Eur Respir J 2002 Aug;20(2):319-24
Screening for alpha1-Pi deficiency in patients with lung diseases.

Wencker M, Marx A, Konietzko N, Schaefer B, Campbell EJ.

Dept of Pneumology, University Hospital, Ruhrlandklinik, Gelsenkirchen, Germany.

In patients with pulmonary emphysema, studies have reported 2-3% of individuals with severe alpha1-Pi deficiency. The aims of this study were to evaluate the accuracy of a new method for quantifying alpha1-Pi through phenotyping from dried blood spots (DBS) and to test the hypothesis that the screening of a population at risk increases the detection rate for severe alpha1-Pi deficiency. The accuracy of phenotyping results from DBS was compared to conventional methods in a total of 555 individuals. In a prospective study 1,060 patients with chronic lung disease were screened for alpha1-Pi deficiency using DBS. The validation of the phenotyping method from DBS showed an accuracy of 100%. Out of 1,060 tested patients, none had a severe PiZ deficiency and only 3 had PiSZ, whilst 36 (3.34%) individuals were identified as heterozygous for PiMS and 39 (3.68%) for PiMZ. No patients with severe alpha1-Pi deficiency could be detected in this population and the frequency of PiMS or PiMZ detected was similar to that of the normal population. Thus, the screening of an unselected population of chronic obstructive pulmonary disease and asthma patients may not detect a large number of individuals with severe alpha1-Pi deficiency.

Publication Types:
Validation Studies

Am J Hum Genet 2002 May;70(5):1229-39

Genomewide linkage analysis of quantitative spirometric phenotypes in severe early-onset chronic obstructive pulmonary disease.

Silverman EK, Palmer LJ, Mosley JD, Barth M, Senter JM, Brown A, Drazen JM, Kwiatkowski DJ, Chapman HA, Campbell EJ, Province MA, Rao DC, Reilly JJ, Ginns LC, Speizer FE, Weiss ST.

Channing Laboratory and Division of Pulmonary and Critical Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.

Chronic obstructive pulmonary disease (COPD) is a common, complex disease associated with substantial morbidity and mortality. COPD is defined by irreversible airflow obstruction; airflow obstruction is typically determined by reductions in quantitative spirometric indices, including forced expiratory volume at 1 s (FEV(1)) and the ratio of FEV(1) to forced vital capacity (FVC). To identify genetic determinants of quantitative spirometric phenotypes, an autosomal 10-cM genomewide scan of short tandem repeat (STR) polymorphic markers was performed in 72 pedigrees (585 individuals) ascertained through probands with severe early-onset COPD. Multipoint variance-component linkage analysis (using SOLAR) was performed for quantitative phenotypes, including FEV(1), FVC, and FEV(1)/FVC. In the initial genomewide scan, significant evidence for linkage to FEV(1)/FVC was demonstrated on chromosome 2q (LOD score 4.12 at 222 cM). Suggestive evidence was found for linkage to FEV(1)/FVC on chromosomes 1 (LOD score 1.92 at 120 cM) and 17 (LOD score 2.03 at 67 cM) and to FVC on chromosome 1 (LOD score 2.05 at 13 cM). The highest LOD score for FEV(1) in the initial genomewide scan was 1.53, on chromosome 12, at 36 cM. After inclusion of 12 additional STR markers on chromosome 12p, which had been previously genotyped in this population, suggestive evidence for linkage of FEV(1) (LOD score 2.43 at 37 cM) to this region was demonstrated. These observations provide both significant evidence for an early-onset COPD-susceptibility locus on chromosome 2 and suggestive evidence for linkage of spirometry-related phenotypes to several other genomic regions. The significant linkage of FEV(1)/FVC to chromosome 2q could reflect one or more genes influencing the development of airflow obstruction or dysanapsis.

Hum Mol Genet 2002 Mar 15;11(6):623-32

Genome-wide linkage analysis of severe, early-onset chronic obstructive pulmonary disease: airflow obstruction and chronic bronchitis phenotypes.

Silverman EK, Mosley JD, Palmer LJ, Barth M, Senter JM, Brown A, Drazen JM, Kwiatkowski DJ, Chapman HA, Campbell EJ, Province MA, Rao DC, Reilly JJ, Ginns LC, Speizer FE, Weiss ST.

Channing Laboratory, Division of Pulmonary and Critical Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, 181 Longwood Avenue, Boston, MA 02115, USA.

Familial aggregation of chronic obstructive pulmonary disease (COPD) has been demonstrated, but linkage analysis of COPD-related phenotypes has not been reported previously. An autosomal 10 cM genome-wide scan of short tandem repeat (STR) polymorphic markers was analyzed for linkage to COPD-related phenotypes in 585 members of 72 pedigrees ascertained through severe, early-onset COPD probands without severe alpha1-antitrypsin deficiency. Multipoint non-parametric linkage analysis (using the ALLEGRO program) was performed for qualitative phenotypes including moderate airflow obstruction [forced expiratory volume at one second (FEV(1)) < 60% predicted, FEV(1)/FVC < 90% predicted], mild airflow obstruction (FEV(1) < 80% predicted, FEV(1)/FVC < 90% predicted) and chronic bronchitis. The strongest evidence for linkage in all subjects was observed at chromosomes 12 (LOD = 1.70) and 19 (LOD = 1.54) for moderate airflow obstruction, chromosomes 8 (LOD = 1.36) and 19 (LOD = 1.09) for mild airflow obstruction and chromosomes 19 (LOD = 1.21) and 22 (LOD = 1.37) for chronic bronchitis. Restricting analysis to cigarette smokers only provided increased evidence for linkage of mild airflow obstruction and chronic bronchitis to several genomic regions; for mild airflow obstruction in smokers only, the maximum LOD was 1.64 at chromosome 19, whereas for chronic bronchitis in smokers only, the maximum LOD was 2.08 at chromosome 22. On chromosome 12p, 12 additional STR markers were genotyped, which provided additional support for an airflow obstruction locus in that region with a non-parametric multipoint approach for moderate airflow obstruction (LOD = 2.13) and mild airflow obstruction (LOD = 1.43). Using a dominant model with the STR markers on 12p, two point parametric linkage analysis of all subjects demonstrated a maximum LOD score of 2.09 for moderate airflow obstruction and 2.61 for mild airflow obstruction. In smokers only, the maximum two point LOD score for mild airflow obstruction was 3.14. These observations provide suggestive evidence that there is a locus on chromosome 12p which contributes to susceptibility to early-onset COPD.

Hum Hered 2001;52(4):223-32

Linkage analysis of alpha 1-antitrypsin deficiency: lessons for complex diseases.

Silverman EK, Mosley JD, Rao DC, Palmer LJ, Province MA, Elston RC, Weiss ST, Campbell EJ.

Channing Laboratory, Brigham and Women's Hospital, Boston, Mass. 02114, USA.

OBJECTIVES: Severe alpha 1-antitrypsin (A1AT) deficiency is the one proven genetic risk factor for chronic obstructive pulmonary disease (COPD). Familial aggregation has been demonstrated for COPD among individuals who do not have A1AT deficiency, but linkage analysis of COPD has not been reported. To investigate the optimal phenotype definitions and analytical methods for the linkage analysis of COPD, we examined a set of 28 A1AT- deficient families containing 155 individuals. We have used the protease inhibitor (PI) type as a genetic marker rather than a disease gene, and we have performed linkage analysis between PI type and serum A1AT level and spirometry-related phenotypes. METHODS: Linkage analysis was performed on the quantitative phenotypes forced expiratory volume at 1 s (FEV(1) as % predicted), the ratio of FEV(1) to forced vital capacity (FEV(1)/FVC as % predicted), and serum A1AT level using the variance component approach in SOLAR, the generalized estimating equation approach in RELPAL, and the model-based classical lod score method in LINKAGE. Linkage analysis with qualitative A1AT and spirometry phenotypes was performed using a model-based method (LINKAGE) and a model-free method (GENEHUNTER). Adjustments for smoking effects were investigated under each method. RESULTS: All of the methods demonstrated linkage of PI type to serum A1AT level. Interestingly, however, the other quantitative phenotypes provided only weak evidence for linkage of PI type to lung disease. Better evidence for linkage of lung disease to PI type was found using a moderate or a mild threshold for the definition of airflow obstruction. CONCLUSIONS: For linkage analysis of spirometry phenotypes in A1AT deficiency, qualitative phenotypes provided stronger evidence for linkage than quantitative phenotypes. Possible contributors to the stronger evidence for linkage to qualitative spirometry phenotypes include the ascertainment scheme and the nonnormality of the pulmonary function data in PI Z subjects. This study provides guidelines for studies of the genetics of COPD unrelated to A1AT deficiency. Copyright 2001 S. Karger AG, Basel

Eur Respir J 2001 Mar;17(3):356-9

Alpha-1-antitrypsin genotyping with mouthwash specimens.

Stockley RA, Campbell EJ.

Dept. of Medicine, Queen Elizabeth Hospital, Birmingham, UK.

Alpha1-antitrypsin (alpha1-AT) deficiency is diagnosed as a two-stage procedure (concentration and phenotype). However the latter does not provide clues to the presence of null genes without family studies and obtaining blood from patients at a distance often proves difficult. The aim of the study was to assess the feasibility of genotyping alpha1-AT using buccal cells. Mouthwash specimens were sent by 84 patients (with a variety of phenotypes of alpha1-antitrypsin) through the post. Deoxyribonucleic acid (DNA) was isolated from buccal cells in each sample and subjected to polymerase chain reaction (PCR) using a genotyping kit to detect the S and Z alleles. Eighty-three of 84 samples received were suitable for amplification. The specific primers successfully identified the S and Z alleles in each case. However, five of the 35 samples obtained from patients thought to be Z allele homozygotes were found to be heterozygotes for another severe deficiency allele. These data confirm the feasibility of "at distance" testing for alpha1-antitrypsin deficiency alleles using buccal cells from mouthwash samples. The results raise the possibility that other deficiency alleles are more common than has previously been suspected.

Thorax 2001 May;56(5):366-72

Assessment of airway neutrophils by sputum colour: correlation with airways inflammation.

Stockley RA, Bayley D, Hill SL, Hill AT, Crooks S, Campbell EJ.

Department of Medicine, Queen Elizabeth Hospital, Edgbaston, Birmingham B15 2TH, UK.

BACKGROUND: Airway inflammation, with recruitment of neutrophils to the airway lumen, results in purulent secretions and a variety of potential adverse consequences for patients with chronic bronchitis and bronchiectasis. We hypothesised that gradations of sputum colour would correlate directly with the myeloperoxidase content of sputum and with various other indicators of the activity and consequences of bronchial diseases. METHODS: To test this hypothesis, we quantified sputum colour by reference to a sensitive nine point colour chart and correlated this assessment with indices of a number of inflammatory mediators in sputum. RESULTS: The results indicate that standardised visual measurements of sputum colour correlated strongly with myeloperoxidase, interleukin 8, leucocyte elastase (both activity and total quantity), sputum volume, protein leak, and secretory leucocyte proteinase inhibitor (p<0.001 for all). In addition, there was a strong direct correlation between leucocyte elastase and both myeloperoxidase (p<0.003) and sputum volume (p<0.001), but a strong negative correlation with secretory leucocyte proteinase inhibitor (p<0.001). CONCLUSIONS: These results indicate that sputum colour graded visually relates to the activity of the underlying markers of bronchial inflammation. The results of this simple visual analysis of sputum provides guidance concerning underlying inflammation and its damaging potential. It also provides a useful scientific tool for improving the monitoring of chronic airways diseases and response to treatment.

J Clin Invest 2000 Dec;106(12):1445-6

Animal models of emphysema: the next generations.

Campbell EJ.

Department of Medicine, University of Utah, 410 Chipeta Way, Room 410, Salt Lake City, Utah 84108, USA.

Publication Types:
Comment

Am J Respir Crit Care Med 2000 Dec;162(6):2152-8

Gender-related differences in severe, early-onset chronic obstructive pulmonary disease.

Silverman EK, Weiss ST, Drazen JM, Chapman HA, Carey V, Campbell EJ, Denish P, Silverman RA, Celedon JC, Reilly JJ, Ginns LC, Speizer FE.

Channing Laboratory, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.

Men have higher prevalence rates of chronic obstructive pulmonary disease (COPD) than women, which has been attributed to the historically higher rates of cigarette smoking in males. However, the increased rates of cigarette smoking in females within the last several decades have been associated with steadily increasing rates of COPD in women. As part of a study of the genetics of severe, early-onset COPD, we assembled a group of 84 probands with severe, early-onset COPD (without severe alpha(1)-antitrypsin deficiency) and 348 of their first-degree relatives. We found a markedly elevated prevalence (71.4%) of females among the early-onset COPD probands. Among the entire group of first-degree relatives of early-onset COPD probands, univariate analysis demonstrated similar spirometric values and bronchodilator responsiveness in males and females; however, among current or ex-smokers, female first-degree relatives had significantly lower FEV(1)/ FVC (81.4 +/- 17.2% in females versus 87.0 +/- 12.9% in males, p = 0.009) and significantly greater bronchodilator responsiveness (expressed as percentage of baseline FEV(1)) (7.7 +/- 9.4% pred in females versus 4.1 +/- 6.4% pred in males, p = 0.002). Female smoking first-degree relatives were significantly more likely to demonstrate profound reductions in FEV(1) (< 40% pred) than male smoking first-degree relatives (p = 0. 03). Multivariate analysis, performed with generalized estimating equations, demonstrated that current or ex-smoking female first-degree relatives had significantly greater risk of FEV(1) < 80% pred (OR 1.91, 95% CI 1.03- 3.54), FEV(1) < 40% pred (OR 3.56, 95% CI 1.08-11.71), and bronchodilator response greater than 10% of baseline FEV(1) (OR 4.74, 95% CI 1.91-11.75). These results suggest that women may be more susceptible to the development of severe COPD.

Am J Med 2000 Sep;109(4):288-95

Association between airway bacterial load and markers of airway inflammation in patients with stable chronic bronchitis.

Hill AT, Campbell EJ, Hill SL, Bayley DL, Stockley RA.

Department of Medicine, Queen Elizabeth Hospital, Birmingham, UK.

PURPOSE: Viable bacteria are often isolated from airway secretions in clinically stable patients with chronic bronchitis. We hypothesized that the number of organisms and bacterial species might be important modulators of airway inflammation. SUBJECTS AND METHODS: We performed quantitative sputum cultures in 160 stable patients [55 with chronic obstructive pulmonary disease (COPD) and normal serum alpha(1)-antitrypsin levels, 62 with COPD and severe alpha(1)-antitrypsin deficiency (PiZ), and 43 with idiopathic bronchiectasis]. The results were related to several indicators of the mechanisms and severity of airway inflammation. RESULTS: Airway bacterial load correlated with sputum myeloperoxidase level, an indirect measure of neutrophil activation and number (r = 0.50, P<0. 001); sputum neutrophil chemoattractants [interleukin-8 level (r = 0. 68, P<0.001) and leukotriene B4 level (r = 0.53, P<0.001)]; sputum leukocyte elastase activity (r = 0.55, P<0.001); and albumin leakage from serum to sputum (r = 0.26, P<0.01). Markers of inflammation increased at bacterial loads of 10(6) to 10(7) colony-forming units per milliliter, and increased progressively with increasing bacterial load. For example, the median (interquartile range) sputum myeloperoxidase level was 0.3 U/mL (0.1 to 0.5 U/mL) for patients who were not colonized or who had mixed normal oropharyngeal flora alone; 0.5 U/mL (0.2 to 0.7 U/mL) for patients with 10(5) to 10(6) colony-forming units per milliliter (P = 0.07); 0.5 U/mL (0.3 to 1.2 U/mL) for patients with 10(6) to 10(7) colony-forming units per milliliter (P<0.01); 0.7 U/mL (0.3 to 1.2 U/mL) for patients with 10(7) to 10(8) colony-forming units per milliliter (P <0.005); and 2.4 U/mL (0.7 to 4.8 U/mL) for patients with 10(8) or greater colony-forming units per milliliter (P<0.0001). The bacterial species influenced airway inflammation; for example, sputum myeloperoxidase activity was greater (P<0.005) in patients colonized with Pseudomonas aeruginosa [median 32 U/mL (interquartile range, 20 to 65 U/mL)] than those colonized with nontypeable Hemophilus influenzae [4 U/mL (2 to 31 U/mL)], which in turn was greater (P = 0.01) than among those colonized with Moraxella catarrhalis [1.1 U/mL (0.6 to 1.8 U/mL)]. We did not find a relation between bacterial load and lung function.CONCLUSIONS: The bacterial load and species contribute to airway inflammation in patients with stable chronic bronchitis. Further studies are required to determine the consequences of bacterial colonization on patient morbidity and decline in lung function.

Publication Types:
Clinical Trial
Controlled Clinical Trial

Immunol 2000 Sep 15;165(6):3366-74

Bioactive proteinase 3 on the cell surface of human neutrophils: quantification, catalytic activity, and susceptibility to inhibition.

Campbell EJ, Campbell MA, Owen CA.

Department of Internal Medicine, University of Utah Health Sciences Center, Salt Lake City, UT 84132, USA.

Although proteinase 3 (PR3) is known to have the potential to promote inflammation and injure tissues, the biologic forms and function of PR3 in polymorphonuclear neutrophils (PMN) from healthy donors have received little attention. In this paper, we show that PMN contain 3.24 +/- SD 0.24 pg of PR3 per cell, and that the mean concentration of PR3 in azurophil granules of PMN is 13.4 mM. Low levels of PR3 are detectable on the cell surface of unstimulated PMN. Exposure of PMN to cytokines or chemoattractants alone induces modest (1.5- to 2.5-fold) increases in cell surface-bound PR3. In contrast, brief priming of PMN with cytokines, followed by activation with a chemoattractant, induces rapid and persistent, 5- to 6-fold increases in cell surface expression of PR3, while causing minimal free release of PR3. Membrane-bound PR3 on PMN is catalytically active against Boc-Alanine-Alanine-Norvaline-thiobenzyl ester and fibronectin, but in marked contrast to soluble PR3, membrane-bound PR3 is resistant to inhibition by physiologic proteinase inhibitors. PR3 appears to bind to the cell surface of PMN via a charge-dependent mechanism because exposure of fixed, activated PMN to solutions having increasing ionic strength results in elution of PR3, HLE, and CG, and there is a direct relationship between their order of elution and their isoelectric points. These data indicate that rapidly inducible PR3 expressed on the cell surface of PMN is an important bioactive form of the proteinase. If PR3 expression on the cell surface of PMN is dysregulated, it is well equipped to amplify tissue injury directly, and also indirectly via the generation of autoantibodies.

Respir Med 2000 Aug;94 Suppl C:S18-21

Alpha1-antitrypsin deficiency: incidence and detection program.

Campbell EJ.

Department of Internal Medicine, University of Utah Health Sciences Center, Salt Lake City 84108, USA.

Most individuals with AAT deficiency have escaped detection by healthcare systems worldwide. The large number of undiagnosed individuals has made it difficult to define the natural history of the clinical disease accurately, and severe ascertainment bias has colored the clinical descriptions of the disease. Most importantly, undetected individuals lose opportunities for important lifestyle changes and preventive therapies. To address this problem, the World Health Organization has recommended that all patients with chronic obstructive lung disease, and all adults and adolescents with asthma, be tested for AAT deficiency. Historically, the AAT Deficiency Detection Center has tested more than 30 000 individuals for the disease, and we have identified more than 1000 cases of AAT deficiency (approximately 30% of the known cases in the United States). Currently, we are implementing methods for determining AAT concentration (level), phenotype, and genotype in specimens of whole blood dried onto filter paper. This full spectrum of robust tests is performed on samples that are easily obtained and shipped to a central laboratory for processing. Wide application of these procedures may help to bring large numbers of presently undiagnosed patients to medical attention.

Eur Respir J 2000 May;15(5):886-90

Airways inflammation in chronic bronchitis: the effects of smoking and alpha1-antitrypsin deficiency.

Hill AT, Bayley DL, Campbell EJ, Hill SL, Stockley RA.

Dept of Medicine, Queen Elizabeth Hospital, Birmingham, UK.

Airways inflammation in chronic bronchitis is thought predominantly to be a direct consequence of neutrophil recruitment and release of elastase in response to factors such as cigarette smoke. The aims of this study were to assess the role of smoking and determine whether the serum elastase inhibitor alpha1-antitrypsin (alpha1AT) influenced the process. Airways inflammation was compared between patients with chronic obstructive bronchitis with (n=39) and without (n=42) severe alpha1AT deficiency. The authors assessed the sputum concentration of the neutrophil chemoattractants interleukin-8 (IL-8) and leukotriene (LT)B4, myeloperoxidase (MPO) as a marker of neutrophil influx, neutrophil elastase activity and its natural inhibitors, alpha1AT and secretory leukoprotease inhibitor (SLPI). Finally serum alpha1AT was measured to determine the degree of protein leakage (sputum sol serum alpha1AT ratio). Compared to current smokers, the exsmokers had a lower concentration of the chemoattractant IL-8 (p<0.05) and a lower MPO concentration, although this failed to reach conventional statistical significance (p=0.06). Patients with alpha1AT deficiency had greater inflammation in the larger airways with increased LTB4 (p<0.005), MPO (p<0.001), neutrophil elastase activity (p<0.01), protein leak (p<0.001), and were found to have a lower anti-proteinase screen with both reduced sputum alpha1AT (p<0.001) and SLPI concentrations (p<0.05). The reduction in sputum interleukin-8 levels in exsmokers may decrease neutrophil influx and thus explain the slower rate of neutrophil mediated progression of lung disease compared to subjects who continue to smoke. Patients with alpha1-antitrypsin deficiency had greater inflammation suggesting that alpha1-antitrypsin plays an important role in protecting the larger airways from the inflammatory effects of elastase activity and may explain their more rapid progression of disease.

Chest 2000 May;117(5 Suppl 1):291S-3S

Bronchial inflammation: its relationship to colonizing microbial load and alpha(1)-antitrypsin deficiency.

Stockley RA, Hill AT, Hill SL, Campbell EJ.

Department of Medicine, Queen Elizabeth Hospital, Birmingham B15 2TH, UK.

Neutrophil elastase is capable of generating many of the features of chronic bronchial disease. In patients with COPD, airways inflammation with neutrophil recruitment and elastase release is positively correlated with colonizing bacterial load in the stable clinical state (p < 0.0005). In addition, alpha(1)-antitrypsin deficiency is associated with a greater neutrophil load, higher elastase activity, leukotriene-B(4) concentration, and serum protein leak than matched patients without deficiency (p < 0.005). These data confirm an effect of bronchial colonization on airways inflammation in COPD and indicate the role of alpha(1)-antitrypsin in its modulation.

Chest 2000 May;117(5 Suppl 1):303S

Quantum proteolysis by neutrophils : implications for pulmonary emphysema in alpha(1)-antitrypsin deficiency

Campbell EJ, Campbell MA, Boukedes SS, Owen CA.

Department of Internal Medicine, University of Utah Health Sciences Center, Salt Lake City, UT.

Chest 2000 May;117(5 Suppl 1):273S-4S

Familial aggregation of severe, early-onset COPD : candidate gene approaches

Silverman EK, Speizer FE, Weiss ST, Chapman HA Jr, Schuette A, Campbell EJ, Reilly JJ Jr, Ginns LC, Drazen JM.

Brigham and Women's Hospital (Drs. Silverman, Speizer, Weiss, Chapman, Reilly, and Drazen, and Mr. Schuette), Massachussetts General Hospital (Dr. Ginns), and Harvard Medical School (Drs. Silverman, Speizer, Weiss, Chapman, Reilly, Gi.

Wound Repair Regen 1999 Nov-Dec;7(6):410-22

Leukocyte proteinases in wound healing: roles in physiologic and pathologic processes.

Barrick B, Campbell EJ, Owen CA.

Department of Internal Medicine, University of Utah School of Medicine,Salt Lake City 84108, USA.

Leukocytes express a number of proteinases which play critical roles in physiologic processes during wound healing. However, if the activity of these proteinases is uncontrolled, they can contribute to devastating tissue injury that can affect most organ systems. Until recently, little was known about the mechanisms by which leukocytes retain the activity of their proteinases within the extracellular space which contains highly effective proteinase inhibitors. Studies of the cell biology of leukocyte proteinases have begun to identify the mechanisms by which proteinases can circumvent the effects of physiologic proteinase inhibitors. Herein, we will review the cell biology of leukocyte proteinases, and we will discuss the mechanisms by which leukocyte proteinases can contribute to physiologic processes occurring during wound healing, as well as their roles in pathologic processes.

Publication Types:

· Review

· Review, Tutorial

Eur Respir J 1999 Nov;14(5):1009-14

Bronchodilator responsiveness and serum total IgE levels in families of probands with severe early-onset COPD.

Celedon JC, Speizer FE, Drazen JM, Weiss ST, Campbell EJ, Carey VJ, Reilly JJ, Ginns L, Silverman EK.

Channing Laboratory, Dept of Medicine, Brigham and Women's Hospital, Boston, MA, USA.

Bronchodilator responsiveness has been associated with a subsequent accelerated decline in forced expiratory volume in one second (FEV1). Therefore, bronchodilator responsiveness and total serum immunoglobulin E(IgE) levels were assessed in 184 adult first-degree relatives of probands with severe early-onset chronic obstructive pulmonary disease (COPD) and a control group. Greater bronchodilator responsiveness was found among current smokers or exsmokers who were first-degree relatives of early-onset COPD probands than in currently or exsmoking controls, expressed as increase in FEV1 as a percentage of baseline (5.8+/-8.1 versus 2.9+/-5.1%, p<0.01), absolute increase in FEV1 from baseline (120+/-130 versus 60+/-110 mL, p<0.05), and increase in FEV1 as a percentage of the predicted value (3.6:4.1 versus 2.2+/-3.9%, p<0.05). However, elevated total serum IgE levels were not found in first-degree relatives of early-onset COPD probands compared with control subjects. The increased bronchodilator responsiveness among currently smoking/exsmoking first-degree relatives of early-onset COPD probands suggests that these individuals may have enhanced susceptibility to the detrimental effects of cigarette smoking.

Am J Respir Crit Care Med 1999 Dec;160(6):1968-75

Evidence for excessive bronchial inflammation during an acute exacerbation of chronic obstructive pulmonary disease in patients with alpha(1)-antitrypsin deficiency (PiZ).

Hill AT, Campbell EJ, Bayley DL, Hill SL, Stockley RA.

Department of Medicine, Queen Elizabeth Hospital, Edgbaston, Birmingham, United Kingdom.

Patients with homozygous (PiZ) alpha(1)-antitrypsin (AAT) deficiency have not only low baseline serum AAT levels (approximately 10 to 15% normal) but also an attenuated acute phase response. They are susceptible to the development of premature emphysema but may also be particularly susceptible to lung damage during bacterial exacerbations when there will be a significant neutrophil influx. The purposes of the present study were to assess the inflammatory nature of acute bacterial exacerbations of chronic obstructive pulmonary disease (COPD) in subjects with AAT deficiency, to compare this with COPD patients without deficiency, and to monitor the inflammatory process and its resolution following appropriate antibacterial therapy. At the start of the exacerbation, patients with AAT deficiency had lower sputum AAT (p < 0.001) and secretory leukoprotease inhibitor (SLPI; p = 0.02) with higher elastase activity (p = 0.02) compared with COPD patients without deficiency. Both groups had a comparable acute phase response as assessed by C-reactive protein (CRP) but the AAT-deficient patients had a minimal rise in serum AAT (to < 6 microM). After treatment with antibiotics, in patients with AAT deficiency, there were significant changes in many sputum proteins including a rise in SLPI levels, and a reduction in myeloperoxidase (MPO) and elastase activity (p < 0. 005 for all measures); the sputum chemoattractants interleukin-8 (IL-8) and leukotriene B(4) (LTB(4)) fell (p < 0.01), and protein leak (sputum/serum albumin ratio) became lower (p < 0.01). The changes were rapid and within 3 d of the commencement of antibiotic therapy the biochemical markers had decreased significantly, but took a variable time thereafter to return to baseline values. In conclusion, patients with AAT deficiency had evidence of increased elastase activity at the start of the exacerbation when compared with nondeficient COPD patients which probably reflects a deficient antiproteinase screen (lower sputum AAT and SLPI). The increased bronchial inflammation at presentation resolved rapidly with 14 d of antibiotic therapy.

J Lab Clin Med 1999 Oct;134(4):341-51

Extracellular proteolysis: new paradigms for an old paradox.

Owen CA, Campbell EJ.

Department of Internal Medicine, University of Utah School of Medicine, Salt Lake City, USA.

Publication Types:

· Review

· Review, Tutorial

Respir Med 1999 Jul;93(7):481-90

Chronic obstructive pulmonary disease, with and without alpha-1-antitrypsin deficiency: management practices in the U.K.

Hill AT, Campbell EJ, Ward AM, Stockley RA.

Department of Medicine, Queen Elizabeth Hospital, Birmingham, U.K.

Alpha-1-antitrypsin deficiency is a common genetic defect associated with the development of severe and rapidly progressive lung disease. This study was undertaken to determine whether respiratory physicians manage patients with alpha-1-antitrypsin (AAT) deficiency differently from patients with chronic obstructive pulmonary disease (COPD) without alpha-1-antitrypsin deficiency. In addition we obtained physicians' views on who should be tested for AAT deficiency. A questionnaire was administered to 88 respiratory physicians based throughout the U.K. (44 in teaching hospitals). The main outcome measures were pulmonary function tests, radiological assessment, frequency of repeat testing, follow-up and screening protocol for alpha-1-antitrypsin deficiency. Subjects with homozygous (PiZ) AAT deficiency were more likely to: 1. have baseline and full pulmonary function testing including dynamic flow rates, static lung volumes, and gas transfer; 2. have more comprehensive assessment with high resolution computed tomography (HRCT) thorax and repeated radiological assessment (with annual chest radiography); 3. be followed-up routinely; and 4. have family members tested for alpha-1-antitrypsin deficiency. Testing remains limited for AAT deficiency and is mainly restricted to young patients with COPD. COPD assessment and management is influenced by the presence of AAT deficiency, which may reflect the poorer prognosis of such patients due to more rapid decline. Assessment and monitoring could be simplified to forced expired manoeuvres, although limited HRCT thorax and tests of gas transfer may prove more sensitive to progression of emphysema. Testing for AAT deficiency in the U.K. remains restricted, which will influence the detection rate for AAT deficiency. A wider policy of testing was advocated by the WHO will detect more patients and also influence our understanding of the natural history of the condition.

J Clin Invest 1999 Aug;104(3):337-44

Quantum proteolysis by neutrophils: implications for pulmonary emphysema in alpha 1-antitrypsin deficiency.

Campbell EJ, Campbell MA, Boukedes SS, Owen CA.

Department of Internal Medicine, University of Utah Health Sciences Center, Salt Lake City 84132, USA.

Traditional enzyme kinetics provide a poor explanation for the increased risk of lung injury in alpha 1-antitrypsin (AAT) deficiency. Millimolar concentrations of leukocyte elastase, when released from single azurophil granules of activated neutrophils, lead to evanescent quantum bursts of proteolytic activity before catalysis is quenched by pericellular inhibitors. Herein, we tested the possibility that quantum proteolytic events are abnormal in AAT deficiency. We incubated neutrophils on opsonized fluoresceinated fibronectin in serum from individuals with various AAT phenotypes, and then measured and modeled quantum proteolytic events. The mean areas of the events in serum from heterozygous individuals (Pi MZ and Pi SZ) were slightly, but significantly, larger than those in serum from normal patients (Pi M). In marked contrast, mean areas of events in serum from AAT-deficient individuals were 10-fold larger than those in serum from normal patients. Diffusion modeling predicted that local elastase concentrations exceed AAT concentrations for less than 20 milliseconds and for more than 80 milliseconds in Pi M and Pi Z individuals, respectively. Thus, quantum proteolytic events are abnormally large and prolonged in AAT deficiency, leading directly to an increased risk of tissue injury in the immediate vicinity of activated neutrophils. These results have potentially important implications for the pathogenesis and prevention of lung disease in AAT deficiency.

J Leukoc Biol 1999 Feb;65(2):137-50

The cell biology of leukocyte-mediated proteolysis.

Owen CA, Campbell EJ.

Department of Internal Medicine, University of Utah School of Medicine, Salt Lake City, USA.

Leukocyte-derived proteinases have the capacity to degrade every component of the extracellular matrix, and thereby play fundamental roles in physiological processes. However, if the activity of these proteinases is uncontrolled or dysregulated, they have the capacity to contribute to tissue injury that potentially affects every organ in the body. Although there is a substantial literature on structure and activity of these proteinases when they are free in solution, until recently there has been little information about the cell biology of proteinases and their inhibitors. Recent studies, however, have identified several mechanisms by which inflammatory cells can degrade extracellular proteins in a milieu that contains high-affinity proteinase inhibitors.

Publication Types:

Review

· Review, Tutorial

Biochim Biophys Acta 1999 Mar 19;1430(2):179-90

Interaction between leukocyte elastase and elastin: quantitative and catalytic analyses.

Morrison HM, Welgus HG, Owen CA, Stockley RA, Campbell EJ.

Department of Internal Medicine, Respiratory and Critical Care, Jewish Hospital at Washington University Medical Center, St. Louis, MO 63110, USA.

Solubilization of elastin by human leukocyte elastase (HLE) cannot be analyzed by conventional kinetic methods because the biologically relevant substrate is insoluble and the concentration of enzyme-substrate complex has no physical meaning. We now report quantitative measurements of the binding and catalytic interaction between HLE and elastin permitted by analogy to receptor-ligand systems. Our results indicated that a limited and relatively constant number of enzyme binding sites were available on elastin, and that new sites became accessible as catalysis proceeded. The activation energies and solvent deuterium isotope effects were similar for catalysis of elastin and a soluble peptide substrate by HLE, yet the turnover number for HLE digestion of elastin was 200-2000-fold lower than that of HLE acting on soluble peptide substrates. Analysis of the binding of HLE to elastin at 0 degrees C, in the absence of significant catalytic activity, demonstrated two classes of binding sites (Kd=9.3x10(-9) M and 2.5x10(-7) M). The higher affinity sites accounted for only 6% of the total HLE binding capacity, but essentially all of the catalytic activity, and dissociation of HLE from these sites was minimal. Our studies suggest that interaction of HLE with elastin in vivo may be very persistent and permit progressive solubilization of this structurally important extracellular matrix component.

Am J Respir Crit Care Med 1998 Jun;157(6 Pt 1):1770-8

Genetic epidemiology of severe, early-onset chronic obstructive pulmonary disease. Risk to relatives for airflow obstruction and chronic bronchitis.

Silverman EK, Chapman HA, Drazen JM, Weiss ST, Rosner B, Campbell EJ, O'DONNELL WJ, Reilly JJ, Ginns L, Mentzer S, Wain J, Speizer FE.

Channing Laboratory, Brigham and Women's Hospital, Boston, MA 02115, USA.

Severe alpha-1-antitrypsin deficiency is the only proven genetic risk factor for chronic obstructive pulmonary disease (COPD). We have assembled a cohort of 44 probands with severe, early-onset COPD, who do not have severe alpha-1-antitrypsin deficiency. A surprisingly high prevalence of females (79.6%) was found. Assessment of the risk to relatives of these early-onset COPD probands for airflow obstruction and chronic bronchitis was performed to determine whether significant familial aggregation for COPD, independent of alpha-1-antitrypsin deficiency, could be demonstrated. First- degree relatives of early-onset COPD probands had significantly lower FEV1 and FEV1/FVC values than control subjects (p < 0.01), despite similar pack-years of smoking. Reduced spirometric values in first-degree relatives of early-onset COPD probands were found only in current or ex-cigarette smokers. The mean FEV1 in current or ex-smoking first-degree relatives was 76.1 +/- 20.9% predicted compared to 89.2 +/- 14.4% predicted in current or ex-smoking control subjects (p < 0.01); in lifelong nonsmokers, the mean FEV1 was 93.4% predicted for both control subjects and first-degree relatives of early-onset COPD probands. Generalized estimating equations, adjusting for age and pack-years of smoking, demonstrated increased odds of reduced FEV1 and chronic bronchitis in current or ex-smoking first-degree relatives of early-onset COPD probands. Using a new method to estimate relative risk from relative odds, we estimate that the relative risks for FEV1 below 60%, FEV1 below 80%, and chronic bronchitis are each approximately three in current or ex-smoking first-degree relatives of early-onset COPD probands. The increased risk to relatives of early-onset COPD probands for reduced FEV1 and chronic bronchitis, limited to current or ex-smokers, suggests genetic risk factor(s) for COPD that are expressed in response to cigarette smoking.

J Immunol 1998 Feb 1;160(3):1436-43

Angiotensin II generation at the cell surface of activated neutrophils: novel cathepsin G-mediated catalytic activity that is resistant to inhibition.

Owen CA, Campbell EJ.

Department of Internal Medicine, University of Utah Health Sciences Center, Salt Lake City 84132, USA.

Human neutrophils express inducible, catalytically active cathepsin G on their cell surface. Herein, we report that membrane-bound cathepsin G on intact neutrophils has potent angiotensin II-generating activity. Membrane-bound cathepsin G on activated neutrophils 1) converts both human angiotensin I and angiotensinogen to angiotensin II; 2) expresses angiotensin II-generating activity equivalent to 8.6 +/- 2.3 (+/-SD) x 10(-18) mol of free cathepsin G (5.2 +/- 1.4 x 10(6) molecules)/cell; and 3) has similar high affinity for angiotensin I compared with free cathepsin G (Km = 5.9 x 10(-4) and 4.6 x 10(-4) M; k(cat) = 4.0 and 2.0/s, respectively). In marked contrast to soluble cathepsin G, membrane-bound enzyme was substantially resistant to inhibition by plasma proteinase inhibitors and converted angiotensin I to angiotensin II even in undiluted plasma. There was a striking inverse relationship between inhibitor size and its effectiveness against membrane-bound cathepsin G activity. Alpha1-antichymotrypsin was a markedly ineffective inhibitor of membrane-bound enzyme (IC50 = 2.18 microM and 1.38 nM when tested against 1 nM membrane-bound and free cathepsin G, respectively). These data indicate that membrane-bound cathepsin G expressed on neutrophils is an inducible and mobile angiotensin II-generating system that may exert potent local vasoactive and chemoattractant properties at sites of inflammation.

Am J Physiol 1997 Mar;272(3 Pt 1):L385-93

Cytokines regulate membrane-bound leukocyte elastase on neutrophils: a novel mechanism for effector activity.

Owen CA, Campbell MA, Boukedes SS, Campbell EJ.

Department of Medicine, University of Utah Health Sciences Center, Salt Lake City 84132, USA.

Membrane-bound leukocyte elastase activity on neutrophils may have potent proinflammatory effects. Herein, we report the effects of tumor necrosis factor-alpha (TNF-alpha), platelet-activating factor (PAF), N-formyl-leucyl-methionyl-phenylalanine (fMLP), and interleukin-8 (IL-8) on membrane-bound elastase expression. TNF-alpha or PAF alone induced only approximately two- to threefold increases in membrane-bound elastase but exhibited marked dose- and time-dependent priming effects for subsequent stimulation with fMLP or IL-8 (up to 20-fold increases in membrane-bound human leukocyte elastase compared with unstimulated cells). Optimally PAF-primed and fMLP-stimulated cells expressed 1.105 +/- 0.25 (SD) x 10(-17) mol [6.65 +/- 1.51 (SD) x 10(6) molecules] membrane-bound elastase activity/cell or approximately 12% of the content of unstimulated cells. Elastase binds to the cell surface by a charge-dependent mechanism since 1) incubation of cells with cationic molecules abrogated agonist-induced upregulation of membrane-bound elastase and 2) elastase was progressively eluted from the cell surface by solutions with increasing ionic strength. Thus interactions between proinflammatory mediators strikingly upregulate membrane-bound elastase on neutrophils, which may promote inflammatory responses and/or contribute to tissue injury.

J Immunol 1996 Sep 15;157(6):2624-31

Quantum proteolysis resulting from release of single granules by human neutrophils: a novel, nonoxidative mechanism of extracellular proteolytic activity.

Liou TG, Campbell EJ.

Department of Medicine, University of Utah Health Sciences Center, Salt Lake City 84132, USA.

Proteinase inhibitors confine the activity of proteolytic enzymes of inflammatory cells, but fail to protect substrates in the immediate pericellular zone. We report quantitative imaging that demonstrates discrete, evanescent, quantized proteolytic events attributable to the release of single azurophil granules from neutrophils. The images provide information about the dynamics of this nonequilibrium system, which is characterized by overwhelmingly high local concentrations of enzymes that rapidly dissipate. With physiologic concentrations of extracellular human leukocyte elastase inhibitors (32.8 microM), the radii of the unit proteolytic events are 1.32 microm (approximately 8 times the radius of the azurophil granule) and are inversely and nonlinearly related to the concentration of proteinase inhibitor that is present in the bathing medium. We have obtained identical results with alpha1-antitrypsin, alpha2m, recombinant secretory leukocyte proteinase inhibitor, and ICI 200,355, and we have found that phagocyte-derived oxidants are not required for the genesis of this catalytic activity. Our results reveal that the enzyme:inhibitor ratio is the primary delimiter of quantized proteolysis in the local microenvironment.