0894-1939/91 $3.00 t .OO Copyright 8 1991 Taylor & Francis
.lmirna/ iflnw\ti,rutiw .Siir,wri,. Volume 4. pp. 423-430 Printed in the CIK All nghts reserved.
Topics and Trends in Surgical Research
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Neutrophil Chemotaxis and Neutrophil Elastase in the Aortic Wall in Patients with Abdominal Aortic Aneurysms JON R. COHEN, M D LEO KEEGAN, M D ISAAC SARFATI, PhD DEBRA DANNA CARL ILARDI, M D LESLIE WISE, M D Departments of Surgery and Pathology Long Island Jewish Medical Center New Hyde Park, NY 11042 Albert Einstein School of Medicine New York, NY I 1042 To test the hypothesis that elastin-derivedpeptides (EDP),fromhuman aortic tissue may he chemotacticfor inflammatorv cells, we studied the chemotuxis of neictrophils and monocytes to EDP derived,from abdominal aortic aneurysm (AAA), aortic occlusive disease (AOD), and control aortas. In addition, we determined ifneictrophils deliver neutrophil elastase to the aorta in vivo h.v staining for neiitrophil elastase (NE) throughout the course of abdominal aortic aneurysms with the monoclonal antibody to human NE. EDP Jrom AAA. AOD, and control tissue demonstrated signljicant chemotactic activityfor both neutrophils and monocytes. AN nrutrophils had u greater attraction to EDPfiom AAA tissue compared to ilOD and control aorta. Neutrophils .from AAA patients were more attracted to EDP qf AAA tissue than were nrutrophils of AOD or control patients attracted to their respective aortic EDP. Neictrophil elastase stained positive in the adventitia and thrornhw throughout the course of the aneurysm, but was not Jbund in the intima, media, or plaque qf the aorta. Abstract
Keywords Neutrophil, elastase, aortic aneurysm.
Introduction Elastin-derived peptides (EDP) produced from human elastin are known to be chemotactic for inflammatory cells in vivo resulting in further elastin proteolysis. These Presented at the Fourth Annual Meeting of the Eastern Vascular Society, May 1990, Boston, MA. Address correspondence to Jon R. Cohen, MD, Department of Surgery, Long Island Jewish Medical Center, New Hyde Park, NY 11042.
423
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findings suggest that extracellular connective tissue macromolecules and their proteolytic fragments (EDP) may play a significant role in directing cell movement into tissues during injury, repair, and turn over. Patients with abdominal aortic aneurysms (AAA) have increased smooth muscle cell elastase,’ increased elastase activity in the aneurysm all,^.^ increased neutrophil ela~tase,~ and increased circulating elastolytica c t i ~ i t y .However, ~.~ to date, the mechanism whereby neutrophil elastase might be delivered to the aorta is unknown. If neutrophil elastase is delivered to the aortic wall, then the questions arise as to whether it continues to be present in the aortic wall, whether it is equally distributed throughout the aorta and aneurysm, and at what specific site within the aortic wall it is localized. The purpose of this study was twofold: ( 1 ) to determine if aortic aneurysm tissue is more or less chemotactic for leukocytes compared with aortic occlusive disease tissue or normal aortic tissue; and (2) if neutrophil elastase is present, to determine its distribution within the aneurysm and specific localization within the aortic wall in patients with abdominal aortic aneurysms.
Materials and Methods Experimental Design Elastin-derived peptides prepared from the digestion of elastin were tested as the chemotactic agent for neutrophils and monocytes from the three different experimental groups as follows: ( 1 ) EDP from AAA tissue was tested against AAA neutrophils and monocytes, (2) EDP from aortic occlusive disease (AOD) patients was tested for chemotactic activity against neutrophils and monocytes from AOD patients, and (3) EDP from standard human elastin was tested for chemotactic activity against neutrophils and monocytes from five healthy human volunteers. The EDP was derived from elastin isolated from the aortic specimen obtained at the time of the surgery. Each patient had his or her own EDP tested to his or her own neutrophils and monocytes such that the five AAA patients and four aortic occlusive disease patients had their own neutrophils and monocytes tested against their own aortic tissue. Because aortic tissue cannot be obtained from healthy human controls, standard human elastin was used as the chemotactic agent when testing against the five healthy human neutrophils and monocytes. The chemotactic activity for each patient’s neutrophils and monocytes against their own aortic EDP was determined and the mean chemotactic activity for the five aneurysm patients was compared with the mean chemotactic activity of the four aortic occlusive disease patients and with the mean chemotactic activity of the control patients. Neutrophils and monocytes from each of the three groups were also tested against EDP from the other groups as follows: Neutrophils and monocytes from AOD and control patients were also tested for chemotaxis to the AAA EDP; the neutrophils and monocytes from AAA and AOD patients were tested for chemotaxis to control EDP; and neutrophils and monocytes from AAA and control patients were tested for chemotaxis to the AOD EDP. With three groups of neutrophils and monocytes and three groups of EDP, the nine possible combinations of comparing the chemotactic activity of neutrophil activity in each group with the different EDP tissue are listed in Table 1. The total neutro-
Neutrophil Chemotaxis and Elastase
425
phi1 chemotactic activity was defined as the sum of the neutrophil chemotactic activity of the three different groups against each type of EDP (column 1, Table 1). The value for the mean neutrophil chemotactic activity of each group was compared with each other group by using the Student t test. In the second part of the study, the monoclonal antibody to neutrophil elastase was used to determine if neutrophil elastase is indeed present within abdominal aortic aneurysm tissue and to determine its distribution along the course of the aneurysm and its localization within the layers of the aortic wall.
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Methods
Preparation of EDP. Elastin peptides'.' were prepared by digestion of elastin with human neutrophil elastase: 500 pg elastin and 20 pL of 1-pg/pL elastase solution in PBS, pH 7.5, were placed in an Eppendorf tube and incubated at 37 "C for 24 h. The elastase was eliminated by affinity chromatography through an aprotinin-agarose column equilibrated with 0.05 M Tris buffer in saline, pH 8.0. The column was regenerated by eluting the elastase with 0.05 M acetate buffer in saline, pH 5.0.' Elastin was isolated from aortic specimens obtained during surgery. It was extracted essentially according to the Lansing pro~edure.'.'~After the adventitia was removed, the remaining tissue was defatted by refluxing in ethanol for 1 h and in acetone for another hour. The defatted tissue was then homogenized and digested with 0.1N NaOH for 1 h at 92 "C. The digest was dialyzed at 4 "C against deionized water (changed twice) for 24 h and lyophilized. Commercially procured human aortic elastin (Elastin Products Company, St Louis, MO) was used as control.
Isolation of Test Cells. Cells were harvested from heparinized peripheral venous blood after gradient Centrifugation": In a 50-mL conical centrifuge tube, 10 mL Histopaque 1077 was carefully placed over an equal volume of Histopaque 1 1 19. Blood, 10 mL diluted one to one with saline, was in turn placed over this. After a 30-min centrifugation at 700g monocytes were collected from the layer between Histopaque 1077 and plasma, neutrophils between Histopaque 1 1 19 and Histopaque 1077. The cells were washed once and centrifuged at 700g for 5 min. If excessive numbers of red blood cells contaminated the neutrophil pellet, the pellet was resuspended in ice-cold lysing solution (NH,Cl, 8.29 g; KHC03, 1.O g; Na,EDTA, 0.372 g; brought up to 1 L and pH 7.2-7.3)'* and recentrifuged immediately for 10 min. After washing once again, the cells were resuspended in saline, counted, centrifuged once more, and finally suspended in the appropriate volume of MEM to make suspensions of 2.5 X lo6 monocytes/mL and 1.5 X lo6 neutrophils/mL. Measurement of Chemotactic Activity. The wells of the chemotactic chamber were filled with EDP solution, 100 pg/mL, or FMLP solution, M (standard) or MEM (blank). A double-membrane technique was used. The lower cellulose ester membrane had a pore size of 0.45 pm. The upper polycarbonate membrane had a pore size of 2 pm for neutrophils and 5 pm for monocytes. In the upper compartment 0.35 mL of cell suspension was added. After incubation, 60 min for monocytes and 120 min for neutrophils at room ambient, the chambers were taken apart and the membrane pairs were removed, stained with hematoxylin, coded, randomized, and then read without reference to the code.13Chemotaxis was quantified as the number of cells at the membrane interface within an eyepiece grid under high dry magnification
426
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(400X). Five random grids were counted for each membrane pair, and the results were expressed as the mean number of cells per grid for each set of triplicates corrected for random cell migration as measured from chambers having only medium in the lower compartment.
Stuining Procediires Paraffin sections were prepared of biopsy specimens taken from five AAA patients who had tube grafts placed. The specimens were taken from four locations:just proximal to the aneurysm (proximal anastomosis), the right aneurysm wall, the left aneurysm wall, and the aortic bifurcation (distal anastomosis). Immunostaining for elastase was done with mouse monoclonal anti-human neutrophil elastase as a primary antibody. Deparaffinized sections of aortic tissue were bathed consecutively in a 1:100 dilution of antibody, anti-mouse antibody, mouse APAAP, substrate solution. Finally, they were counterstained with nonalcoholic Gill hematoxylin. Staining for elastin was done by the elastic Van Gieson procedure. Deparaffinized sections were hydrated to distilled water, stained for 30 min (10% alcoholic hematoxylin in 80%ethanol, I5 mL; absolute ethanol, 15 mL; Lugol’s iodine, 12 mL; 10%ferric chloride, 12 mL), decolorized with 2% ferric chloride, fixed for 1 min with 5% sodium thiosulfate, and counterstained with Van Gieson stain (97.5 mL saturated picric acid and 2.5 mL 1% acid fuchsin). Materials Phosphate-buffered saline (PBS), N-formyl-met-leu-phe (FMLP), aprotinin-agarose, Histopaque 1077, and Histopaque I 1 19 (ficoll-sodium diatrizoate mixtures ofdensities 1.077 and 1.1 19, respectively) were obtained from Sigma Chemical Company (St Louis, MO). Minimum Essential Medium (MEM) was prepared by mixing alphamodified Eagle’s minimum essential medium (Sigma) with 10% heat-inactivated fetal bovine serum (Sigma). Human neutrophil elastase (HNE) was obtained from Calbiochem (San Diego, CA); antibodies and alkaline phosphatase/anti-alkaline phosphatase (APAAP) kits from Dako Corporation (Santa Barbara, CA); and stains from Fisher Scientific (Springfield,NJ). All common reagents were analytical grade. Modified Boyden chambers were bought from Duremberg Company (Pasadena, CA); polycarbonate filters from Nucleopore Corporation (Pleasanton, CA); and Gelman Metricel (mixed cellulose esters) membranes from Baxter Scientific (Edison, NJ).
Results Elastin-derived peptides from all sources displayed significant chemotactic activity for both neutrophils and monocytes. Significant differences between the rate of chemotaxis were as follows ( P < .05, Student’s t test) (Table 1):( I ) The overall number of neutrophils attracted by AAA-EDP ( 141.4 neutrophils/high-power field) was greater than the overall number of neutrophils attracted by AOD-EDP ( 126.8 neutrophils/ high-power field) (Fig 1). (2) The overall number of neutrophils attracted by AAAEDP ( 14 1.4 neutrophils/high-power field) was greater than the overall number of neutrophils attracted by control EDP ( 1 I 1.2 neutrophils/high-power field) (Fig 1). (3) AAA-EDP attracted AAA neutrophils (56.2 neutrophils/high-power field) to a greater degree than they attracted control neutrophils (39.2 neutrophils/high-power field). (4) AAA-EDP attracted AAA neutrophils (56.2 neutrophils/high-power field)
Neutrophil Chemotaxis and Elastase
42 7
Table 1 Comparison of Chemotactic Activity of Neutrophils in Each Group with EDP Tissue
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AAA-EDP AOD-EDP Control-EDP
Total Neutrophils
AAA Neutrophils
AOD Neutrophils
141.4 (a) 126.8 (b) 1 1 I .2 (c)
56.2 L 14 (d) 46.2 L 10 41.2 k 20
46.0 10 42.8 I I (f) 38.8 f 8
* *
Control Neutrophils 39.2 f 4 (e) 37.2 9 31.2 k 7 (g)
*
Nofe.Neutrophils/interface/high-powerfield (400X); mean of five random grids. Significant (P< .05, Student t test) observations were ( 1) total neutrophils attracted by AAA-EDP (a) was greater than total neutrophils attracted by AOD-EDP (b) and Control-EDP (c); (2) AAAEDP attracted AAA neutrophils (d) more than control neutrophils (e);(3)AAA-EDP attracted AAA neutrophils (d) greater than AOD-EDP attracted AOD neutrophils (f) or control-EDP attracted control neutrophils (g). Values are + standard deviation.
greater than AOD-EDP attracted AOD neutrophils (42.8 neutrophils/high-power field) (Fig 2). (5) AAA-EDP attracted AAA neutrophils (56.2 neutrophils/high-power field)to a greater degree than control EDP attracted control neutrophils (3 1.2 neutrophils/high-power field) (Fig 2). Monocytes were chemotactic to all three types of EDP, but were not different in their rates of chemotaxis to any of the three different types of aortic tissue. The tissue sections of all five patients at all four biopsy sites except one (patient 4, left lateral wall, Table 2) stained positive for the monoclonal antibody to human neutrophil elastase within neutrophils in the adventitia and thrombus (Table 2). However, neutrophil elastase was not detected within the intima, media, or plaque except for an occasional neutrophil that stained positive in the vasa vasorum that extended into the media (Table 2). No neutrophil elastase stained positive outside of neutrophils and no neutrophil elastase was found in association with the fragmentation of elastin seen in the media (Table 2). NEUTllOPtllLS / t1.P. FIELD
IGO
I
I
i
1 4 0 ~-
-!
.-.I
1 2 0 -100
I
. -
a0 1
--
0
1
. I
1AAA-EDP
AOD-EDP
Figure 1. Total neutrophils versus aortic EDP. All neutrophils had a greater attraction for AAA elastin-derived peptides (EDP) than for aortic occlusive disease (AOD) or control EDP.
J. R. Cohen et al.
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428
AAA-EDP W I
AOD-EDP T
H
CONTROL-EDP
N E U T I I O I ’ I I I L S
Figure 2. Neutrophil chemotaxis: EDP in each group. AAA neutrophils had a greater attraction to AAA elastin-derived peptides (EDP) than aortic occlusive disease (AOD) neutrophils or control neutrophils had for their respective EDP.
Discussion The data from this study indicate that neutrophils from patients with abdominal aortic aneurysms have a greater affinity for aneurysmal elastin-derived peptides than neutrophils from aortic occlusive disease patients or control neutrophils have for their respective elastin-derived peptides (Fig 2). In addition, all types of neutrophils had a greater attraction to the elastin-derived peptides of aneurysmal tissue than to elastin-derived peptides from aortic occlusive disease or control aortas (Fig 1). The data also indicate that neutrophils and monocytes are highly chemotactic to elastinderived peptides from human aortic elastin, confirming previous observations. Monocytes, although chemotactic to all elastin-derived peptides from the different tissues, did not behave differently in their chemotaxis to the three different types of aortic tissue study. The major difference between the aortic tissue of patients with aneurysms compared with the aortic tissue of aortic occlusive disease and control patients is the disorganization and fragmentation of the elastin within the media. I4 We speculate Table 2 Neutrophil Elastase Monoclonal Antibody Positive Staining in the Aortic Wall of AAA Patients
PROX DISTAL RLW LLW
Adventitia
Media
Intima
Plaque
Thrombus
515 515 515 415
015 115 1/ 5 015
015 015
1/ 5 015 015 015
115 215 215 215
015 015
Note. n = 5 patients, number of positive stains of a possible five patients. PROX, proximal extension of AAA; DISTAL, distal extension of AAA; RLW, right lateral wall of AAA; LLW, left lateral wall of AAA.
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429
that it is this very difference that makes the aneurysmal tissue more chemotactic for neutrophils compared with the others. The elastin-derived peptides are based on a fixed dry weight of elastin (500 mg) from each of the different experimental groups. It is therefore possible that, because aneurysmal tissue elastin is already fragmented and disorganized, digestion by elastase to yield the EDP may be different or more complete compared with aortic occlusive disease or control tissue. It is possible, however, that some other chemotactic factor that co-purifies with the EDP is responsible for the increased chemotaxis. If, indeed, the chemotactic agent is EDP, then the continuous exposure of elastin fragments to the circulation from aneurysmal tissue in vivo may act as a strong chemotactic stimulus compared with the aortic tissue of patients with aortic occlusive disease. The difference in the behavior of neutrophils and their attraction to elastin fragments of aneurysmal tissue provides a mechanism whereby neutrophil elastase could be delivered to the aorta. Because patients with abdominal aortic aneurysms have increased neutrophil elastase in their circulating neutrophils,’ these elastase-laden neutrophils would deliver more proteolytic enzymes more often than in patients with aortic occlusive disease. Patients with atherosclerosis have continuing destruction and repair of their aortic tissue. This continuing turnover and production of proteolytic fragments and elastin peptides results in the recruitment of several types of cells, including neutrophils. The presence of neutrophil elastase along the course of the infrarenal aortic aneurysm indicates that neutrophils are present but have no specific predilection for different sites. The absence of neutrophil elastase in the media in areas of elastin disorganization and fragmentation, seen commonly in the aortic wall of patients with abdominal aortic aneurysm^,'^ is interesting. One can speculate that aortic smooth muscle elastase, which is also elevated in patients with aortic aneurysms, may be the primary enzyme responsible for elastin degradation and fragmentation within the media because neutrophil elastase is conspicuously absent. In fact, identification of the properties of the elastase found in AAA tissue indicates that smooth muscle cell elastase may play the major role.2 Based on the above observations, we propose that, during the atherosclerotic process of aortic wall breakdown and repair, an increased amount of proteolytic enzymes are delivered to the aorta in aneurysm patients by elastase-laden neutrophils. The increased amount of proteolytic enzymes delivered by these neutrophils, combined with increases in local smooth muscle cell elastase and increased serum proteolytic activity, all contribute to a chronic increase in aortic elastin breakdown, weakening of the aortic wall, and aneurysmal dilation. This hypothesis suggests that abdominal aortic aneurysms are indeed a variant of atherosclerosis that could occur only if atherosclerosisis present and would account for the fact that abdominal aortic aneurysms always coexist with aortic atherosclerosis.
References 1. Senior RM, Griffin GL, Mecham RP. Chemotactic activity of elastin derived peptides. J
Clin Invert. 1980;66:859-862. 2. Cohen JR, Mandell C, Wise L. Characterization of human aortic elastase found in patients with abdominal aortic aneurysms. Surg Gyn Obstet. 1987;165:301-304. 3. Cohen JR, Mandell C, Chang J, Wise L. Elastin metabolism ofthe infrarenal aorta. J Vusc Surg 1988;7:210-214.
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4. Campa JS, Greenhalgh RM, Powell JT. Elastin degradation in abdominal aortic aneurysms. Atherosclerosis. 1987;65:13-2 1. 5. Cannon DJ, Read RC. Blood elastolytic activity in patients with aortic aneurysm. Ann Thorac Surg. 1982;34:10-15. 6 . Busutil RW, Rinderbreicht H, Flesher A, Carmack C. Elastase activity: the role of elastase in aortic aneurysm formation. Research. 1982;32:214-2 17. 7. Neuman RE, Ainsworth AL. A simplified procedure for the assay of leukocyte chemotaxis. J Reticulo Endothel SOC.1980;28:305-312. 8. Martodam RR, Baugh RJ, Twumasi DY, Liener IE. A rapid procedure for the large scale purification of elastase and cathepsin G from human sputum. Prep Biochem. 1979;9:15-3 I. 9. Lansing AI, Rosenthal TB, Alex M, Dempsey EW. The structure and chemical characterization of elastic fibers as revealed by elastase and by electron microscopy. Anat Rec. 1952;114:555-575. 10. Giro MG, Davison JM. Elastin. Mefhods Enzymol. 1988;163:656-673. 1 1. Boyum A. Isolation of mononuclear cells and granulocytes from human blood. Scand J Clin Cab Invest. 1968;21(Suppl 97):77-89. 12. English D,Andersen BR. Singlestep separation of red blood cells: granulocytes and mononuclear leukocytes on discontinuous density gradient of ficoll-hypaque. J Immitnol Methods. 1974;5:249. 13. Campbell PB.An improved method for the in vitro evaluation of monocytes leukotaxis. J Lab CIin Med. 1977;90:381-388. 14. Tilson MD. Histochemistry of aortic elastin in patients with nonspecific abdominal aortic aneurysmal disease. Arch Surg. 1988;123:503-505.
.lmirna/ iflnw\ti,rutiw .Siir,wri,. Volume 4. pp. 423-430 Printed in the CIK All nghts reserved.
Topics and Trends in Surgical Research
J Invest Surg Downloaded from informahealthcare.com by Osaka University on 12/01/14 For personal use only.
Neutrophil Chemotaxis and Neutrophil Elastase in the Aortic Wall in Patients with Abdominal Aortic Aneurysms JON R. COHEN, M D LEO KEEGAN, M D ISAAC SARFATI, PhD DEBRA DANNA CARL ILARDI, M D LESLIE WISE, M D Departments of Surgery and Pathology Long Island Jewish Medical Center New Hyde Park, NY 11042 Albert Einstein School of Medicine New York, NY I 1042 To test the hypothesis that elastin-derivedpeptides (EDP),fromhuman aortic tissue may he chemotacticfor inflammatorv cells, we studied the chemotuxis of neictrophils and monocytes to EDP derived,from abdominal aortic aneurysm (AAA), aortic occlusive disease (AOD), and control aortas. In addition, we determined ifneictrophils deliver neutrophil elastase to the aorta in vivo h.v staining for neiitrophil elastase (NE) throughout the course of abdominal aortic aneurysms with the monoclonal antibody to human NE. EDP Jrom AAA. AOD, and control tissue demonstrated signljicant chemotactic activityfor both neutrophils and monocytes. AN nrutrophils had u greater attraction to EDPfiom AAA tissue compared to ilOD and control aorta. Neutrophils .from AAA patients were more attracted to EDP qf AAA tissue than were nrutrophils of AOD or control patients attracted to their respective aortic EDP. Neictrophil elastase stained positive in the adventitia and thrornhw throughout the course of the aneurysm, but was not Jbund in the intima, media, or plaque qf the aorta. Abstract
Keywords Neutrophil, elastase, aortic aneurysm.
Introduction Elastin-derived peptides (EDP) produced from human elastin are known to be chemotactic for inflammatory cells in vivo resulting in further elastin proteolysis. These Presented at the Fourth Annual Meeting of the Eastern Vascular Society, May 1990, Boston, MA. Address correspondence to Jon R. Cohen, MD, Department of Surgery, Long Island Jewish Medical Center, New Hyde Park, NY 11042.
423
J Invest Surg Downloaded from informahealthcare.com by Osaka University on 12/01/14 For personal use only.
424
J . R. Cohm et al.
findings suggest that extracellular connective tissue macromolecules and their proteolytic fragments (EDP) may play a significant role in directing cell movement into tissues during injury, repair, and turn over. Patients with abdominal aortic aneurysms (AAA) have increased smooth muscle cell elastase,’ increased elastase activity in the aneurysm all,^.^ increased neutrophil ela~tase,~ and increased circulating elastolytica c t i ~ i t y .However, ~.~ to date, the mechanism whereby neutrophil elastase might be delivered to the aorta is unknown. If neutrophil elastase is delivered to the aortic wall, then the questions arise as to whether it continues to be present in the aortic wall, whether it is equally distributed throughout the aorta and aneurysm, and at what specific site within the aortic wall it is localized. The purpose of this study was twofold: ( 1 ) to determine if aortic aneurysm tissue is more or less chemotactic for leukocytes compared with aortic occlusive disease tissue or normal aortic tissue; and (2) if neutrophil elastase is present, to determine its distribution within the aneurysm and specific localization within the aortic wall in patients with abdominal aortic aneurysms.
Materials and Methods Experimental Design Elastin-derived peptides prepared from the digestion of elastin were tested as the chemotactic agent for neutrophils and monocytes from the three different experimental groups as follows: ( 1 ) EDP from AAA tissue was tested against AAA neutrophils and monocytes, (2) EDP from aortic occlusive disease (AOD) patients was tested for chemotactic activity against neutrophils and monocytes from AOD patients, and (3) EDP from standard human elastin was tested for chemotactic activity against neutrophils and monocytes from five healthy human volunteers. The EDP was derived from elastin isolated from the aortic specimen obtained at the time of the surgery. Each patient had his or her own EDP tested to his or her own neutrophils and monocytes such that the five AAA patients and four aortic occlusive disease patients had their own neutrophils and monocytes tested against their own aortic tissue. Because aortic tissue cannot be obtained from healthy human controls, standard human elastin was used as the chemotactic agent when testing against the five healthy human neutrophils and monocytes. The chemotactic activity for each patient’s neutrophils and monocytes against their own aortic EDP was determined and the mean chemotactic activity for the five aneurysm patients was compared with the mean chemotactic activity of the four aortic occlusive disease patients and with the mean chemotactic activity of the control patients. Neutrophils and monocytes from each of the three groups were also tested against EDP from the other groups as follows: Neutrophils and monocytes from AOD and control patients were also tested for chemotaxis to the AAA EDP; the neutrophils and monocytes from AAA and AOD patients were tested for chemotaxis to control EDP; and neutrophils and monocytes from AAA and control patients were tested for chemotaxis to the AOD EDP. With three groups of neutrophils and monocytes and three groups of EDP, the nine possible combinations of comparing the chemotactic activity of neutrophil activity in each group with the different EDP tissue are listed in Table 1. The total neutro-
Neutrophil Chemotaxis and Elastase
425
phi1 chemotactic activity was defined as the sum of the neutrophil chemotactic activity of the three different groups against each type of EDP (column 1, Table 1). The value for the mean neutrophil chemotactic activity of each group was compared with each other group by using the Student t test. In the second part of the study, the monoclonal antibody to neutrophil elastase was used to determine if neutrophil elastase is indeed present within abdominal aortic aneurysm tissue and to determine its distribution along the course of the aneurysm and its localization within the layers of the aortic wall.
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Methods
Preparation of EDP. Elastin peptides'.' were prepared by digestion of elastin with human neutrophil elastase: 500 pg elastin and 20 pL of 1-pg/pL elastase solution in PBS, pH 7.5, were placed in an Eppendorf tube and incubated at 37 "C for 24 h. The elastase was eliminated by affinity chromatography through an aprotinin-agarose column equilibrated with 0.05 M Tris buffer in saline, pH 8.0. The column was regenerated by eluting the elastase with 0.05 M acetate buffer in saline, pH 5.0.' Elastin was isolated from aortic specimens obtained during surgery. It was extracted essentially according to the Lansing pro~edure.'.'~After the adventitia was removed, the remaining tissue was defatted by refluxing in ethanol for 1 h and in acetone for another hour. The defatted tissue was then homogenized and digested with 0.1N NaOH for 1 h at 92 "C. The digest was dialyzed at 4 "C against deionized water (changed twice) for 24 h and lyophilized. Commercially procured human aortic elastin (Elastin Products Company, St Louis, MO) was used as control.
Isolation of Test Cells. Cells were harvested from heparinized peripheral venous blood after gradient Centrifugation": In a 50-mL conical centrifuge tube, 10 mL Histopaque 1077 was carefully placed over an equal volume of Histopaque 1 1 19. Blood, 10 mL diluted one to one with saline, was in turn placed over this. After a 30-min centrifugation at 700g monocytes were collected from the layer between Histopaque 1077 and plasma, neutrophils between Histopaque 1 1 19 and Histopaque 1077. The cells were washed once and centrifuged at 700g for 5 min. If excessive numbers of red blood cells contaminated the neutrophil pellet, the pellet was resuspended in ice-cold lysing solution (NH,Cl, 8.29 g; KHC03, 1.O g; Na,EDTA, 0.372 g; brought up to 1 L and pH 7.2-7.3)'* and recentrifuged immediately for 10 min. After washing once again, the cells were resuspended in saline, counted, centrifuged once more, and finally suspended in the appropriate volume of MEM to make suspensions of 2.5 X lo6 monocytes/mL and 1.5 X lo6 neutrophils/mL. Measurement of Chemotactic Activity. The wells of the chemotactic chamber were filled with EDP solution, 100 pg/mL, or FMLP solution, M (standard) or MEM (blank). A double-membrane technique was used. The lower cellulose ester membrane had a pore size of 0.45 pm. The upper polycarbonate membrane had a pore size of 2 pm for neutrophils and 5 pm for monocytes. In the upper compartment 0.35 mL of cell suspension was added. After incubation, 60 min for monocytes and 120 min for neutrophils at room ambient, the chambers were taken apart and the membrane pairs were removed, stained with hematoxylin, coded, randomized, and then read without reference to the code.13Chemotaxis was quantified as the number of cells at the membrane interface within an eyepiece grid under high dry magnification
426
J. R. C‘ohen el a/.
J Invest Surg Downloaded from informahealthcare.com by Osaka University on 12/01/14 For personal use only.
(400X). Five random grids were counted for each membrane pair, and the results were expressed as the mean number of cells per grid for each set of triplicates corrected for random cell migration as measured from chambers having only medium in the lower compartment.
Stuining Procediires Paraffin sections were prepared of biopsy specimens taken from five AAA patients who had tube grafts placed. The specimens were taken from four locations:just proximal to the aneurysm (proximal anastomosis), the right aneurysm wall, the left aneurysm wall, and the aortic bifurcation (distal anastomosis). Immunostaining for elastase was done with mouse monoclonal anti-human neutrophil elastase as a primary antibody. Deparaffinized sections of aortic tissue were bathed consecutively in a 1:100 dilution of antibody, anti-mouse antibody, mouse APAAP, substrate solution. Finally, they were counterstained with nonalcoholic Gill hematoxylin. Staining for elastin was done by the elastic Van Gieson procedure. Deparaffinized sections were hydrated to distilled water, stained for 30 min (10% alcoholic hematoxylin in 80%ethanol, I5 mL; absolute ethanol, 15 mL; Lugol’s iodine, 12 mL; 10%ferric chloride, 12 mL), decolorized with 2% ferric chloride, fixed for 1 min with 5% sodium thiosulfate, and counterstained with Van Gieson stain (97.5 mL saturated picric acid and 2.5 mL 1% acid fuchsin). Materials Phosphate-buffered saline (PBS), N-formyl-met-leu-phe (FMLP), aprotinin-agarose, Histopaque 1077, and Histopaque I 1 19 (ficoll-sodium diatrizoate mixtures ofdensities 1.077 and 1.1 19, respectively) were obtained from Sigma Chemical Company (St Louis, MO). Minimum Essential Medium (MEM) was prepared by mixing alphamodified Eagle’s minimum essential medium (Sigma) with 10% heat-inactivated fetal bovine serum (Sigma). Human neutrophil elastase (HNE) was obtained from Calbiochem (San Diego, CA); antibodies and alkaline phosphatase/anti-alkaline phosphatase (APAAP) kits from Dako Corporation (Santa Barbara, CA); and stains from Fisher Scientific (Springfield,NJ). All common reagents were analytical grade. Modified Boyden chambers were bought from Duremberg Company (Pasadena, CA); polycarbonate filters from Nucleopore Corporation (Pleasanton, CA); and Gelman Metricel (mixed cellulose esters) membranes from Baxter Scientific (Edison, NJ).
Results Elastin-derived peptides from all sources displayed significant chemotactic activity for both neutrophils and monocytes. Significant differences between the rate of chemotaxis were as follows ( P < .05, Student’s t test) (Table 1):( I ) The overall number of neutrophils attracted by AAA-EDP ( 141.4 neutrophils/high-power field) was greater than the overall number of neutrophils attracted by AOD-EDP ( 126.8 neutrophils/ high-power field) (Fig 1). (2) The overall number of neutrophils attracted by AAAEDP ( 14 1.4 neutrophils/high-power field) was greater than the overall number of neutrophils attracted by control EDP ( 1 I 1.2 neutrophils/high-power field) (Fig 1). (3) AAA-EDP attracted AAA neutrophils (56.2 neutrophils/high-power field) to a greater degree than they attracted control neutrophils (39.2 neutrophils/high-power field). (4) AAA-EDP attracted AAA neutrophils (56.2 neutrophils/high-power field)
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Table 1 Comparison of Chemotactic Activity of Neutrophils in Each Group with EDP Tissue
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AAA-EDP AOD-EDP Control-EDP
Total Neutrophils
AAA Neutrophils
AOD Neutrophils
141.4 (a) 126.8 (b) 1 1 I .2 (c)
56.2 L 14 (d) 46.2 L 10 41.2 k 20
46.0 10 42.8 I I (f) 38.8 f 8
* *
Control Neutrophils 39.2 f 4 (e) 37.2 9 31.2 k 7 (g)
*
Nofe.Neutrophils/interface/high-powerfield (400X); mean of five random grids. Significant (P< .05, Student t test) observations were ( 1) total neutrophils attracted by AAA-EDP (a) was greater than total neutrophils attracted by AOD-EDP (b) and Control-EDP (c); (2) AAAEDP attracted AAA neutrophils (d) more than control neutrophils (e);(3)AAA-EDP attracted AAA neutrophils (d) greater than AOD-EDP attracted AOD neutrophils (f) or control-EDP attracted control neutrophils (g). Values are + standard deviation.
greater than AOD-EDP attracted AOD neutrophils (42.8 neutrophils/high-power field) (Fig 2). (5) AAA-EDP attracted AAA neutrophils (56.2 neutrophils/high-power field)to a greater degree than control EDP attracted control neutrophils (3 1.2 neutrophils/high-power field) (Fig 2). Monocytes were chemotactic to all three types of EDP, but were not different in their rates of chemotaxis to any of the three different types of aortic tissue. The tissue sections of all five patients at all four biopsy sites except one (patient 4, left lateral wall, Table 2) stained positive for the monoclonal antibody to human neutrophil elastase within neutrophils in the adventitia and thrombus (Table 2). However, neutrophil elastase was not detected within the intima, media, or plaque except for an occasional neutrophil that stained positive in the vasa vasorum that extended into the media (Table 2). No neutrophil elastase stained positive outside of neutrophils and no neutrophil elastase was found in association with the fragmentation of elastin seen in the media (Table 2). NEUTllOPtllLS / t1.P. FIELD
IGO
I
I
i
1 4 0 ~-
-!
.-.I
1 2 0 -100
I
. -
a0 1
--
0
1
. I
1AAA-EDP
AOD-EDP
Figure 1. Total neutrophils versus aortic EDP. All neutrophils had a greater attraction for AAA elastin-derived peptides (EDP) than for aortic occlusive disease (AOD) or control EDP.
J. R. Cohen et al.
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AAA-EDP W I
AOD-EDP T
H
CONTROL-EDP
N E U T I I O I ’ I I I L S
Figure 2. Neutrophil chemotaxis: EDP in each group. AAA neutrophils had a greater attraction to AAA elastin-derived peptides (EDP) than aortic occlusive disease (AOD) neutrophils or control neutrophils had for their respective EDP.
Discussion The data from this study indicate that neutrophils from patients with abdominal aortic aneurysms have a greater affinity for aneurysmal elastin-derived peptides than neutrophils from aortic occlusive disease patients or control neutrophils have for their respective elastin-derived peptides (Fig 2). In addition, all types of neutrophils had a greater attraction to the elastin-derived peptides of aneurysmal tissue than to elastin-derived peptides from aortic occlusive disease or control aortas (Fig 1). The data also indicate that neutrophils and monocytes are highly chemotactic to elastinderived peptides from human aortic elastin, confirming previous observations. Monocytes, although chemotactic to all elastin-derived peptides from the different tissues, did not behave differently in their chemotaxis to the three different types of aortic tissue study. The major difference between the aortic tissue of patients with aneurysms compared with the aortic tissue of aortic occlusive disease and control patients is the disorganization and fragmentation of the elastin within the media. I4 We speculate Table 2 Neutrophil Elastase Monoclonal Antibody Positive Staining in the Aortic Wall of AAA Patients
PROX DISTAL RLW LLW
Adventitia
Media
Intima
Plaque
Thrombus
515 515 515 415
015 115 1/ 5 015
015 015
1/ 5 015 015 015
115 215 215 215
015 015
Note. n = 5 patients, number of positive stains of a possible five patients. PROX, proximal extension of AAA; DISTAL, distal extension of AAA; RLW, right lateral wall of AAA; LLW, left lateral wall of AAA.
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that it is this very difference that makes the aneurysmal tissue more chemotactic for neutrophils compared with the others. The elastin-derived peptides are based on a fixed dry weight of elastin (500 mg) from each of the different experimental groups. It is therefore possible that, because aneurysmal tissue elastin is already fragmented and disorganized, digestion by elastase to yield the EDP may be different or more complete compared with aortic occlusive disease or control tissue. It is possible, however, that some other chemotactic factor that co-purifies with the EDP is responsible for the increased chemotaxis. If, indeed, the chemotactic agent is EDP, then the continuous exposure of elastin fragments to the circulation from aneurysmal tissue in vivo may act as a strong chemotactic stimulus compared with the aortic tissue of patients with aortic occlusive disease. The difference in the behavior of neutrophils and their attraction to elastin fragments of aneurysmal tissue provides a mechanism whereby neutrophil elastase could be delivered to the aorta. Because patients with abdominal aortic aneurysms have increased neutrophil elastase in their circulating neutrophils,’ these elastase-laden neutrophils would deliver more proteolytic enzymes more often than in patients with aortic occlusive disease. Patients with atherosclerosis have continuing destruction and repair of their aortic tissue. This continuing turnover and production of proteolytic fragments and elastin peptides results in the recruitment of several types of cells, including neutrophils. The presence of neutrophil elastase along the course of the infrarenal aortic aneurysm indicates that neutrophils are present but have no specific predilection for different sites. The absence of neutrophil elastase in the media in areas of elastin disorganization and fragmentation, seen commonly in the aortic wall of patients with abdominal aortic aneurysm^,'^ is interesting. One can speculate that aortic smooth muscle elastase, which is also elevated in patients with aortic aneurysms, may be the primary enzyme responsible for elastin degradation and fragmentation within the media because neutrophil elastase is conspicuously absent. In fact, identification of the properties of the elastase found in AAA tissue indicates that smooth muscle cell elastase may play the major role.2 Based on the above observations, we propose that, during the atherosclerotic process of aortic wall breakdown and repair, an increased amount of proteolytic enzymes are delivered to the aorta in aneurysm patients by elastase-laden neutrophils. The increased amount of proteolytic enzymes delivered by these neutrophils, combined with increases in local smooth muscle cell elastase and increased serum proteolytic activity, all contribute to a chronic increase in aortic elastin breakdown, weakening of the aortic wall, and aneurysmal dilation. This hypothesis suggests that abdominal aortic aneurysms are indeed a variant of atherosclerosis that could occur only if atherosclerosisis present and would account for the fact that abdominal aortic aneurysms always coexist with aortic atherosclerosis.
References 1. Senior RM, Griffin GL, Mecham RP. Chemotactic activity of elastin derived peptides. J
Clin Invert. 1980;66:859-862. 2. Cohen JR, Mandell C, Wise L. Characterization of human aortic elastase found in patients with abdominal aortic aneurysms. Surg Gyn Obstet. 1987;165:301-304. 3. Cohen JR, Mandell C, Chang J, Wise L. Elastin metabolism ofthe infrarenal aorta. J Vusc Surg 1988;7:210-214.
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4. Campa JS, Greenhalgh RM, Powell JT. Elastin degradation in abdominal aortic aneurysms. Atherosclerosis. 1987;65:13-2 1. 5. Cannon DJ, Read RC. Blood elastolytic activity in patients with aortic aneurysm. Ann Thorac Surg. 1982;34:10-15. 6 . Busutil RW, Rinderbreicht H, Flesher A, Carmack C. Elastase activity: the role of elastase in aortic aneurysm formation. Research. 1982;32:214-2 17. 7. Neuman RE, Ainsworth AL. A simplified procedure for the assay of leukocyte chemotaxis. J Reticulo Endothel SOC.1980;28:305-312. 8. Martodam RR, Baugh RJ, Twumasi DY, Liener IE. A rapid procedure for the large scale purification of elastase and cathepsin G from human sputum. Prep Biochem. 1979;9:15-3 I. 9. Lansing AI, Rosenthal TB, Alex M, Dempsey EW. The structure and chemical characterization of elastic fibers as revealed by elastase and by electron microscopy. Anat Rec. 1952;114:555-575. 10. Giro MG, Davison JM. Elastin. Mefhods Enzymol. 1988;163:656-673. 1 1. Boyum A. Isolation of mononuclear cells and granulocytes from human blood. Scand J Clin Cab Invest. 1968;21(Suppl 97):77-89. 12. English D,Andersen BR. Singlestep separation of red blood cells: granulocytes and mononuclear leukocytes on discontinuous density gradient of ficoll-hypaque. J Immitnol Methods. 1974;5:249. 13. Campbell PB.An improved method for the in vitro evaluation of monocytes leukotaxis. J Lab CIin Med. 1977;90:381-388. 14. Tilson MD. Histochemistry of aortic elastin in patients with nonspecific abdominal aortic aneurysmal disease. Arch Surg. 1988;123:503-505.
