J Stroke Cerebrouasc Dis 1995;5:166-171 © 1995 National Stroke Association
Cytokine and Superoxide Production in Clinical Stroke Wayne M. Clark, M.D., Jeffrey D. Lauten, Nancy B. Beamer, M.s., and Bruce M. Coull, M.D.
Cytokines appear to play an important rol e in a variety of central nervous system (CNS) diseases and may be involved in ischemia. Activated leukocytes are a major source of cytokines and other inflammatory mediators that may directly injure ischemic CNS tissue. To investigate the importance of these substances in clinical stroke, we compared spontaneous secretion of IL-1~, IL-6,IL-8, and superoxide production from neutrophils (PMN) and mononuclear cells (MNC) isolated from either acute stroke patients (n "" 10) or matched controls (n "" 10). Cyto kine production from 5 X 106 cells incubated for 24 h was determined by EUSA. MNC from acute stroke patients secreted significantly less IL-1~, IL-6,and IL-8than MNC from control patients. No significant cytokine production was detected from PMN . Superoxide production from 1.25 X 105 cells was determined over 1 h measuring cytochrome C reduction. There was a trend toward higher superoxide production in PMN from acute stroke patients, whereas the superoxide production in MNC from acute stroke patients was significantly higher than that of control MNC. This difference persisted even when various agonists were added preincubation. This study suggests that MNC sponlaneous cylokine secretion is reduced in acute stroke patients, whereas superoxide production is increased. This decreased cytokine production may be secondary to the effects of a cytokine inhibitory factor. Key Words: Cytokines-Stroke-Brain-Interleukins.
There is increasing evidence that inflammatory responses play an important role in the pathogenesis of several central nervous system (CNS) diseases. Many of these inflammatory responses are mediated by interleukins, a multifunctional subclass of cytokines. The pro-inflammatory interleukins, including IL-1, IL-6, and IL-8, can influence the function and synthesis of other cytokines via a complex cytokine network (1). These pro-inflammatory interleukins are produced by a variety of cells including leukocytes and
From the Department of Neurology, Oregon Stroke Center, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Road, Portland, OR, U.S.A. Address correspondence and reprint requests to Dr. W. M. Clark at Department of Neurology Ll04, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Road, Portland, OR 97201, U.S.A. 166
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microglia and appear to influence CNS infiltration by leukocytes and may modulate CNS cellapoptosis, differentiation, and proliferation (1). Increased levels of IL-1,IL-6,and IL-8 have been detected in cerebrospinal fluid or CNS tissue sections in brain injury (2), Alzheimer's disease (3), multiple sclerosis (4), CNS lupus (5), and CNS infections (6). Recent studies also suggest that the pro-inflammatory interleukins may be involved in stroke potentiation and recovery. IL-l induces glial cell proliferation and promotes leukocyte endothelial adhesion via increased intercellular adhesion molecule expression (7). Increased levels of IL-1 have been observed following experimental forebrain ischemia (7), and treatment with an antagonist to IL-l (ILl-ra) has been demonstrated to reduce experimental eNS ischemic injury (8). Increased endogenous production ofILlra has also been observed in ischemic hippocampal
CYfOKINE PRODUCTION IN STROKE
neurons (9). IL-8 mediates leukocyte adhesion and emigration and stimulates the release of enzymes from neutrophils (10). There is increasing evidence that these leukocyte functions are involved in CNS reperfusion injury (11). IL-6also stimulates hepatocytes to synthesis fibrinogen and other prothrombotic proteins (1). We have recently demonstrated that plasma fibrinogen and IL6 levels are higher in stroke patients than in matched controls (12). However, since interleukins are active in very low (picomolar) concentrations, we have not been able to detect plasma levels of the other proinflammatory interleukins in stroke patients using standard ELISAtechniques. Since mononuclear cells (Mr'{C) are a major source of interleukin production, an alternate strategy is to assess the secretion of cytokines from isolated MNC. An additional advantage of this method is that the production of other inflammatory mediators, including oxygen-free radicals, can be concurrently assessed. This strategy was recently utilized by Rudick et al. to detect changes in cytokine production in multiple sclerosis patients (13). In our study, we compared the secretion ofIL-1, IL-6, IL-8, and superoxide in leukocytes isolated from acute stroke patients to that of leukocytes from matched control subjects.
Methods Study Participants Subjects were recruited from the wards and outpatient clinics of the Portland Veterans Administration Hospital and the Oregon Health Sciences University into two groups: patients with an acute stroke (n = 10, cytokine, and n = 11, superoxide), and healthy control subjects balanced for age with the stroke group (n = 10). Subjects were eligible forrecruitment into the stroke group if they had an ischemic stroke resulting in a significant neurologic deficit (at least four points on NIH Stroke Scale) in the previous 5 days. Volunteers of similar age and sex comprising the control group denied the presence of risk factors for stroke, were currently nonsmoking, and were not taking prescription medication other than aspirin. All subjects gave written informed consent to participate and undergo phlebotomy by members of the neurology staff. Fasting blood samples were drawn (days 3-5 poststroke) from an antecubital vein by venipuncture with minimal stasis into vacutainers (Becton-Dickinson, Rutherford, NJ) and were anticoagulated with sodium heparin. Routine laboratory measurements were performed by laboratories of the
Clinical Pathology Department of Oregon Health Sciences University. Plasma IL-6 levels were determined using the sandwich ELISA protocol described below.
Isolation of Cells Human neutrophils (PMN) and MNC were isolated using a modified Boyum density gradient centrifugation method (14). At room temperature, heparinized blood was carefully layered over Monopoly Resolving Media (1CN Biomedicals, Inc., Los Angeles, CA) and Histopaque 1077 (Sigma, St. Louis, MO), then centrifuged at 500 g until two distinct leukocyte bands appeared. The upper band contained MNC, and the lower band contained PMN. The MNC and PMN were removed independently, washed twice with CA2 and M g2-free phosphate buffered saline (PBS) and counted in a hemacytometer. A slide differential count (nonspecific esterase) of the MNC band revealed 95% MNC with a morphologic analysis revealing approximately 30% monocytes. Evaluation of the PMN band revealed about a 2% MNC contamination.
Measurement of Cytokine Secretion MNC and PMN were adjusted to 4 X 10 6 cell/ml in Earles Balanced Salt Solution/25 mM HEPES (Sigma Chemical Co., St. Louis, MO). One milliterofeach cell population was added to a 24-well, tissue-culture, treated polystyrene dish (Corning) or a 15-ml Teflon chamber (Savillex, Minnetonka, MN). Cells were incubated at 37°C for 24 h, after which the conditioned medium was removed, aliquoted, and stored at - 20°C. The levels ofIL-1~, IL-6, and lL-8 in the conditioned medium were measured by sandwich ELISAs following the manufacturer's protocols (IL-1~, AMAC Inc., Westbrook, ME;IL-6,IL-8,R&D Systems, Minneapolis, MN). Samples of media were diluted, and concentrations of cytokines were determined against standard curves with the following ranges: IL-1/3, 15.6-1,000 pg/ml; IL-6, 3.13-300 pg/ml; and IL-8, 93.8-6,000 pg/ml. Previous investigations have shown that adherence to plastic wells increases MNC activation and cytokine production (15). To determine if cell adhesion affected the observed differences in cytokine secretion between stroke and control patients, we compared the spontaneous secretion of nonadherent cells (Teflon wells) to that of adherent cells (plastic wells).
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Measurement of Superoxide Production Samples from the same PMN and MNC populations used above were adjusted to 5 X 106 cells/ml in DPBS containing Mg2 and Ca 2 and assayed for superoxide production over 60 min via the superoxidedependent reduction of ferricytochrome C (16). The following was added to a 96-well microplate: 25 ul of either PMN or MNC at 5 X 10 6 cell/ml, 100 III of ferricytochrome C at 2.5 mg/ml (Sigma, St. Louis, MO), 100 III DPBS/Ca2M g2, and 25 III of either superoxide dismutase at 1 mg/ml (Sigma) or recombinant human IL-1[3, IL-6, or IL-8 (R&D Systems, Minneapolis, MN) at 20 ng/ml. We added the various cytokines to the samples to determine their influence on superoxide production. The optical density was read at 550 nm, and the plate was incubated at 37°C 5% CO 2 for 60 min, after which the final optical density was determined. The wells containing superoxide dismutase were subtracted as blanks to eliminate any superoxide-independent reduction of ferricytochrome C. Additional wells containing cytokines were also assayed with superoxide dismutase, and no superoxide-independent reduction of ferricytochrome C was induced. Using the extinction coefficient of ferricytochrome C (21.1 mM-l cm -1) the number of moles of superoxide (° 2- ) generated was determined by the following equation: nmols O 2-/125,000 cells = change in OD 550 nm X 23.9. Differences among groups were assessed by analysis of variance, and when there was a significant difference (p < 0.05) among groups, the p value was determined by Bonferroni-corrected post-hoc t tests.
Results A total of21 patients were evaluated. There was no significant difference among age, sex, and aspirin usage between the two groups. Hematologic values for the groups are shown in Table 1. There was no significant difference in hematocrit between the groups; however, serum fibrinogen, total leukocyte counts, and total monocyte count (total WBC X percent monocytes) were higher in the stroke group compared to controls. Significantly higher levels of plasma IL-6 were also found in the stroke group. We did not measure plasma IL-8 or IL-1[3 in this study, but we previously found that levels of these interleukins were undetectable in plasma from stroke patients. For all three cytokines, secretion by PMN was always less than 2% of the corresponding MNC well value. Since the PMN samples contained up to a 2% MNC con168
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Table 1.
Hematologic profile (mean ± SD) Group
Descriptor
Stroke (n = 10)
Hematocrit (%) (mean ± SD) Leukocyte (X 103) Total monocytes (X 10 3) Fibrinogen (mg/dl) Plasma IL-6 (pg/ml)
10 ± 4.3" 0.73 ± 0.1" 348 ± 56" 11.5 ± 10"
44
±6
Control (n = 10) 45
±4
4.8 ± 1.2 0.51 ± 0.2 252 ± 44 2.6 ± 1.3
"p < 0.05 higher than control.
tamination, there did not appear to be any significant PMN cytokine secretion for either group. Figure 1 shows the results of cytokine secretion by MNC at 24 h. Overall, incubation in plastic wells produced significantly higher cytokine values than Teflon well incubation in the control group but not the stroke group. Lower levels of MNC secretion at 24 h were seen for all three cytokines in the stroke group compared to controls. For IL 1[3, the levels in the stroke group (n = 10) were 21,240 ± 5,112 (mean ± SE) pg/ml per 4 X 106 cells (plastic) and 11,921 ± 2,434 pg/ml (Teflon), and the levels in the control group (n = 10) were 47,822 ± 13,250 pg/ml (plastic) and 27,906 ± 6,949 pg/ml (Teflon) (p < 0.05) (see Fig. I, top). For IL-6, the stroke group (n = 10) had levels of 8,663 ± 1,856 pg/ml (plastic) and 9,332 ± 1,500 (Tef1011); control group, (n = 10) 15,623 ± 2,157 pg/ml (plastic) (p < 0.05) and 14,166 ± 2,210 pg/ml (Teflon) (see Fig. I, center). For IL-B, the stroke group (n = 10) had levels of 174,455 ± 40,178 pg/ml (plastic) and 193,589 ± 27,056 pg/ml (Teflon); control group (n = 10),279,207 ± 37,310 pg/ml (plastic) (p < 0.05) and 214,840 ± 29,747 pg/ml (Teflon) (see Fig. I, bottom). Overall, there was a significant negative correlation (r = -0.38) between the MNC IL-6 secretion values and the corresponding plasma IL-6 level. The results of superoxide production for PMN and MNC are shown in Fig. 2. For PMN, there was a nonsignificant trend toward higher superoxide production in the stroke group (n = 11),0.44 ± 0.10 (mean ± SE) nmol O 2-/125,000 cells, compared to that for the control group (n = 10),0.30 ± 0.04 nmol. For MNC, production in the stroke group (n = 11) 0.302 ± 0.04 nmol was significantly higher compared to the control group (n = 10) 0.09 ± 0.05 nmol (p < 0.01). The addition of 20 ng/ml of IL-1[3 to the incubation produced a marked increase in superoxide production by both cells types, whereas the addition of equivalent amounts of IL-6 and IL-8 had no effect.
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Figure 1. Spontaneous MNC inierleukin secretion (mean ± SE). MNC isolated from stroke (n = 10) or control (n = 10) patients were cultured either on Teflon [open bar) or plastic (black bar), and media wereassessed for interleukin secretion after24 h: IL-IB (top), IL-6 (center), and IL-B (bottom).
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There is increasing evidence that leukocytes and inflammatory mediators play a pathologic role in CNS ischemic injury. Clarification of the relationship between leukocytes and their secreted mediators during ischemia may provide clues for future therapeutic interventions. In this study, we found that cytokine
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Figure 2. Superoxide production by PMN andMNC (mean ± SE). Cells wereisolated from stroke {n = 11) orcontrol (n = 10) patients andwereincubated onplastic. Superoxide production was assessed at 1 h forthefollowing conditions: nostimulation or after thepreincubation addition of20 ngtml of 11-1~, IL-6, orIL-B. Top: PMN. Bottom: MNC.
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creased in monocytes from patients with active MS compared to stable MS (13). There are several potential theories for this decreased MNC cytokine secretion following an acute stroke. First, there appears to be a "pro-inflammatory cytokine cascade" with IL-1 release leading to increased production of the other cytokines (10). The ability of IL-1 receptor antagonists to block the production of IL-6 and IL-8 supports this cascade theory (18). Other cytokines including IL-4, IL-10, and transforming growth factor have been termed anti-inflammatory cytokines, since these inhibit IL-1 production and decrease experimental inflammation (19). In our study, it is possible that after an initial increase, IL-1 production has now been downregulated either by anti-inflammatory cytokines or increased levels of the naturally occurring IL-1 receptor antagonist. A negative feedback loop may also exist with the increased concentration of plasma interleukins inhibiting further MNC cytokine secretion . Another possible explanation is that the MNC from patients with an acute stroke have relatively exhausted their cytokine supply. FinaIly, the observed increased plasma IL-6 level in stroke patients may actuaIly be secondary to a non-MNC source. Although superoxide production by monocytes and PMNs is needed for infection defense, the release of free radicals during reperfusion is felt to be detrimental to ischemic neuronal cells (11). In this study, we found increased superoxide production in stroke MNC with a trend toward increased production in PMNs. Since superoxide production is widely recognized to increase with cell activation, our findings suggest that leukocytes from stroke patients have an increased level of activation. This is in agreement with our prior report that PMN adhesion is increased in stroke patients (20). It also suggests that the decreased cytokine secretion observed in the MNC of our stroke patients is not due to a generalized decreased level of leukocyte activation. One prior study by Grau et al. (21) found a decreased level of PMN superoxide production in acute stroke patients compared to controls, but MNC production was not examined in this study. In our study, we used an MNC population and did not isolate the pure monocyte subset. We chose to use MNC to avoid the in vitro activation that can occur during pure monocyte isolation. Since cytokines are felt to be produced predominantly by monocytes, one possible explanation for our findings is that there was a lower percentage of monocytes in the isolated MNC from the stroke group. Although we did not do a differential analysis on all of the MNC isolations, our isolation technique has previously produced a consistent, approximately 30% monocyte recovery in 170
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both stroke and control subjects. Further, there was no difference in the percentage of peripheral monocytes between the stroke and control groups, and the total number of monocytes was actually higher in the stroke group. Finally, since monocytes are also responsible for MNC superoxide production, the higher MNC cell superoxide production observed in the stroke group makes it unlikely that there were less monocytes present in the stroke MNC isolates. We conclude that changes in cytokine and superoxide production occur during acute stroke. These findings support the active role of leukocyte and inflammatory mediators in CNS ischemia. Further studies are needed to investigate the time course of these changes and the role of various anti-inflammatory mediators. Acknowledgment: This study was supported in part by a National Stroke Association!Allied Signal Inc. Award, an NIH:CIDA award to Dr. Clark, and NINDS grant 2P01 NS17493-08 to Dr. Coull.
References 1. Benveniste EN. Inflammatory cytokines within the central nervous system: sources, function, and mechanism of action. Am J PhysioI1992;263:Cl-16. 2. Higgins GA, Olschowska ]A. Induction of interleukin113 mRNA in adult rat brain. Mol Brain Res 1991 ;9:1438. 3. Griffin WST, Stanley LC, ling C, et al. Brain interleukin 1 and S-100 immunoreactivity are elevated in Down synd rome and Alzheimer disease. Proc Natl Acad Sci USA 1986;7611.
4. Merrill ]E, Strom SR, Ellison GW, Myers LW. In vitro study of mediators of inflammation in multiple sclerosis. J cu« lmmunol 1989;9:84-96. 5. Hirohata S, Miyamoto T. Elevated levels of interleukin6 in cerebrospinal fluid from patients with systemic lupus erythematosus and central nervous system involvement. Arthritis Rheum 1990 ;33 :644-9. 6. Gijbels K, VanDamme L Proost P, Put W, Carton H, Billiau A. Interleukin 6 production in the central nervous system during experimental autoimmune encephalomyelitis. Eur J IlIlmunoI1990;20:233-5. 7. Minami M, Kuraishi Y,Yabuuchi K, Yamazaki A, Satoh M. Induction of interleukin-Ijl mRNA in rat brain after transient forebrain ischemia. J Neurochem 1991;58: 390-2. 8. ReIton ]K, Rothwell N]. Interleukin-l receptor antagonist inhibits ischaemic and excitotoxic neuronal damage in the rat. Brain Res BuI/1992;29 :243-6. 9. Licinio ], Wong M, Gold PW. Localization of interleukin-1 receptor antagonist mRNA in rat brain. Endocrinology 1991;129:562-4.
10. Dinarello C, Gelfand ], Wolff S. Anticytokine strategies in the treatment of the systemic inflammatory response syndrome. JAMA 1993;269 :1829-35.
CYfOKlNE PRODUCTION IN STROKE
11. Hallenbeck JM, Dutka AJ. Background review and current concepts of reperfusion injury. Arch Neuro11990; 47:1245-54. 12. Coull BM, Beamer NB, Clark WM. Elevated plasma interleukin-S (IL-6) in acute stroke. Stroke 1993;24:183. 13. Rudick RA,RansohoffRM. Cytokine secretion by multiple sclerosis monocytes: relationship to disease activity. Arch NellroI1992;49:265-70. 14. Boyum A. Isolation of mononuclear cells and granulocytes from human blood. Scand J Clin Lab Invest 1968; 97:77. 15. Kasahara K, Strieter RM, Chensue SW, Standiford TJ, KunkelSL Mononuclear cell adherence induces neutrophil chemotactic factor/interleukin-8 gene expression. J Leukocyte BioI1991;50:287-95. 16. Pick E, Mizel D. Rapid microassays for the measurement of superoxide and hydrogen peroxide production by microphages in culture using an automatic
17.
18.
19. 20. 21.
enzyme immunoassay reader. J Jnl/IIIIII Methods 1981; 46:211-26. Coull B, Beamer N, de Garmo P, et a1. Chronic blood hyperviscosity in subjects with acute stroke, transient ischemic attack, and risk factors for stroke. Stroke 1991; 22:162-8. POTat R, Poutsiaka DD, Miller LC, Granowitz EV, Dinarello CA. Interleukin-l (Ilol) receptor blockade reduces endotoxin and Borrelia burdorferi·stimulated 11.-8 synthesis in human mononuclear cells. FASEB J 1992; 6:482-6. Dinarello CA. Interleukin-I and interleukin-1 antagonism. Blood 1991;77:1627-52. Clark WM, Coull BM, Corliss L, et a1. Role of leukocyte adhesion in clinical stroke. J Stroke Cerebrovasc Dis 1991;2:80-4. Grau A, Berger E,Sung K,Schmid-Schonbein G. Granulocyte adhesion, deforrnability, and superoxide formation in acute stroke. Stroke 1992;23:33-9.
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Cytokine and Superoxide Production in Clinical Stroke Wayne M. Clark, M.D., Jeffrey D. Lauten, Nancy B. Beamer, M.s., and Bruce M. Coull, M.D.
Cytokines appear to play an important rol e in a variety of central nervous system (CNS) diseases and may be involved in ischemia. Activated leukocytes are a major source of cytokines and other inflammatory mediators that may directly injure ischemic CNS tissue. To investigate the importance of these substances in clinical stroke, we compared spontaneous secretion of IL-1~, IL-6,IL-8, and superoxide production from neutrophils (PMN) and mononuclear cells (MNC) isolated from either acute stroke patients (n "" 10) or matched controls (n "" 10). Cyto kine production from 5 X 106 cells incubated for 24 h was determined by EUSA. MNC from acute stroke patients secreted significantly less IL-1~, IL-6,and IL-8than MNC from control patients. No significant cytokine production was detected from PMN . Superoxide production from 1.25 X 105 cells was determined over 1 h measuring cytochrome C reduction. There was a trend toward higher superoxide production in PMN from acute stroke patients, whereas the superoxide production in MNC from acute stroke patients was significantly higher than that of control MNC. This difference persisted even when various agonists were added preincubation. This study suggests that MNC sponlaneous cylokine secretion is reduced in acute stroke patients, whereas superoxide production is increased. This decreased cytokine production may be secondary to the effects of a cytokine inhibitory factor. Key Words: Cytokines-Stroke-Brain-Interleukins.
There is increasing evidence that inflammatory responses play an important role in the pathogenesis of several central nervous system (CNS) diseases. Many of these inflammatory responses are mediated by interleukins, a multifunctional subclass of cytokines. The pro-inflammatory interleukins, including IL-1, IL-6, and IL-8, can influence the function and synthesis of other cytokines via a complex cytokine network (1). These pro-inflammatory interleukins are produced by a variety of cells including leukocytes and
From the Department of Neurology, Oregon Stroke Center, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Road, Portland, OR, U.S.A. Address correspondence and reprint requests to Dr. W. M. Clark at Department of Neurology Ll04, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Road, Portland, OR 97201, U.S.A. 166
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microglia and appear to influence CNS infiltration by leukocytes and may modulate CNS cellapoptosis, differentiation, and proliferation (1). Increased levels of IL-1,IL-6,and IL-8 have been detected in cerebrospinal fluid or CNS tissue sections in brain injury (2), Alzheimer's disease (3), multiple sclerosis (4), CNS lupus (5), and CNS infections (6). Recent studies also suggest that the pro-inflammatory interleukins may be involved in stroke potentiation and recovery. IL-l induces glial cell proliferation and promotes leukocyte endothelial adhesion via increased intercellular adhesion molecule expression (7). Increased levels of IL-1 have been observed following experimental forebrain ischemia (7), and treatment with an antagonist to IL-l (ILl-ra) has been demonstrated to reduce experimental eNS ischemic injury (8). Increased endogenous production ofILlra has also been observed in ischemic hippocampal
CYfOKINE PRODUCTION IN STROKE
neurons (9). IL-8 mediates leukocyte adhesion and emigration and stimulates the release of enzymes from neutrophils (10). There is increasing evidence that these leukocyte functions are involved in CNS reperfusion injury (11). IL-6also stimulates hepatocytes to synthesis fibrinogen and other prothrombotic proteins (1). We have recently demonstrated that plasma fibrinogen and IL6 levels are higher in stroke patients than in matched controls (12). However, since interleukins are active in very low (picomolar) concentrations, we have not been able to detect plasma levels of the other proinflammatory interleukins in stroke patients using standard ELISAtechniques. Since mononuclear cells (Mr'{C) are a major source of interleukin production, an alternate strategy is to assess the secretion of cytokines from isolated MNC. An additional advantage of this method is that the production of other inflammatory mediators, including oxygen-free radicals, can be concurrently assessed. This strategy was recently utilized by Rudick et al. to detect changes in cytokine production in multiple sclerosis patients (13). In our study, we compared the secretion ofIL-1, IL-6, IL-8, and superoxide in leukocytes isolated from acute stroke patients to that of leukocytes from matched control subjects.
Methods Study Participants Subjects were recruited from the wards and outpatient clinics of the Portland Veterans Administration Hospital and the Oregon Health Sciences University into two groups: patients with an acute stroke (n = 10, cytokine, and n = 11, superoxide), and healthy control subjects balanced for age with the stroke group (n = 10). Subjects were eligible forrecruitment into the stroke group if they had an ischemic stroke resulting in a significant neurologic deficit (at least four points on NIH Stroke Scale) in the previous 5 days. Volunteers of similar age and sex comprising the control group denied the presence of risk factors for stroke, were currently nonsmoking, and were not taking prescription medication other than aspirin. All subjects gave written informed consent to participate and undergo phlebotomy by members of the neurology staff. Fasting blood samples were drawn (days 3-5 poststroke) from an antecubital vein by venipuncture with minimal stasis into vacutainers (Becton-Dickinson, Rutherford, NJ) and were anticoagulated with sodium heparin. Routine laboratory measurements were performed by laboratories of the
Clinical Pathology Department of Oregon Health Sciences University. Plasma IL-6 levels were determined using the sandwich ELISA protocol described below.
Isolation of Cells Human neutrophils (PMN) and MNC were isolated using a modified Boyum density gradient centrifugation method (14). At room temperature, heparinized blood was carefully layered over Monopoly Resolving Media (1CN Biomedicals, Inc., Los Angeles, CA) and Histopaque 1077 (Sigma, St. Louis, MO), then centrifuged at 500 g until two distinct leukocyte bands appeared. The upper band contained MNC, and the lower band contained PMN. The MNC and PMN were removed independently, washed twice with CA2 and M g2-free phosphate buffered saline (PBS) and counted in a hemacytometer. A slide differential count (nonspecific esterase) of the MNC band revealed 95% MNC with a morphologic analysis revealing approximately 30% monocytes. Evaluation of the PMN band revealed about a 2% MNC contamination.
Measurement of Cytokine Secretion MNC and PMN were adjusted to 4 X 10 6 cell/ml in Earles Balanced Salt Solution/25 mM HEPES (Sigma Chemical Co., St. Louis, MO). One milliterofeach cell population was added to a 24-well, tissue-culture, treated polystyrene dish (Corning) or a 15-ml Teflon chamber (Savillex, Minnetonka, MN). Cells were incubated at 37°C for 24 h, after which the conditioned medium was removed, aliquoted, and stored at - 20°C. The levels ofIL-1~, IL-6, and lL-8 in the conditioned medium were measured by sandwich ELISAs following the manufacturer's protocols (IL-1~, AMAC Inc., Westbrook, ME;IL-6,IL-8,R&D Systems, Minneapolis, MN). Samples of media were diluted, and concentrations of cytokines were determined against standard curves with the following ranges: IL-1/3, 15.6-1,000 pg/ml; IL-6, 3.13-300 pg/ml; and IL-8, 93.8-6,000 pg/ml. Previous investigations have shown that adherence to plastic wells increases MNC activation and cytokine production (15). To determine if cell adhesion affected the observed differences in cytokine secretion between stroke and control patients, we compared the spontaneous secretion of nonadherent cells (Teflon wells) to that of adherent cells (plastic wells).
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Measurement of Superoxide Production Samples from the same PMN and MNC populations used above were adjusted to 5 X 106 cells/ml in DPBS containing Mg2 and Ca 2 and assayed for superoxide production over 60 min via the superoxidedependent reduction of ferricytochrome C (16). The following was added to a 96-well microplate: 25 ul of either PMN or MNC at 5 X 10 6 cell/ml, 100 III of ferricytochrome C at 2.5 mg/ml (Sigma, St. Louis, MO), 100 III DPBS/Ca2M g2, and 25 III of either superoxide dismutase at 1 mg/ml (Sigma) or recombinant human IL-1[3, IL-6, or IL-8 (R&D Systems, Minneapolis, MN) at 20 ng/ml. We added the various cytokines to the samples to determine their influence on superoxide production. The optical density was read at 550 nm, and the plate was incubated at 37°C 5% CO 2 for 60 min, after which the final optical density was determined. The wells containing superoxide dismutase were subtracted as blanks to eliminate any superoxide-independent reduction of ferricytochrome C. Additional wells containing cytokines were also assayed with superoxide dismutase, and no superoxide-independent reduction of ferricytochrome C was induced. Using the extinction coefficient of ferricytochrome C (21.1 mM-l cm -1) the number of moles of superoxide (° 2- ) generated was determined by the following equation: nmols O 2-/125,000 cells = change in OD 550 nm X 23.9. Differences among groups were assessed by analysis of variance, and when there was a significant difference (p < 0.05) among groups, the p value was determined by Bonferroni-corrected post-hoc t tests.
Results A total of21 patients were evaluated. There was no significant difference among age, sex, and aspirin usage between the two groups. Hematologic values for the groups are shown in Table 1. There was no significant difference in hematocrit between the groups; however, serum fibrinogen, total leukocyte counts, and total monocyte count (total WBC X percent monocytes) were higher in the stroke group compared to controls. Significantly higher levels of plasma IL-6 were also found in the stroke group. We did not measure plasma IL-8 or IL-1[3 in this study, but we previously found that levels of these interleukins were undetectable in plasma from stroke patients. For all three cytokines, secretion by PMN was always less than 2% of the corresponding MNC well value. Since the PMN samples contained up to a 2% MNC con168
J STROKE CEREBROVASC DIS, VOL. 5, NO.3,
1995
Table 1.
Hematologic profile (mean ± SD) Group
Descriptor
Stroke (n = 10)
Hematocrit (%) (mean ± SD) Leukocyte (X 103) Total monocytes (X 10 3) Fibrinogen (mg/dl) Plasma IL-6 (pg/ml)
10 ± 4.3" 0.73 ± 0.1" 348 ± 56" 11.5 ± 10"
44
±6
Control (n = 10) 45
±4
4.8 ± 1.2 0.51 ± 0.2 252 ± 44 2.6 ± 1.3
"p < 0.05 higher than control.
tamination, there did not appear to be any significant PMN cytokine secretion for either group. Figure 1 shows the results of cytokine secretion by MNC at 24 h. Overall, incubation in plastic wells produced significantly higher cytokine values than Teflon well incubation in the control group but not the stroke group. Lower levels of MNC secretion at 24 h were seen for all three cytokines in the stroke group compared to controls. For IL 1[3, the levels in the stroke group (n = 10) were 21,240 ± 5,112 (mean ± SE) pg/ml per 4 X 106 cells (plastic) and 11,921 ± 2,434 pg/ml (Teflon), and the levels in the control group (n = 10) were 47,822 ± 13,250 pg/ml (plastic) and 27,906 ± 6,949 pg/ml (Teflon) (p < 0.05) (see Fig. I, top). For IL-6, the stroke group (n = 10) had levels of 8,663 ± 1,856 pg/ml (plastic) and 9,332 ± 1,500 (Tef1011); control group, (n = 10) 15,623 ± 2,157 pg/ml (plastic) (p < 0.05) and 14,166 ± 2,210 pg/ml (Teflon) (see Fig. I, center). For IL-B, the stroke group (n = 10) had levels of 174,455 ± 40,178 pg/ml (plastic) and 193,589 ± 27,056 pg/ml (Teflon); control group (n = 10),279,207 ± 37,310 pg/ml (plastic) (p < 0.05) and 214,840 ± 29,747 pg/ml (Teflon) (see Fig. I, bottom). Overall, there was a significant negative correlation (r = -0.38) between the MNC IL-6 secretion values and the corresponding plasma IL-6 level. The results of superoxide production for PMN and MNC are shown in Fig. 2. For PMN, there was a nonsignificant trend toward higher superoxide production in the stroke group (n = 11),0.44 ± 0.10 (mean ± SE) nmol O 2-/125,000 cells, compared to that for the control group (n = 10),0.30 ± 0.04 nmol. For MNC, production in the stroke group (n = 11) 0.302 ± 0.04 nmol was significantly higher compared to the control group (n = 10) 0.09 ± 0.05 nmol (p < 0.01). The addition of 20 ng/ml of IL-1[3 to the incubation produced a marked increase in superoxide production by both cells types, whereas the addition of equivalent amounts of IL-6 and IL-8 had no effect.
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production was significantly decreased, whereas superoxide production was increased in MNC isolated from stroke patients compared to controls. The stroke group also had significantly higher leukocyte counts, fibrinogen levels, and serum IL-6 levels. We have previously reported similarly increased leukocyte, fibrinogen, and IL-6 values in stroke patients in a larger cross-sectional study (12,17). Despite a concurrent elevated level of plasma IL-6, we observed lower levels of IL-6, IL-l~, and IL-S secretion in MNC from stroke patients compared to matched controls. Although, to our knowledge, this is the first report of cytokine secretion in stroke patients, similar findings have been observed in patients with multiple sclerosis (MS). Rudick et al. (13) found that the monocyte secretion of TNF-a and IL-l ~ were de-
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Figure 1. Spontaneous MNC inierleukin secretion (mean ± SE). MNC isolated from stroke (n = 10) or control (n = 10) patients were cultured either on Teflon [open bar) or plastic (black bar), and media wereassessed for interleukin secretion after24 h: IL-IB (top), IL-6 (center), and IL-B (bottom).
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There is increasing evidence that leukocytes and inflammatory mediators play a pathologic role in CNS ischemic injury. Clarification of the relationship between leukocytes and their secreted mediators during ischemia may provide clues for future therapeutic interventions. In this study, we found that cytokine
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Figure 2. Superoxide production by PMN andMNC (mean ± SE). Cells wereisolated from stroke {n = 11) orcontrol (n = 10) patients andwereincubated onplastic. Superoxide production was assessed at 1 h forthefollowing conditions: nostimulation or after thepreincubation addition of20 ngtml of 11-1~, IL-6, orIL-B. Top: PMN. Bottom: MNC.
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W. M. CLARK IT AL
creased in monocytes from patients with active MS compared to stable MS (13). There are several potential theories for this decreased MNC cytokine secretion following an acute stroke. First, there appears to be a "pro-inflammatory cytokine cascade" with IL-1 release leading to increased production of the other cytokines (10). The ability of IL-1 receptor antagonists to block the production of IL-6 and IL-8 supports this cascade theory (18). Other cytokines including IL-4, IL-10, and transforming growth factor have been termed anti-inflammatory cytokines, since these inhibit IL-1 production and decrease experimental inflammation (19). In our study, it is possible that after an initial increase, IL-1 production has now been downregulated either by anti-inflammatory cytokines or increased levels of the naturally occurring IL-1 receptor antagonist. A negative feedback loop may also exist with the increased concentration of plasma interleukins inhibiting further MNC cytokine secretion . Another possible explanation is that the MNC from patients with an acute stroke have relatively exhausted their cytokine supply. FinaIly, the observed increased plasma IL-6 level in stroke patients may actuaIly be secondary to a non-MNC source. Although superoxide production by monocytes and PMNs is needed for infection defense, the release of free radicals during reperfusion is felt to be detrimental to ischemic neuronal cells (11). In this study, we found increased superoxide production in stroke MNC with a trend toward increased production in PMNs. Since superoxide production is widely recognized to increase with cell activation, our findings suggest that leukocytes from stroke patients have an increased level of activation. This is in agreement with our prior report that PMN adhesion is increased in stroke patients (20). It also suggests that the decreased cytokine secretion observed in the MNC of our stroke patients is not due to a generalized decreased level of leukocyte activation. One prior study by Grau et al. (21) found a decreased level of PMN superoxide production in acute stroke patients compared to controls, but MNC production was not examined in this study. In our study, we used an MNC population and did not isolate the pure monocyte subset. We chose to use MNC to avoid the in vitro activation that can occur during pure monocyte isolation. Since cytokines are felt to be produced predominantly by monocytes, one possible explanation for our findings is that there was a lower percentage of monocytes in the isolated MNC from the stroke group. Although we did not do a differential analysis on all of the MNC isolations, our isolation technique has previously produced a consistent, approximately 30% monocyte recovery in 170
J STROKE CEREBROVASC DIS, VOL. 5, NO.3,
1995
both stroke and control subjects. Further, there was no difference in the percentage of peripheral monocytes between the stroke and control groups, and the total number of monocytes was actually higher in the stroke group. Finally, since monocytes are also responsible for MNC superoxide production, the higher MNC cell superoxide production observed in the stroke group makes it unlikely that there were less monocytes present in the stroke MNC isolates. We conclude that changes in cytokine and superoxide production occur during acute stroke. These findings support the active role of leukocyte and inflammatory mediators in CNS ischemia. Further studies are needed to investigate the time course of these changes and the role of various anti-inflammatory mediators. Acknowledgment: This study was supported in part by a National Stroke Association!Allied Signal Inc. Award, an NIH:CIDA award to Dr. Clark, and NINDS grant 2P01 NS17493-08 to Dr. Coull.
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CYfOKlNE PRODUCTION IN STROKE
11. Hallenbeck JM, Dutka AJ. Background review and current concepts of reperfusion injury. Arch Neuro11990; 47:1245-54. 12. Coull BM, Beamer NB, Clark WM. Elevated plasma interleukin-S (IL-6) in acute stroke. Stroke 1993;24:183. 13. Rudick RA,RansohoffRM. Cytokine secretion by multiple sclerosis monocytes: relationship to disease activity. Arch NellroI1992;49:265-70. 14. Boyum A. Isolation of mononuclear cells and granulocytes from human blood. Scand J Clin Lab Invest 1968; 97:77. 15. Kasahara K, Strieter RM, Chensue SW, Standiford TJ, KunkelSL Mononuclear cell adherence induces neutrophil chemotactic factor/interleukin-8 gene expression. J Leukocyte BioI1991;50:287-95. 16. Pick E, Mizel D. Rapid microassays for the measurement of superoxide and hydrogen peroxide production by microphages in culture using an automatic
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