion was significantly higher in the CF patients compared with controls (CF versus CTL: Media P
