SUBJECTS AND METHODS

Patients and controls

We enrolled 55 patients with acute stroke (within 24 hours after the onset) who were admitted to the Department of Neurology of the University of Milan at the San Gerardo Hospital, in Monza, Italy, from January 1996 through December 1997. The study was approved by the Ethic Committee of San Gerardo Hospital. Excluded from the study were patients with history of recent (within 3 months before admission) infection, concurrent major cardiac, renal, hepatic, autoimmune and cancerous diseases, recent (within 1 year) history of prior transient ischemic attacks, stroke or head trauma, and signs of acquired infection after admission. Patients taking immunosuppressive or anti-inflammatory drugs were also excluded. Ticlopidine or heparin were admitted for secondary stroke prevention. In preliminary experiments, we tested these drugs and found them not active on cytokine release or cytokine assays (data not shown).

All patients underwent complete physical and neurologic examination at entry and stroke severity was assessed by the National Institutes of Health Stroke Scale (Brott et al., 1989) at the onset of stroke and then at days 2, 4, 10, 30, and 90 after stroke. All patients were examined with computerized tomography of the brain within 24 hours and at 5 days after stroke. The volume of ischemia or hemorrhage was determined from standard computed tomography scans by calculating the volume of an ellipsoid containing the lesion (Pullicino et al., 1997) and also using the "Cavalieri Direct Estimator," as previously described (Clatterbuck et al., 1997). Both measurements were corrected to in vivo dimensions using the analog scale found on the hard copy computed tomography image, had good interrater agreement and were highly correlated to each other (r = 0.99, P < .0001). Blood cell count, including monocytes and platelets, and analysis of erythrocyte sedimentation rate, C-reactive protein, fibrinogen, protein C and S, antiphospholipid antibodies were performed in all patients at entry and at 10 days after stroke.

Twenty healthy control subjects, without known stroke risk factors (blood donors of the San Gerardo Hospital), balanced for age with the stroke group, were also selected for analysis of cytokine release. Demographic and clinical characteristics of patients and controls are shown in Table 1. Because nine patients were lost for intercurrent infections and six patients died, 40 of 55 (73%) completed the study. Among the studied patients, 29 had ischemic and 11 had hemorragic brain lesions; their data were analyzed both together and as separate groups.

Table 1 - Demographic and clinical characteristics of patients and controls.

Full table

For cytokine measurements studies, patients' blood was serially drawn, after informed consent, through the antecubital vein within 48 hours and at days 4, 10, 30, and 90 from stroke onset. Seven milliliters were immediately used for blood cell stimulation studies, 3 mL of serum were also stored for analysis of cytokine levels.

Blood samples from controls were collected with a similar procedure at only one time point because preliminary experiments have shown that in healthy subjects cytokine release remains stable during serial venipuncture.

Whole blood stimulation

Experiments were performed as described by Fantuzzi et al. (1995) with minor modifications. Heparinized whole blood obtained from stroke patients and healthy controls was immediately processed. Blood was diluted 1:2 (v/v) in RPMI 1640 medium (without bovine serum), plated in 96-well tissue culture plates (200L/well), and incubated for 4 hours at 37°C and 5% CO₂, in the presence of 0.01 nmol/L endotoxin (LPS: phenol-extracted preparation from Escherichia coli 055: B5 [Sigma] dissolved in sterile pyrogen-free saline) or the same volume of pyrogen-free saline. After incubation, whole blood was centrifuged and supernatants collected and stored at -20°C until assay.

Cytokine assays

For each patient, assays were performed when sample collection had been completed (day 90 from stroke). Eight patients and four controls were processed in the same assay and samples were run in triplicate.

Interleukin-6 bioassay. IL-6 was measured in cell supernatant as hybridoma growth factor using the 7TD1 cell line as previously described (De Simoni et al., 1990). Results are expressed as units per milliliter in comparison with a reference curve obtained in each experiment using recombinant human IL-6 (Immunex, Seattle, WA). Reference curves obtained were comparable in all experiments. One unit in the 7TD1 assay corresponded to 1 pg human recombinant IL-6. The sensitivity of the assay was 50 U/mL.

TNF- bioassay. TNF- was measured in cell supernatant by cytotoxicity on L929 cells in the presence of 0.001 mol/L of actinomycin D as previously described (Terrazzino et al., 1997). TNF- levels were calculated using recombinant human TNF- (BASF/Knoll, Ludwigshafen, Germany; specific activity 107 U/mg) and expressed as nanograms per milliliter. The sensitivity of the bioassay was 5 pg/mL.

IL-6 and TNF- immunoassays. For the quantitative measurement of IL-6 and TNF- in serum we used both the bioassay and the Immulite Automated Immunoassay System (DPC, Cirrus, New Jersey, USA). With this method, a poly-styrene bead solid phase (Immulite Test Unit) is coated with a monoclonal antibody specific for human TNF- or IL-6, with no cross-reactivity to other cytokines. Sensitivity of the assay was 1.7 pg/mL for TNF- and 1 pg/mL for IL-6.

Statistical analysis

The significance of cytokine production in patients compared to controls was analyzed by one-way analysis of variance (Dunnett's test). Difference between ischemic and hemorragic patient samples at single time points was evaluated by Student's t-test.

Statistic analysis of correlations between cytokine values and brain lesion volume, stroke severity, outcome and peripheral inflammatory indexes was performed with the Pearson's correlation test.

RESULTS

Fig. 1 shows the time course of IL-6 release from blood cells of stroke patients. After stimulation, the release was significantly higher than that from controls' cells at all time points from days 1 to 2 after stroke until 1 month, and it was still 127% of controls after 3 months, although no longer statistically different. The highest release was observed at day 4 after stroke (224%, P < .01 versus controls), but at 30 days it was still about 160% of the controls. IL-6 was not detectable when blood cells were not exposed to LPS.

Figure 1.

Interleukin-6 bioactivity in supernatants of blood cells stimulated with lipopolysaccharide (0.01 nmol/L at 37°C for 4 hours). Data are obtained from stroke patients (n = 40) and aged-matched healthy control subjects, n = 20) and are expressed as the mean SD. *P < .05, **P < .01, Dunnett's test.

Full figure and legend (85K)

TNF- release, shown in Fig. 2, was significantly increased from days 1 to 2 up to 3 months after stroke. There was no peak response, the mean increase being about three-fold compared to controls from days 1 to 2 through day 30. At 3 months TNF- values were still 226% of controls. As for IL-6, TNF- was not detectable in nonstimulated supernatants.

Figure 2.

Tumor necrosis factor- bioactivity in supernatants of blood cells stimulated with lipopolysaccharide. For other details see Fig. 1.

Full figure and legend (77K)

When ischemic and hemorragic strokes were analyzed separately, a difference in the time-course of IL-6 release became evident (Fig. 3). In fact, IL-6 increased earlier in ischemic compared to hemorragic patients, the difference being evident at days 1 to 2 and 4, and disappearing from day 10. Thus, the peak response appeared at day 4 in the ischemic and at day 10 in the hemorragic stroke. No difference could be shown in the time-course of TNF- release in the two patient groups (data not shown).

Figure 3.

Interleukin-6 bioactivity in supernatants of blood cells stimulated with lipopolysaccharide (0.01 nmol/L at 37°C for 4 hours). Comparison between ischemic (n = 29) and hemorragic (n = 11) stroke patients. Data are expressed as the meanSD. *P < .05, **P < .01 versus hemorragic patients at a given time point, Student's t-test.

Full figure and legend (56K)

Analysis of possible correlations between the increase of cytokine release and stroke severity at the onset (expressed by the National Institutes of Health Stroke Scale score), residual damage (expressed by National Institutes of Health Stroke Scale score at 3 months) or clinical improvement (measured as a difference of the two scores) did not reveal any significant correlation (data not shown). Moreover, no relation between cytokine increase and the volume of ischemic or hemorragic lesion was observed (data not shown).

Finally, cytokine release was neither related to peripheral indeces of inflammation such as erythrocyte sedimentation rate, C-reactive protein, fibrinogen, nor to the number of leukocytes or monocytes. Antiphospholipid antibodies have been observed in one hemorragic and three ischemic patients; the increase in cytokine release in these patients was similar to that of other patients.

Analysis of serum levels of circulating cytokines by bioassays did not show detectable levels neither in controls nor in stroke patients. On the contrary, immunological analysis by Immulite showed increased concentrations of IL-6 at all time points, with peaks at 4 and 10 days (Table 2). TNF- serum levels were only slightly and not significantly increased at day 1 to 2, and were similar to controls at later times. No correlation has been found between the release from blood cells measured by bioassay and serum levels of cytokines measured by immunological assay (data not shown). Serum cytokine levels were also unrelated to stroke size and severity (data not shown).

Table 2 - Serum IL-6 and TNF- concentrations in stroke patients and controls measured by Immulite.

Full table

DISCUSSION

In this article we show for the first time that blood cells from patients with acute stroke (ex vivo) release larger amounts of IL-6 and TNF- after exposure to LPS, a trigger of cytokine production.

This finding may answer some questions and methodologic problems recently raised in the literature. Our results suggest that activated peripheral blood cells should be a major source of cytokines described in various studies (Fassbender et al., 1994; Beamer et al., 1995; Kim et al., 1996; Intiso et al., 1997; Elneihoum et al., 1996; DeGraba et al., 1996) in plasma or serum of stroke patients; accordingly, cytokine leakage from infarcted brain or from CSF should not represent the main contributor to circulating cytokines. This is in line with a previous study showing no correlation between CSF and serum cytokine levels early after stroke, when they are higher in CSF than in serum (Tarkowski et al., 1995); in the same study the correlation appeared only from day 21 to day 90 after stroke, when their gradient indicated a blood to CSF flow (Tarkowski et al., 1995). Our data of protracted release of cytokines from peripheral blood cells favors this interpretation.

Moreover, we observed increased serum IL-6 levels by immunological assay, but not by bioassay, that yielded undetectable concentration for both cytokines. For IL-6, but not for TNF, this could be due to the different sensitivity of the assays, the immunoassay being more sensitive than the 7TD1 bioassay. The different results obtained with the two assays could also indicate that most of the immunomaterial detected in serum is related to cytokine fragments or monomers without biological activity (Turtinen and Juran, 1998), and that release studies are more reliable than serum measurements.

The normal number of leukocytes and in particular of monocytes (the major source of blood cytokines) and the lack of correlation between monocyte counts and cytokine release indicate that the increased release is linked to blood cell activation and not to monocytosis.

After clearing these methodologic issues, we like to discuss the physiopathologic and clinical relevance of our finding. First of all, we observed that IL-6 release is activated in ischemic and hemorragic strokes with a different pattern. Thus, the different brain tissue reactions may play a specific role in the activation of peripheral blood cells. This is in line with the different expression of cytokine mRNA observed in brain tissue from patients who died at various times after cerebral ischemia or hemorrhage (Tomimoto et al., 1996). These observations indicate that specific brain modifications after stroke trigger peripheral immunologic activation.

The mechanism whereby a CNS lesion such as brain ischemia or hemorrhage activates peripheral blood cells is still unclear, although the existance of a signaling pathway from the brain to the periphery has been clearly shown (De Simoni et al., 1990; 1993; 1995; 1997). Experimental data show that an inflammatory stimulus (such as IL-1 or endotoxin) delivered in the brain efficiently induces inflammatory cytokine production in the periphery (De Simoni et al., 1993; 1995). The present data indicate that a peripheral immune response is elicited by stroke and further underlines the brain-immune system communication pathway in this type of CNS lesion. Because we did not find a correlation between the lesion volume or stroke severity and cytokine release, it is possible that even small lesions may activate peripheral immune system.

On the other hand, it has been repeatedly shown that peripheral cytokines may affect CNS by several mechanisms (De Simoni, 1997). Moreover, leukocytes and monocytes recruited from the periphery have been shown in brain infarcts (Kochanek and Hallenbeck, 1992; Wang et al., 1993), and a direct passage of cytokines from peripheral blood to CNS has been proposed (Banks et al., 1994; Gutierrez et al., 1993). Thus, the possibility that peripherally produced cytokines may in turn affect the brain should also be considered.

The role of cytokines in stroke is intriguing and it is difficult to establish a role of each individual inflammatory cytokine in the pathogenesis of the ischemic process, given their diverse functions and the multiplicity of various cytokine activation. Several experimental studies suggest a role for IL-6 and TNF- in the pathogenesis of CNS diseases and in neuronal damage, possible mechanisms being the inhibition of glutamate uptake and the facilitation of excitotoxicity (Ye and Sontheimer, 1996; Fine et al., 1996). On the other hand, both cytokines display also protective actions in the brain (Bruce et al., 1996) and possess neurotrophic activity in vitro, an effect which may be direct and/or mediated by neurotrophic factors (Hama et al., 1989; Gadient et al., 1990; Yamada and Hatanaka, 1994; Kossmann et al., 1996).

In line with these neurotrophic activities, our demonstration of a protracted activation of blood cells after acute stroke raises the possibility that cytokines released from these cells are involved not only in the mechanisms of acute reaction to brain infarct, but also in the mechanisms of plasticity and repair. Recently, the importance of cytokines and growth factors as signaling mechanisms not only in stroke recovery, but also as protection from further insults (ischemic tolerance or preconditioning) has been underscored (Chen and Simon, 1997; Hallenbeck, 1997; Mattson, 1997).

We have interpreted the increase in TNF- levels observed throughout the study as a consequence of the stroke event; however, data in the literature suggest that an alternative explanation should also be considered. Experimental data show that genetically stroke prone/hypertensive rats produce more TNF- in response to a provocative dose of LPS intravenous than control animals free of these risk factors (Hallenbeck et al., 1991). Thus, high TNF- levels in stroke patients could be the result of a greater susceptibility of blood cells to inflammatory stimuli present before the stroke event. The data open the possibility that genetic differences and/or stroke risk factors may contribute to the marked and long-lasting TNF- production observed. Persistent inflammatory response has been recently shown in stroke survivors, associated with increased risk for recurrent vascular events (Beamer et al., 1998).

Finally, our findings show the possibility to analyze the peripheral immunologic activation in patients with brain lesions such as stroke, with the aim of investigating their involvement in the mechanisms of stroke damage and repair.

References

1. Banks WA, Kastin AJ & Gutierrez EG. (1994) Penetration of interleukin-6 across the blood-brain barrier. Neurosci Lett 179: 53−56. | Article | PubMed | ChemPort |
2. Beamer NB, Coull BM, Clark WM, Hazel JS & Silberger JR. (1995) Interleukin-6 and interleukin-1 receptor antagonist in acute stroke. Ann Neurol 37: 800−804. | Article | PubMed | ChemPort |
3. Beamer NB, Coull BM, Clark WM, Briley DP, Wynn M & Sexton G. (1998) Persistent inflammatory response in stroke survivors. Neurology 50: 1722−1728. | PubMed | ChemPort |
4. Brott T, Adams HP, Olinger CP, Marler JR, Barsan WG, Biller J, Spilker J, Holleran R, Eberle R, Hertzberg V, Rorick M, Moomaw CJ & Walker M. (1989) Measurements of acute cerebral infarction: a clinical examination scale. Stroke 20: 7864−7870.
5. Bruce AJ, Boling W, Kindy MS, Peschon J, Kraemer PJ, Carpenter MK, Holtsberg FW & Mattson MP. (1996) Altered neuronal and microglial responses to excitotoxic and ischemic brain injury in mice lacking TNF receptors. Nature Med 2: 7788−7794.
6. Buttini M, Sauter A & Boddeke HWGM. (1994) Induction of interleukin-1 mRNA after focal cerebral ischaemia in the rat. Mol Brain Res 23: 126−134. | Article | PubMed | ChemPort |
7. Chen J & Simon R. (1997) Ischemic tolerance in the brain. Neurology 48: 306−311. | PubMed | ChemPort |
8. Clark WM. (1997) Cytokines and reperfusion injury. Neurology 49: 4S10−4S14.
9. DeGraba T, Penix L, McCarron R, Kelly B, Dutka A & Hallenbeck J. (1996) Profile of inflammation following acute ischemic stroke in humans. Cerebrovasc Dis 6: 10.
10. De Simoni MG, Sironi M, De Luigi A, Manfridi A, Mantovani A & Ghezzi P. (1990) Intracerebroventricular injection of interleukin-1 induces high circulating levels of interleukin-6. J Exp Med 171: 1773−1778. | Article | PubMed | ChemPort |
11. De Simoni MG, De Luigi A, Gemma L, Sironi M, Manfridi A & Ghezzi P. (1993) Modulation of systemic interleukin-6 induction by central interleukin-1. Am J Physiol 265 (Regulatory Integrative Comp Physiol 34): R739−R742. | PubMed | ChemPort |
12. De Simoni MG, Del Bo R, De Luigi A, Simard S & Forloni G. (1995) Central endotoxin induces different patterns of interleukin (IL)-1b and IL-6 messenger ribonucleic acid expression and IL-6 secretion in the brain and periphery. Endocrinology 136: 897−902. | Article | PubMed | ChemPort |
13. De Simoni MG. (1997) Two-way communication pathways between the brain and the immune system. A review. Neurosci Res Commun 21: 3163−3172.
14. De Simoni MG, Terreni L, Chiesa R, Mangiarotti F & Forloni GL. (1997) Interferon- potentiates IL-6 and TNF, but not IL-1 induced by endotoxin in the brain. Endocrinology 138: 125220−125226.
15. Elneihoum AM, Falke MD, Axelsson L, Lundberg EBS, Lindgarde F & Ohlsson K. (1996) Leukocyte activation detected by increased plasma levels of inflammatory mediators in patients with ischemic cerebrovascular diseases. Stroke 27: 1734−1738. | PubMed | ChemPort |
16. Fantuzzi G, Di Santo E, Sacco S, Benigni F & Ghezzi P. (1995) Role of the hypothalamus-pituitary-adrenal axis in the regulation of TNF production in mice. J Immunol 155: 3552−3555. | PubMed | ChemPort |
17. Fassbender K, Rossal S, Kammer T, Daffertshofer M, Wirth S, Dollman M & Hennerici M. (1994) Proinflammatory cytokines in serum of patients with acute cerebral ischemia: kinetics of secretion and relation to the extent of brain damage and outcome of disease. J Neurol Sci 122: 135−139. | Article | PubMed | ChemPort |
18. Fine SM, Angel RA, Perry SW, Epstein LG, Rothstein JD, Dewhurst S & Gelbard HA. (1996) Tumour necrosis factor- inhibits glutamate uptake by primary human astrocytes. J Biol Chem 271: 2615303−2615306.
19. Gadient RA, Cron KC & Otten U. (1990) Interleukin-1b and tumor necrosis factor- synergistically stimulate nerve growth factor (NGF) release from cultured rat astrocytes. Neurosci Lett 117: 335−340. | Article | PubMed | ChemPort |
20. Grau AJ, Buggle F, Becher H, Zimmermann E, Spiel M, Fent T, Maiwald M, Werle E, Zorn M, Hengel H & Hacke W. (1998) Recent bacterial and viral infection is a risk factor for cerebrovascular ischemia. Neurology 50: 196−203. | PubMed | ChemPort |
21. Gutierrez EG, Banks WA & Kastin AJ. (1993) Murine tumor necrosis factor alpha is transported from blood to brain in the mouse. J Neuroimmunol 47: 169−176. | Article | PubMed | ChemPort |
22. Hallenbeck JM, Dutka AJ, Vogel SN, Heldman E, Doron DA & Feuerstein G. (1991) Lipopolysaccharide-induced production of tumor necrosis factor activity in rats with and without risk factors for stroke. Brain Res 541: 115−120. | Article | PubMed | ChemPort |
23. Hallenbeck JM. (1997) Cytokines, macrophages, and leukocytes in brain ischemia. Neurology 49: S5−S9. | PubMed | ChemPort |
24. Hama T, Miyamoto M, Tsukui H, Nishio C & Hatanaka H. (1989) Interleukin-6 as a neurotrophic factor for promoting the survival of cultured basal forebrain cholinergic neurons from postnatal rats. Neurosci Lett 104: 340−344. | Article | PubMed | ChemPort |
25. Intiso D, Cioffi R, Lagioia G, Checchia De Ambrosio C, Apollo F, Di Viesti P, Fogli D, Simone P & Tonali P. (1997) TNF- in acute ischemic stroke. Europ J Neurol 4: S82.
26. Kim JS. (1996) Cytokines and adhesion molecules in stroke and related diseases. J Neurol Sci 137: 69−78. | Article | PubMed | ChemPort |
27. Kim JS, Yoon SS, Kim YH & Ryu JS. (1996) Serial measurement of interleukin-6, transforming growth factor-, and S-100 protein in patients with acute stroke. Stroke 27: 1553−1557. | PubMed | ChemPort |
28. Kochanek PM & Hallenbeck JM. (1992) Polymorphonuclear leukocytes and monocytes/macrophages in the pathogenesis of cerebral ischemia and stroke. Stroke 23: 1367−1379. | PubMed | ISI | ChemPort |
29. Kossmann T, Hans V, Imhof HG, Trentz O & Morganti-Kossmann MC. (1996) Interleukin-6 released in human cerebrospinal fluid following traumatic brain injury may trigger nerve growth factor production in astrocytes. Brain Res 713: 143−152. | Article | PubMed | ChemPort |
30. Liu T, Clark RK, Mc Donnell MS, Young PR, White RF, Barone FC & Feuerstein GZ. (1994a) Tumor necrosis factor-expression in ischemic neurons. Stroke 25: 1481−1488. | PubMed | ISI | ChemPort |
31. Mattson MP. (1997) Neuroprotective signal transduction: relevance to stroke. Neurosci Biobehav Rev 21: 2193−2206.
32. Minami M, Kuraishi Y, Yabuuchi K, Yamazaki A & Satoh M. (1993) Induction of interleukin-1 in rat brain after transient forebrain ischemia. J Neurochem 58: 390−392.
33. Tarkowski E, Rosengren L, Blomstrand C, Wikkelso C, Jensen C, Ekholm S & Tarkowski A. (1995) Early intrathecal production of interleukin-6 predicts the size of brain lesion in stroke. Stroke 26: 81393−81398.
34. Terrazzino S, Perego C, De Luigi A & De Simoni MG. (1997) Interleukin-6, tumor necrosis factor and corticosterone induction by central endotoxin in aged Sprague-Dawley rats. Life Sci 61: 695−701. | Article | PubMed | ChemPort |
35. Tomimoto H, Akiguchi I, Wakita H, Kinoshita A, Ikemoto A, Nakamura S & Kimura J. (1996) Glial expression of cytokines in the brains of cerebrovascular disease patients. Acta Neuropathol 92: 281−287. | Article | PubMed | ChemPort |
36. Turtinen LW & Juran BD. (1998) Standardization and comparison of an XTT-based TNF- bioassay and a TNF- ELISA. Biotechniques 24: 232−238. | PubMed | ChemPort |
37. Wang X, Yue TL, Young PR, Barone FC & Feuerstein GZ. (1995) Expression of interleukin-6, c-fos, and zif268 mRNAs in rat ischemic cortex. J Cereb Blood Flow Metab 15: 166−171. | PubMed | ChemPort |
38. Wang PY, Kao CH, Mui MY & Wang SJ. (1993) Leukocytic infiltration in acute hemispheric stroke. Stroke 24: 236−240. | PubMed | ChemPort |
39. Yamada M & Hatanaka H. (1994) Interleukin-6 protects cultured of hippocampal neurons against glutamate-induced cell death. Brain Res 643: 173−180. | PubMed | ISI | ChemPort |
40. Ye ZC & Sontheimer H. (1996) Cytokine modulation of glial glutamate uptake: a possible involvement of nitric oxide. Neuroreport 7: 132181−132185.

Acknowledgements

The authors thank Drs. Ada De Luigi (Mario Negri Institute) and Elisabetta Raggi ("E. Medea" Institute) for their contributions to part of this study.