Clin Biochem, Vol. 25, pp. 2 8 5 - 2 8 8 , 1992 Printed in the USA. All rights reserved.

0009-9120/92 $5.00 + .00 Copyright c 1992 The Canadian Society of Clinical Chemists.

Superoxide Anion Production and Intracellular Free Calcium Levels in Resting and Stimulated Polymorphonuclear Leukocytes Obtained From Healthy and Arteriosclerotic Subjects of Various Ages ATTILA MOHACSI, TAMAS FOLOP, JR., BERTALAN KOZLOVSZKY, ILDIKO SERES, and ANDRAS LEOVEY First Department of Internal Medicine, First Department of Surgery, University Medical School of Debrecen, Hungary It has been established that phagocytic cells are integral components of advanced arteriosclerotic plaques but their role in plaque formation remains unclear. Therefore, toxic agents, such as superoxide anion produced by polymorphonuclear leukocytes (PMNLs) were studied in a clinically defined group of arteriosclerotic patients suffering from obliterative arteriosclerosis of the lower legs. Owing to a close correlation between 02generation and calcium, the intracellular free calcium concentrations of PMNLs were measured in a resting state and after stimulation with various agents, for example, opsonized zymosan (OZ), the chemotactic peptide f-met-leu-phe (FMLP), and the calcium ionophore A23187. Healthy aged-matched controls were employed. The patients were divided into two age groups: 30-59 years and 60-80 years. We found that in the younger group of arteriosclerotic patients, superoxide anion production and intracellular free calcium concentrations were increased even in the resting state, and only a slight increase was observed after stimulation compared with healthy controls. Granulocyte responses seemed to be similar, independent of the patient's age, to those found in healthy elderly subjects. Arteriosclerosis appears to be associated with an early aging process expressing marked alterations that are greater than those associated with normal aging.

KEY WORDS: polymorphonuclear cells; superoxide anion; intracellular free calcium; arteriosclerosis; aging process. Introduction

eukocytes and their metabolites are important L mediators of inflammation and vascular wall injury (1,2). The biologically active oxygen species, for example, superoxide anion, H202 and especially HOC1, may also be involved in aging processes (3,4), Correspondence: Dr. Attila Moh(~csi, First Department of Internal Medicine, University of Debrecen, P.O. Box 19, H-4012, Debrecen, Hungary. Manuscript received May 1, 1990; revised June 3, 1991 and February 1, 1992; accepted February 21, 1992. CLINICAL BIOCHEMISTRY, VOLUME 25, AUGUST 1992

but their role in the progression of arteriosclerosis has not yet been accepted. It appears that the initial step in arteriosclerotic plaque formation is endothelial damage, but the cause of this injury remains unknown. In vitro studies have shown that lysis of SlCr-labeled endothelial cells (EC) can be generated by leukocyte-derived oxygen metabolites; thus respiratory burst products may be one of the factors inducing plaque formation (5-7). Besides monocytes having a well-known role in plaque formation, polymorphonuclear leukocytes (PMNLs) might also be sources of oxygen metabolites. The adherence of PMNLs to endothelial cells is amplified by the action of different cytokines, most prominently interleukin-1 and tumor necrosis factor-alpha (8). These cytokines elicit adhesion of PMNLs to EC and induce production of chemotactic factors (9-11). Therefore, the aim of our present study was to investigate whether the respiratory oxygen species (ROS) production by PMNLs is altered in a welldefined arteriosclerotic group, namely occlusive arterial disease of the lower limbs. Postreceptor signal transduction events were also investigated by measurement of intracellular free calcium and ROS at rest and after stimulation with opsonized zymosan (OZ), N-formyl-methionyl-leucyl-phenylalanine (FMLP) and calcium ionophore (A231sT). The opsonized zymosan stimulates the Fc receptor, but its postreceptor signal transduction is largely unknown, although phospholipase A2 (PLA2) is an integral component of the Fc receptor (12). PLA2 has a stimulatory effect on the respiratory burst oxidase. FMLP mainly activates the cells through phosphatidylinositol breakdown, increasing the intracellular free calcium {Ca2+}i (13). This, with calmodulin as second messenger, plays a key role in the receptorstimulated biochemical responses, for example, the production of ROS. A231s 7 acts directly on calcium metabolism, ultimately leading to the same 285

MOHACSI, FULOP, KOZLOVSZKY,SERES, AND LEOVEY

cellular response as receptor stimulation, with O~ generation (14).

140 120

Methods

100

PATIENTS

80

PMNLs were obtained from 78 patients referred to our institution with symptoms due to obliterative arteriosclerosis of the lower limbs. The diagnosis was confirmed by Doppler m e a s u r e m e n t of ankle/ arm pressure index. Smoking habits were investigated by a questionnaire: 20 were nonsmokers and 58 were smokers (on average more t han 15 cigarettes/day for more t h a n 10 years). The diagnosis of diabetes mellitus and m e a s u r e m e n t of the systemic arterial blood pressure were based on conventional examinations. F o ur t een patients of 78 suffered from diabetes mellitus (mean fasting blood glucose level 9.8 - 2.0 mmol/L) and 65 of 78 patients had high blood pressure for more t han ten years (on average 170 -+ 15 mm Hg systolic and 100 - 20 mm Hg diastolic blood pressure). Patients were divided into two age groups: 3 0 - 5 9 years (4 women, 35 men, m e a n age 50.6 -+ 6.3 years) and 6 0 - 8 0 years (6 women and 33 men, m ean age 65.7 -+ 1.2 years). All patients gave fully informed consent. Ten healthy, nonsmoking middle-aged (3 women, 7 men, m e a n age 45 -+ 3.5 years) and 10 healthy, nonsmoking elderly subjects (4 women, 6 men, mean age 67.2 - 6.5 years) served as controls.

60 40 20 0

middle aged H Balal

elderly H ~

OZ

middle aged AT8 ~

FMLP

~

elderly AT8 A23187

Figure 1 - - O~ generation in resting and stimulated PMNLs obtained from control and arteriosclerotic subjects of various ages. The stimulants were as follows: OZ = l0 s particles/mL opsonized zymosan; FMLP = 10-s mol/L; Ca ionophore (A23~ST) = 10-6 mol/L. Each column represents the mean -+ SD. ratory (19). In brief, the fluorescence was me a su re d with a Hitachi MPF 4 spectrofluorimeter, excitation wavelength 339 nm and emission w avel e n g th 492 nm at 37 °C. The sat urat i on fluorescence (Fm~x) was measured by addition of digitonin to the cell suspension; the m i n i m u m fluorescence (Fmin) was measured in medium containing 2 mM EGTA and Tris to take the pH above 8.4. The {Ca2+}i w a s calculated by the equation:

PMNLs

{Ca2 + }i = Kd(F - F m i n ) / ( F m ~ x - F ) where Kd = 115 nM at 37 °C and F is the fluorescence of the resting cell.

These were separated by Ficoll-Hypaque density centrifugation (15); judged by morphological criteria, the PMNLs suspension was 95% pure, and 97% of the cells were viable by the T r y p a n Blue dye exclusion test. Cell suspensions were made in Hank's balanced salt solution with different cell densities. All incubations were performed in CO2 (5%), air (95%), humidity (95%) at 37 °C. P a r a m e t e r s of resting PMNLs were determined immediately after cell separation.

nmol 0~13 x 10e cells/mL 140 - -

0 2

120

Statistical analysis Student's t-test was used for comparison between the clinical groups.

no10

PRODUCTION

100

This was determined according to the method of Babior e t al. (16) as already described (17). In brief: the r e d u c t i o n of c y t o c h r o m e C (Sigma type III) which can be inhibited by superoxide dismutase was measured at 550 nm in suspensions of 3 × 106 cells/ mL. PMNLs were stimulated with l 0 s OZ particles, 10 - s mol/L F M L P and 10 -6 mol/L calcium ionophore, A231s 7. MEASUREMENT OF CYTOSOLICFREE CALCIUM

PMNLs were loaded with Quin 2(AM), a calciumi n d icatin g f l u o r e s c e n t i n t r a c e l l u l a r dye (Calbioc h em, L u c e r n e , S w i t z e r l a n d ) a c c o r d i n g to t h e method of Tsien (18) slightly modified in our labo286

n-58 80

sd

n.20

40 20 0 Smokers ATS

Non emokers ATS Basal

~

OZ

I - ~ FMLP

Control m

A23187

Figure 2 - - The effect of smoking on the O~ generation in PMNLs obtained from arteriosclerotic patients and control subjects. Stimulation was carried out as described in the legend to Figure 1. Each column represents the mean -+ SD. CLINICAL BIOCHEMISTRY, VOLUME 25, AUGUST 1992

SUPEROXIDE ANION PRODUCTION AND INTRACELLULARFREE CALCIUM TABLE 1 The Cytosolic Free Calcium Levels (nmol/L) in Resting State and After Stimulation With FMLP (10 -s mol/L) Controls

Arteriosclerotic

Age (years)

Resting (n = 10)

FMLP (n = 10)

Increase (%)

Resting (n = 39)

FMLP (n = 39)

Increase (%)

30-59 60-79

131 +- 30 269 -+ 70a

201 + 10 280 --- 30

153 104

205 -+ 42a 254 - 40~

245 -+ 12 270 -+ 18

119 106

Each value represents the mean --- SD of determinations carried out in triplicate on each subject. a Significant increase compared to values of middle aged controls (p < 0.01).

Results A significant difference was found in the basal levels of 0 2 (5.6 -+ 0.18 vs 30.2 -+ 8.1 nmol/3 x 106 cells, p < 0.01) between the two age groups of healthy subjects (Figure 1). The basal level of 0 2 in PMNLs of middle-aged arteriosclerotic patients was quite similar to t h a t of h e a l t h y elderly (28.8 +- 7.3 nmol/3 x 106 cells). In middle-aged controls the 0 2 production was markedly increased by stimulation with OZ, FMLP and the calcium ionophore A231sT, while stimulation was less effective in PMNLs obtained from elderly controls as well as in both age groups of arteriosclerotic patients. To further examine this question, the effects of diabetes mellitus, hypertension, hyperlipidemia, and smoking on ROSgenerating capacity were investigated. No significant influence of these factors could be demonstrated except for smoking which amplified the basal generation of active oxygen species in arteriosclerotic patients (p < 0.015) but did not affect the response to the various stimulations (Figure 2). Intracellular free-calcium concentrations of resting PMNLs are presented in Table 1. It is apparent " • t h a t {Ca 2 + }i is increased with age. The percentage 2+ increase of {Ca }i induced by FMLP was less in elderly controls and arteriosclerotic patients of both groups, compared to middle-aged controls.

Discussion The aging process m a y be simply the sum of deleterious effects of ROS as proposed in the free radical theory of aging (3,4). A close relationship between age and arteriosclerosis has been known for a long time. Endothelial cells m a y play a crucial role in plaque formation as the target of the free radical products of PMNLs ( 5 - 7 , 2 0 - 2 2 ) . Several factors such as interleukin-1, endotoxin, and leukotrienes enhance the adhesion of PMNLs to endothelial cells resulting in their injury (8). These data suggest a possible role of P M N L s in t h e p a t h o g e n e s i s of arteriosclerosis. In our laboratory we confirmed t h a t the production of 0 2 and H202 is increased with aging (17,23). Modified activation of respiratory burst via specific receptors was also found with aging (23). Similar alterations were found in middle-aged arterioscleCLINICALBIOCHEMISTRY,VOLUME 25, AUGUST 1992

rotic patients as in elderly controls. No statistically significant differences were demonstrated between elderly controls and elderly patients suffering from obliterative arteriosclerosis of the legs, whereas the results of middle-aged arteriosclerotic patients differed significantly from those of middle-aged controls. These observations are consistent with the finding t h a t lipid peroxidation is raised in the elderly and in patients with arteriosclerosis. ROS m a y initiate the peroxidation of lipids in cell membranes and the nonenzymatic oxidative cleavage of low density lipoproteins (11). The calcium dependency of the respiratory burst can be explained as a complex positive-feedback system. Accumulation of calcium in the cells and in the arterial wall may be linked to the acceleration of arteriosclerotic changes, as shown by 45Ca kinetic studies in experimental animals (24). The enlargement of the intracellular calcium pool in aortic cells has also been demonstrated (24). Our {Ca 2 ÷}i measurements show t h a t the intracellular free-calcium level of PMNLs is increased in elderly controls and in arteriosclerotic patients of both age groups. The raised intracellular free-calcium, with calmodulin as second messenger, could have significant influences on cellular functions, such as production of active oxygen species, chemotaxis, and release of intralysosomal enzymes such as elastase. The intracellular free-calcium was demonstrated to increase with aging (19). In arteriosclerotic patients this enhancement occurs at an earlier age compared to healthy subjects. These data support our observation t h a t middle-aged arteriosclerotic patients are exposed to an accelerated aging process. Acknowledgements The authors are greatly indebted for the excellent technical assistance of Mrs. M. FOlbp, Mrs. M. Nagy and Mrs. G. Mozga.

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CLINICAL BIOCHEMISTRY, VOLUME 25, AUGUST 1992