The Calcium Messenger System and the Kinetics of Elastase Release from Human Neutrophils in Patients with Abdominal Aortic Aneurysms Jon R. Cohen, MD, Glenn Faust. MD, Noel Tcncnbaum, MD, Isaac Sarfati, PhD, ~ Paula Rogowsky, Leslie Wise, MD, Nett)H y d ~Ptrrk. N m York
Aortic elastase and neutrophil elastase is higher in patients with abdominal aortic aneurysms. The purpose of this study was to determine if these proteolytic elevations occur after abdominal aortic aneurysms have been repaired. Specifically, we studied the stimulation and inhibition of elastase degranulation from neutrophils in postoperative abdominal aortic aneurysm patients compared to aortic occlusive disease patients. Neutrophil elastase was determined in postoperative abdominal aortic aneurysm and aortic occlusive disease patients in response to calcium and the lonophore A23187. Inhibition of elastase release was determined with the calcium channel blocking agent Verapamil. Neutrophil elastase secretion was significantly higher in the abdominal aortic aneurysm patients (47%) versus aortic occlusive disease (20%) (p < .05), while the effect of Verapamil in blocking this response was significantly lower in the abdominal aortic aneurysm patients (14%) compared to aortic occlusive disease patients (27%) (p < .02). The time for degranulationto occur was longer in the abdominalaortic aneurysm patients (14.7 minutes) versus aortic occlusive disease patients (3.5 minutes), but the rate of secretion was not different between the two groups. These data indicate that, (1) neutrophils secrete more elastase in responseto a calcium stimulus in abdominal aortic aneurysm patients; (2) it takes longer to secrete the increased amount of elastase in abdominal aortic aneurysm patients since the rate of secretion is similar between the two groups; and (3) Verapamil blocks elastase secretion ineffectively in abdominal aortic aneurysm patients. We conclude that the proteolytic alterations in abdominal aortic aneurysm patients are more likely a primary event and not a response to the abdominal aortic aneurysm and that Verapamil is a poor drug to use to medically manipulate the proteasesystem in abdominalaortic aneurysm patients. (Ann Vasc Surg 1990;4:570-574). KEY WORDS: Elastase, aortic; elastase, neutrophil; Verapamil; calcium channel blockers; abdominal aortic aneurysms; aortic occlusive disease.
Abdominal aortic aneurysms (AAA) are usually described as variants of atherosclerosis which grow until From the Department of Sirrgery, Long Island Je\jish Medical Center and the Albert Einstein School o j Medicine, New Hyde Park, New York. Presented at the meeting of The Societv j b r Vascular Surgetyllnternutional Societv f o r Cardiovascirlar Sirrgery, June 21, 1989, New York, New York. Reprint requests: Jon R . Cohen, MD, Depnrtment of Surgery, Long Island Jewish Medical Center, New Hyde Park, New York 11042.
eventual rupture. However, recent clinical observations and biochemical data now suggest that aneurysmal pathogenesis may be much more complex than traditionally believed and more likely related to a connective tissue disorder with alterations in systemic elastin metabolism [ I]. Biochemical data that lends support to a systemic alteration in elastin metabolism in AAA patients includes increased leukocyte elastase [2], increased aortic elastase [ I ,3], increased pancreatic elastase [41, imbalance of elastase and alpha-l-antitrypsin in the aortic wall, and an increase in elastin urine
570
VOLUME4 No 6 - 1990
57 I
ELAS7ASE RELEASF; I N AAA.5
metabolites 151. Although both aortic elastase tissue levels and neutrophil elastase are higher in patients with AAAs, one can argue that the increased proteolytic activity is a response to the AAA and not the primary event. Further investigation into the mechanism of elastase release from lysosomes reveals that the translocation of calcium plays a predominant role in the enzyme release [6,7] (Fig. I ) . It is now well established that the calcium messenger system, by which extracellular messages regulate cell function. plays a major role in activating neutrophils [6]. In addition, the translocation of calcium by the lonophore A23 187 stimulates the release of neutrophil elastase [6,8]. The calcium channel blocker Verapamil in some cases is capable of suppressing this stimulation of elastase release 161. Using a model for the intracellular calcium stimulation of elastase release, the purpose of this study was to determine if the stimulation and inhibition of elastase degranulation from neutrophils in postoperative AAA patients is altered.
MATERIALS AND METHODS Neutrophil elastase degranulation and secretion is highly sensitive to and increases in response to elevations in intracellular calcium [6-81. Neutrophil elastase secretion was determined in patients with postoperative AAA and aortic occlusive disease in response to incubation with calcium and the Ionophore A23187. Eleven AAA patients and 10 aortic occlusive disease patients were studied. The mean postoperative period at the time of blood drawing for all patients was six months. Neutrophil preparation and elastase assay
Approximately l0cc of whole blood was obtained by venipuncture and mixed with lOcc of normal saline. Neutrophil cell suspensions were obtained from hypaque gradient centrifugation. Neutrophil permeabilization was achieved with 100 microliters of A23187 (200 micromolar) incubated at 37" centigrade. After incubation, the suspension was centrifuged at 17000 RPMs for five minutes. The pellets were washed with calcium free buffer and centrifuged twice. Right-angle light scattering
The rate of neutrophil elastase release was measured by right-angle light scattering. The latter is a function of granule circulation, and its decline mirrors the kinetics of elastase release [7,9,10]. Neutrophils were resuspended in buffer ( K C l , 100 mM; NaCl 20 mM: HEPES, 30 mM) to half the original
ca++
A23187
\
ELASTASE
Ca"
1
Fig. 1. Release of neutrophil elastase as a result of an increase in intracellular calcium. 1. lonophore A23187 stimulates calcium translocation into cell. 2. Endoplasmic reticulum produce secondary messengers inositol triphosphate and diacylglycerol. 3. Secondary messengers stimulate lysosomal release of elastase. 4. Elastase is released at cell membrane.
blood volume. Ca ionophore A23187 solution, 200 uM in dimethyl sulfoxide (DMSO), was added to make the concentration 5 mM, and the suspension was incubated with agitation at 37°C for 30 minutes. It was centrifuged at 700 x g for five minutes, the cells were washed twice with saline and finally suspended in buffer. Two ml of buffer were added to a cuvette and the cuvette was placed in the holder of a Turner spectrofluorometer (model #430) thermostated at 37°C. Excitation and emission wave lengths were both set at 380 nm. The fluorometer was hooked to a Gilford 6050 recorder. After a steady baseline (b,) was obtained, the cuvette was replaced by one containing the cell suspension and a new higher baseline (b,) was established. CaCI, was then added. Percent degranulation was calculated from the height of the plot (h) as 100 (b2-h)/(b2-b,)l. Elastase release
Elastase release was measured under various conditions and was calculated as (1) a fraction of total elastase contained in the cells: (2) a fraction of total protein: and (3) as the amount per million cells. The cells were resuspended in Hanks' balanced salt solution (HBSS) and counted. When necessary, the volume of the suspension was adjusted to make it in the order of 10' cellsiml. The suspension was aliquoted to five different tubes to which one of the following was added: Tube 1-50 pl HBSS: Tube 2-25 pl A23187 solution to make the final concentration 2 pM: Tube 3-Verapamil HCI to make the final concentration 50 pM: and Tube 4-A23 187 and
512
ANNALS OF VASCIJLAK SUKGEKY
ELASTASE RELEASE I N AAAs
Verapamil. The mixtures were agitated at 37°C for 30 minutes, then centrifuged, and the supernate was assayed for elastase activity. In Tube 5 the cells were incubated for 30 minutes with 20 mg NaCl and 25 p1 Triton X-100 per ml. This treatment caused complete disruption of the cells. After centrifugation, the supernate of this fifth tube was used to determine total elastase activity and total protein assays. The percent elastase release was calculated for A23187 alone (Tube 2iTube 5 ) , Verapamil alone (Tube 3/Tube 5 ) and A23187 with Verapamil (Tube 4/Tube 5). Elastase activity was measured by a variation of previously described techniques [ 1 1 ,I 21, using N-methoxysuccinyl-ala-ala-Lpro-L-valp-nitroanilaide as a substrate: 100 pl of sample and 25 pI of substrate solution (25 mM in DMSO) were added to a cuvette containing 1.5 ml of 0.2 M Tris buffer, pH 8.0. The release of p-nitroaniline was monitored by readings of the optical density (O.D.) at 410 nm with a Gilford Stasar 11 spectrophotometer. The slope of the regression line, O.D. vs. time, was taken as the measure of elastase activity. Total protein was determined by the Lowery method [6]: 5 ml Cu tartrate reagent (50 ml of 2% Na2C0, in 0. I M NaOH, 1 ml of 1% Na tartrate and 1 ml of 0.5% CuSO,), are mixed with 0.1 ml sample. After 10 minutes, 0.5 ml Folin-Ciocalteu’s reagent (diluted 35 to 100) was added. After 30 minutes the O.D. is measured at 700 nm.
Statistical methods
The mean values of both groups of patients were compared using the Student’s t-test. The Student’s t-test was also applied to compare the groups for the time to degranulation (minutes) and for the rate of secretion (ng/min).
8 OCCLUSIVE (N=IO)
=
AAA (N=II)
Fig. 2. Percent of elastase secreted of total cellular elastase in l o 6 cells with A23187. Neutrophil elastase secreted from AAA patients was significantly higher than in aortic occlusive patients.
DISCUSSION Lysosomal enzymes are stored within neutrophils in active form after their synthesis and have potential deleterious effects if released. The proteolysis that results from these enzymes is balanced by circulating antiproteases, such as alpha-l-antitrypsin. Unrestricted release of elastase represents the pathophysiological event by which lung tissue is destroyed in emphysema [ 131. Recent evidence indicates that a similar proteolytic process may be occurring in the aorta of patients with abdominal aortic aneurysms and may be the reason why some patients develop AAAs and others aortic occlusive disease. This imbalance between the proteolytic enzyme elastase and its major inhibitor, alpha-lantitrypsin, has been correlated with the different types of infrarenal aortic pathology [ I ] . Recent evidence also indicates that the release of lysosomal enzymes such as elastase is dependent on the translocation of calcium [14] (Fig. 1). This calcium messenger system plays a major role as an extracellular messenger in regulating cell function and activating neutrophils [ 15,161. The major trans-
.-
RESULTS
p = .02
Q
30
Neutrophil elastase secretion was significantly W higher (p < .05) in the AAA patients (47.6%) when k compared to the aortic occlusive patients (19.8%)(Fig. I 20 2 2, Table I). Verapamil was significantlyless effective in blocking elastase release in the AAA patients (14.8%) 8 10 than in the aortic occlusive disease (OCC) patients (27.4%) (p < .02) (Fig. 3, Table I). The time for total 0 degranulation to occur in the AAA patients was signifOCCLUSIVE ( N = l O l AAA (N=lll icantly longer (14.7 minutes) when compared to the OCC patients (3.5 minutes) (p < .009) (Fig. 4, Table I). The rate of secretion was higher in the occlusive group Fig. 3. Percent of neutrophil elastase inhibited by but not significantly different from the AAA group Verapamil was significantly higher in aortic occlusive (Table I). patients.
-
VOLUME4 No 6 - 1990
ELA STASl< R E L E A SE I N A A A .$
573
TABLE 1.
In presence of A23187 Elastase secreted ng/m%proteinilo, cells Elastase secreted ngil0 cells Yo secreted of total elastase 1O6 cells In presences of A23187 and Verapamil Elastase secreted ngimg proteinil O6 cells Elastase secreted mg/106 cells o/o secreted of total elastase 1O6 cells Time degranulation (min) Rate of secretion (nghin)
Occlusive fn=lOl
AAA (n=lll
P values Student‘s t-test
18.69 6.39 19.8
25.5 10.84 47.6
p < 0.05 p < .02 p < .05
8.39 2.85 27.4 3.5 + 1.8 5.7 + 6
18.2 7.77 14.8 14.7 + 6.2 2.9 2.2
p < .05 p < .02 p < .02 p = ,009 p = .33
ducing event that occurs as a result of an increase in intracellular calcium through this messenger system is the stimulation of inositol-triphosphate and diacylglycerol 117-191. In addition, the translocation of calcium intracellularly can be stimulated by the calcium Ionophore A23 187 with subsequent degranulation and release of lysosomal elastase [6,8]. This model has given us the opportunity to specifically study the neutrophil elastase secretion in patients with abdominal aortic aneurysms and compare them to patients with aortic occlusive disease. Our data indicate that neutrophil elastase secretion is significantly greater in the AAA patients. However, the effectiveness of Verapamil in blocking this response is significantly lower in the AAA patients. The time for degranulation to occur was longer in the AAA patients, but the rate of secretion was not different between the two groups. Since the rate of secretion is similar between the two groups and AAA patients secrete more elastase, it makes sense that it takes longer to secrete the increased amount of elastase in the AAA patients. Cannon [2] and Busuttil [3] originally reported increased neutrophil elastase in preoperative blood specimens in patients with AAAs. The increase in neutrophil elastase in our AAA patients represents postoperative data after the AAA have long been repaired (six 25 I
I 20.9
:2 0 1
T
I-
OCCLUSIVE (N=10)
=
p
=
,009
+
months). The implication of this observation is that increased circulating proteolytic activity is present in AAA patients regardless of the presence of the aneurysm itself. The observation that Verapamil was ineffective in blocking the calcium-mediated stimulation of elastase secretion in the AAA patients is consistent with previous observations. In other tissues, such as muscle and nerve. Verapamil can suppress cell function that is mediated by calcium translocation [20]. This inhibitory effect occurs by blocking the voltage-sensitive calcium channels 12 I], which are not present within neutrophils [22]. These channels, however, cannot be blocked in neutrophils (since they do not exist) by Verapamil. Verapamil’s effect in neutrophils is known to be through a direct intracellular site of action independent of calcium influx [6,23]. Based upon our data, Verapamil has little effect on regulating the mechanism for elastase release in neutrophils in AAA patients.
CONCLUSIONS We conclude that the increased proteolytic activity, specifically the increased neutrophil elastase in AAA patients, is occurring pre- and postoperatively in these patients and more likely is a primary event and not a response to the presence of the abdominal aortic aneurysm. We also conclude that, although elastase release is controlled to a great extent by the intracellular concentration of calcium, Verapamil, as a calcium channel blocker, is ineffective in neutrophils. It is, therefore, unlikely to medically manipulate the release of neutrophil elastase in AAA patients.
REFERENCES A A A (N.11)
Fig. 4. Time to degranulation in lo6 cells was significantly longer in AAA patients.
1. COHEN JR. MANDELL C . CHANG JB, et al. Elastin
metabolism of infrarenal aorta. J Vnsc Surg 1988;7:210-214. 2 . CANNON DJ, READ RC. Blood elastolytic activity in patients with aortic aneurysm. Ann Thoruc Surg 198834:5-10. 3 . HUSUTTIL RW. REINDERBRIECHT H, FLESHERA, et
574
E LA S 7 ASE R E L W SE I N A A A s
al. Elastase activity: the role of elastase in aortic aneurysm formation. J Sirrg Res 1982;32:214. 4. H U N T E R G C , PEREZ-LIZZANO E, MAJUMDAR A N , et al. Involvement of pancreatic proteases in human abdominal aortic aneurysms. Clin R e s 1984:34:28. 5 . COHEN JR, STEIN T A , DIETZEK A, el al. Urinary L-Valyl proline in patients with aortic aneurysms. S i i r g Gynecol Ohsrer 1988. In Press. 6. KOKOT K , TESCHNER M. SCHAEFER RM. et al. Stimulation and inhibition of elastase release from human neutrophil-dependence on the calcium messenger system. Miri Elc’ct Merub 1987:/3: 189-195. 7. SMOLEN JE. STOEHR SJ. TRAYNOR AE. et al. The kinetics of secretion from permeabilized human neutrophils: release of elastase and correlations with other granule constituents and right angle light scatter. J Larik Bio 1987:4/:813. 8. POSTMA DD. FOLKERS B. SLUITEK HJ. et al. Discrepancies in release of elastase from human peripheral polymononuclear leukocytes. I Rr.\p Dis 1986:69:217-224. 9. S K L A R LA. OADES ZG. FINNEY DA. Neutrophil degranulation detected by right angle light scattering: spectroscopic methods suitable for simultaneous analyses of degranulation or shape change. elastase release. and cell aggregation. J Irnrniiiwl 1984:/33: 1483-1487. 10. S K L A R LA. MC N E I L V , JESAITIS AJ. et al. A continuous, spectroscopic analysis of elastase secretion by neutrophils . I B i d Cherri I9 8?:257(/O) 5 4 7 1-5475. 1 1 . BIETH J. SPIESS B. WERMUTH CG. The synthesis and analytical use of a highly sensitive and convenient substrate of elastabe. Bioc./7rm M e d 1974:1/:350L357. 12. BELLON G , O O Y A M A T . HOKNBECK W. et al. Isolation and partial characterization of an elastase-type enzyme from
13. 14.
15.
16. 17. 18. 19.
20.
21. 22.
23.
ANNALS OF VASCULAK SURGEKY
human arterial wall by lima bean trypsin inhibitor affinity chromatography. Arti2i.v 1980;7:290-302. STOCKLEY RA. Alpha-I-antitrypsin and the pathogenesis of emphysema. L i i n g 1987:/65:61-77. S H O W E L L H. NACCACHE P. SHA’AFI R, et al. The eRects of extracellular K‘. and Ca++ on lysosomal enzymes secretion from polymorphonuclear leukocytes. J Imrniino/ I977 :/ /0:804-8 1 1. PRENTKI M. WOLLHEIM C, L E W P. Ca’ ’ homeostasis in permeabilized human neutrophils. J Biol Clirrn 1984:259: 13777-1 3782. POZZAN T. L E W 1’. WOLLHEIM C. et al. Is cytosolic free calcium regulating neutrophil activation? S c , i o r c , c l 1983;22/: 1413-1415. RASMUSSEN H. The calcium messenger system. Nevi, Ei7gl J Mcti 1986:24:109&1101. BERRIDGE M . Calcium: a universal second messenger. 7riciri,glr I985 :24:79-90. BEKKIDGE M. lntracellular signaling through inositoltriphosphate and diacylglycerol. Cheiti H o p p e - S e y l ~ r1986; 36 7: 447456, FLECKENSTEIN A. Specific pharmacology of calcium in myocardium. cardiac pacemakers, and vascular smooth muscle. Auri Rei. P h t i r r ~ t i c ~ oTo.ric,o/ l 1977;/7:149-166. SCHRAMM M. TOWART R. Modulation of calcium channel function by drugs. Life Sci l985:37: 1843-1860. LEW P. WOLLHEIM C, SERGER R. et al. Cytosolic free calcium changes induced by chemotactic peptide in neutrophils from patient with granulomatous disease. Blood 1984: 63:23 1-233, GREENBERG J. FEINBEKG H, W E N C E L J. et al. Fluoride stimulation of canine neutrophils: the role in calcium binding. Proc. So(. E x p Biol Med 1982:/70:333-340.
Aortic elastase and neutrophil elastase is higher in patients with abdominal aortic aneurysms. The purpose of this study was to determine if these proteolytic elevations occur after abdominal aortic aneurysms have been repaired. Specifically, we studied the stimulation and inhibition of elastase degranulation from neutrophils in postoperative abdominal aortic aneurysm patients compared to aortic occlusive disease patients. Neutrophil elastase was determined in postoperative abdominal aortic aneurysm and aortic occlusive disease patients in response to calcium and the lonophore A23187. Inhibition of elastase release was determined with the calcium channel blocking agent Verapamil. Neutrophil elastase secretion was significantly higher in the abdominal aortic aneurysm patients (47%) versus aortic occlusive disease (20%) (p < .05), while the effect of Verapamil in blocking this response was significantly lower in the abdominal aortic aneurysm patients (14%) compared to aortic occlusive disease patients (27%) (p < .02). The time for degranulationto occur was longer in the abdominalaortic aneurysm patients (14.7 minutes) versus aortic occlusive disease patients (3.5 minutes), but the rate of secretion was not different between the two groups. These data indicate that, (1) neutrophils secrete more elastase in responseto a calcium stimulus in abdominal aortic aneurysm patients; (2) it takes longer to secrete the increased amount of elastase in abdominal aortic aneurysm patients since the rate of secretion is similar between the two groups; and (3) Verapamil blocks elastase secretion ineffectively in abdominal aortic aneurysm patients. We conclude that the proteolytic alterations in abdominal aortic aneurysm patients are more likely a primary event and not a response to the abdominal aortic aneurysm and that Verapamil is a poor drug to use to medically manipulate the proteasesystem in abdominalaortic aneurysm patients. (Ann Vasc Surg 1990;4:570-574). KEY WORDS: Elastase, aortic; elastase, neutrophil; Verapamil; calcium channel blockers; abdominal aortic aneurysms; aortic occlusive disease.
Abdominal aortic aneurysms (AAA) are usually described as variants of atherosclerosis which grow until From the Department of Sirrgery, Long Island Je\jish Medical Center and the Albert Einstein School o j Medicine, New Hyde Park, New York. Presented at the meeting of The Societv j b r Vascular Surgetyllnternutional Societv f o r Cardiovascirlar Sirrgery, June 21, 1989, New York, New York. Reprint requests: Jon R . Cohen, MD, Depnrtment of Surgery, Long Island Jewish Medical Center, New Hyde Park, New York 11042.
eventual rupture. However, recent clinical observations and biochemical data now suggest that aneurysmal pathogenesis may be much more complex than traditionally believed and more likely related to a connective tissue disorder with alterations in systemic elastin metabolism [ I]. Biochemical data that lends support to a systemic alteration in elastin metabolism in AAA patients includes increased leukocyte elastase [2], increased aortic elastase [ I ,3], increased pancreatic elastase [41, imbalance of elastase and alpha-l-antitrypsin in the aortic wall, and an increase in elastin urine
570
VOLUME4 No 6 - 1990
57 I
ELAS7ASE RELEASF; I N AAA.5
metabolites 151. Although both aortic elastase tissue levels and neutrophil elastase are higher in patients with AAAs, one can argue that the increased proteolytic activity is a response to the AAA and not the primary event. Further investigation into the mechanism of elastase release from lysosomes reveals that the translocation of calcium plays a predominant role in the enzyme release [6,7] (Fig. I ) . It is now well established that the calcium messenger system, by which extracellular messages regulate cell function. plays a major role in activating neutrophils [6]. In addition, the translocation of calcium by the lonophore A23 187 stimulates the release of neutrophil elastase [6,8]. The calcium channel blocker Verapamil in some cases is capable of suppressing this stimulation of elastase release 161. Using a model for the intracellular calcium stimulation of elastase release, the purpose of this study was to determine if the stimulation and inhibition of elastase degranulation from neutrophils in postoperative AAA patients is altered.
MATERIALS AND METHODS Neutrophil elastase degranulation and secretion is highly sensitive to and increases in response to elevations in intracellular calcium [6-81. Neutrophil elastase secretion was determined in patients with postoperative AAA and aortic occlusive disease in response to incubation with calcium and the Ionophore A23187. Eleven AAA patients and 10 aortic occlusive disease patients were studied. The mean postoperative period at the time of blood drawing for all patients was six months. Neutrophil preparation and elastase assay
Approximately l0cc of whole blood was obtained by venipuncture and mixed with lOcc of normal saline. Neutrophil cell suspensions were obtained from hypaque gradient centrifugation. Neutrophil permeabilization was achieved with 100 microliters of A23187 (200 micromolar) incubated at 37" centigrade. After incubation, the suspension was centrifuged at 17000 RPMs for five minutes. The pellets were washed with calcium free buffer and centrifuged twice. Right-angle light scattering
The rate of neutrophil elastase release was measured by right-angle light scattering. The latter is a function of granule circulation, and its decline mirrors the kinetics of elastase release [7,9,10]. Neutrophils were resuspended in buffer ( K C l , 100 mM; NaCl 20 mM: HEPES, 30 mM) to half the original
ca++
A23187
\
ELASTASE
Ca"
1
Fig. 1. Release of neutrophil elastase as a result of an increase in intracellular calcium. 1. lonophore A23187 stimulates calcium translocation into cell. 2. Endoplasmic reticulum produce secondary messengers inositol triphosphate and diacylglycerol. 3. Secondary messengers stimulate lysosomal release of elastase. 4. Elastase is released at cell membrane.
blood volume. Ca ionophore A23187 solution, 200 uM in dimethyl sulfoxide (DMSO), was added to make the concentration 5 mM, and the suspension was incubated with agitation at 37°C for 30 minutes. It was centrifuged at 700 x g for five minutes, the cells were washed twice with saline and finally suspended in buffer. Two ml of buffer were added to a cuvette and the cuvette was placed in the holder of a Turner spectrofluorometer (model #430) thermostated at 37°C. Excitation and emission wave lengths were both set at 380 nm. The fluorometer was hooked to a Gilford 6050 recorder. After a steady baseline (b,) was obtained, the cuvette was replaced by one containing the cell suspension and a new higher baseline (b,) was established. CaCI, was then added. Percent degranulation was calculated from the height of the plot (h) as 100 (b2-h)/(b2-b,)l. Elastase release
Elastase release was measured under various conditions and was calculated as (1) a fraction of total elastase contained in the cells: (2) a fraction of total protein: and (3) as the amount per million cells. The cells were resuspended in Hanks' balanced salt solution (HBSS) and counted. When necessary, the volume of the suspension was adjusted to make it in the order of 10' cellsiml. The suspension was aliquoted to five different tubes to which one of the following was added: Tube 1-50 pl HBSS: Tube 2-25 pl A23187 solution to make the final concentration 2 pM: Tube 3-Verapamil HCI to make the final concentration 50 pM: and Tube 4-A23 187 and
512
ANNALS OF VASCIJLAK SUKGEKY
ELASTASE RELEASE I N AAAs
Verapamil. The mixtures were agitated at 37°C for 30 minutes, then centrifuged, and the supernate was assayed for elastase activity. In Tube 5 the cells were incubated for 30 minutes with 20 mg NaCl and 25 p1 Triton X-100 per ml. This treatment caused complete disruption of the cells. After centrifugation, the supernate of this fifth tube was used to determine total elastase activity and total protein assays. The percent elastase release was calculated for A23187 alone (Tube 2iTube 5 ) , Verapamil alone (Tube 3/Tube 5 ) and A23187 with Verapamil (Tube 4/Tube 5). Elastase activity was measured by a variation of previously described techniques [ 1 1 ,I 21, using N-methoxysuccinyl-ala-ala-Lpro-L-valp-nitroanilaide as a substrate: 100 pl of sample and 25 pI of substrate solution (25 mM in DMSO) were added to a cuvette containing 1.5 ml of 0.2 M Tris buffer, pH 8.0. The release of p-nitroaniline was monitored by readings of the optical density (O.D.) at 410 nm with a Gilford Stasar 11 spectrophotometer. The slope of the regression line, O.D. vs. time, was taken as the measure of elastase activity. Total protein was determined by the Lowery method [6]: 5 ml Cu tartrate reagent (50 ml of 2% Na2C0, in 0. I M NaOH, 1 ml of 1% Na tartrate and 1 ml of 0.5% CuSO,), are mixed with 0.1 ml sample. After 10 minutes, 0.5 ml Folin-Ciocalteu’s reagent (diluted 35 to 100) was added. After 30 minutes the O.D. is measured at 700 nm.
Statistical methods
The mean values of both groups of patients were compared using the Student’s t-test. The Student’s t-test was also applied to compare the groups for the time to degranulation (minutes) and for the rate of secretion (ng/min).
8 OCCLUSIVE (N=IO)
=
AAA (N=II)
Fig. 2. Percent of elastase secreted of total cellular elastase in l o 6 cells with A23187. Neutrophil elastase secreted from AAA patients was significantly higher than in aortic occlusive patients.
DISCUSSION Lysosomal enzymes are stored within neutrophils in active form after their synthesis and have potential deleterious effects if released. The proteolysis that results from these enzymes is balanced by circulating antiproteases, such as alpha-l-antitrypsin. Unrestricted release of elastase represents the pathophysiological event by which lung tissue is destroyed in emphysema [ 131. Recent evidence indicates that a similar proteolytic process may be occurring in the aorta of patients with abdominal aortic aneurysms and may be the reason why some patients develop AAAs and others aortic occlusive disease. This imbalance between the proteolytic enzyme elastase and its major inhibitor, alpha-lantitrypsin, has been correlated with the different types of infrarenal aortic pathology [ I ] . Recent evidence also indicates that the release of lysosomal enzymes such as elastase is dependent on the translocation of calcium [14] (Fig. 1). This calcium messenger system plays a major role as an extracellular messenger in regulating cell function and activating neutrophils [ 15,161. The major trans-
.-
RESULTS
p = .02
Q
30
Neutrophil elastase secretion was significantly W higher (p < .05) in the AAA patients (47.6%) when k compared to the aortic occlusive patients (19.8%)(Fig. I 20 2 2, Table I). Verapamil was significantlyless effective in blocking elastase release in the AAA patients (14.8%) 8 10 than in the aortic occlusive disease (OCC) patients (27.4%) (p < .02) (Fig. 3, Table I). The time for total 0 degranulation to occur in the AAA patients was signifOCCLUSIVE ( N = l O l AAA (N=lll icantly longer (14.7 minutes) when compared to the OCC patients (3.5 minutes) (p < .009) (Fig. 4, Table I). The rate of secretion was higher in the occlusive group Fig. 3. Percent of neutrophil elastase inhibited by but not significantly different from the AAA group Verapamil was significantly higher in aortic occlusive (Table I). patients.
-
VOLUME4 No 6 - 1990
ELA STASl< R E L E A SE I N A A A .$
573
TABLE 1.
In presence of A23187 Elastase secreted ng/m%proteinilo, cells Elastase secreted ngil0 cells Yo secreted of total elastase 1O6 cells In presences of A23187 and Verapamil Elastase secreted ngimg proteinil O6 cells Elastase secreted mg/106 cells o/o secreted of total elastase 1O6 cells Time degranulation (min) Rate of secretion (nghin)
Occlusive fn=lOl
AAA (n=lll
P values Student‘s t-test
18.69 6.39 19.8
25.5 10.84 47.6
p < 0.05 p < .02 p < .05
8.39 2.85 27.4 3.5 + 1.8 5.7 + 6
18.2 7.77 14.8 14.7 + 6.2 2.9 2.2
p < .05 p < .02 p < .02 p = ,009 p = .33
ducing event that occurs as a result of an increase in intracellular calcium through this messenger system is the stimulation of inositol-triphosphate and diacylglycerol 117-191. In addition, the translocation of calcium intracellularly can be stimulated by the calcium Ionophore A23 187 with subsequent degranulation and release of lysosomal elastase [6,8]. This model has given us the opportunity to specifically study the neutrophil elastase secretion in patients with abdominal aortic aneurysms and compare them to patients with aortic occlusive disease. Our data indicate that neutrophil elastase secretion is significantly greater in the AAA patients. However, the effectiveness of Verapamil in blocking this response is significantly lower in the AAA patients. The time for degranulation to occur was longer in the AAA patients, but the rate of secretion was not different between the two groups. Since the rate of secretion is similar between the two groups and AAA patients secrete more elastase, it makes sense that it takes longer to secrete the increased amount of elastase in the AAA patients. Cannon [2] and Busuttil [3] originally reported increased neutrophil elastase in preoperative blood specimens in patients with AAAs. The increase in neutrophil elastase in our AAA patients represents postoperative data after the AAA have long been repaired (six 25 I
I 20.9
:2 0 1
T
I-
OCCLUSIVE (N=10)
=
p
=
,009
+
months). The implication of this observation is that increased circulating proteolytic activity is present in AAA patients regardless of the presence of the aneurysm itself. The observation that Verapamil was ineffective in blocking the calcium-mediated stimulation of elastase secretion in the AAA patients is consistent with previous observations. In other tissues, such as muscle and nerve. Verapamil can suppress cell function that is mediated by calcium translocation [20]. This inhibitory effect occurs by blocking the voltage-sensitive calcium channels 12 I], which are not present within neutrophils [22]. These channels, however, cannot be blocked in neutrophils (since they do not exist) by Verapamil. Verapamil’s effect in neutrophils is known to be through a direct intracellular site of action independent of calcium influx [6,23]. Based upon our data, Verapamil has little effect on regulating the mechanism for elastase release in neutrophils in AAA patients.
CONCLUSIONS We conclude that the increased proteolytic activity, specifically the increased neutrophil elastase in AAA patients, is occurring pre- and postoperatively in these patients and more likely is a primary event and not a response to the presence of the abdominal aortic aneurysm. We also conclude that, although elastase release is controlled to a great extent by the intracellular concentration of calcium, Verapamil, as a calcium channel blocker, is ineffective in neutrophils. It is, therefore, unlikely to medically manipulate the release of neutrophil elastase in AAA patients.
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Fig. 4. Time to degranulation in lo6 cells was significantly longer in AAA patients.
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