The role of cortisol in immunosuppression in subarachnoid haemorrhage.

BACKGROUND: We sought to determine the extent to which cortisol suppressed innate and T cell-mediated cytokine production and whether it could be involved in reducing peripheral cytokine production following subarachnoid haemorrhage (SAH). METHODS: Whole blood from healthy controls, patients with SAH and healthy volunteers was stimulated with lipopolysaccharide (LPS), to stimulate innate immunity, or phytohaemagglutinin (PHA), to stimulate T cell-mediated immunity. Varying concentrations of cortisol were included, with or without the cortisol antagonist RU486. Concentration of interleukin-6 (IL-6), IL-1ß and tumour necrosis factor-alpha) TNFα were determined as a measure of innate immunity. IL-6, IL-17 (interferon gamma) IFNƔ and IL-17 were determined as an indicator of T cell-mediated immunity. RESULTS: Suppression of innate responses to LPS was apparent in whole blood from SAH patients, relative to healthy controls, and TNFα production was inversely correlated with plasma cortisol concentration. Cytokine production in whole blood from healthy volunteers was inhibited by cortisol concentrations from 0.33 µM, or 1 µM and above, and these responses were effectively reversed by the cortisol antagonist RU-486. In SAH patients, RU-486 reversed suppression of innate TNF-α and IL-6 responses, but not IL-1ß or T cell-mediated responses. CONCLUSION: These data suggest that cortisol may play a role in reducing innate, but not T cell-mediated immune responses in patients with injuries such as SAH and that cortisol antagonists could be effective in boosting early innate responses.

Does previous stroke modify the relationship between inflammatory biomarkers and clinical endpoints in CKD patients?

BACKGROUND: Chronic kidney disease (CKD) is an independent risk factor for stroke. Stroke is also an independent risk factor for worse CKD outcomes and inflammation may contribute to this bidirectional relationship. This study aims to investigate inflammatory biomarkers in patients with non-dialysis CKD (ND-CKD) with and without stroke. METHODS: A propensity matched sample from > 3000 Salford Kidney Study (SKS) patients, differentiated by previous stroke at study recruitment, had stored plasma analyzed for interleukin- 6 (IL-6), Von Willebrand Factor (VWF) and C-reactive protein (CRP). Multivariable cox regression analysis investigated associations between inflammation and death, end-stage renal disease (ESRD) and future non-fatal cardiovascular events (NFCVE). RESULTS: A total of 157 previous stroke patients were compared against 162 non-stroke patients. There were no significant differences in inflammatory biomarkers between the two groups. Previous stroke was associated with greater mortality risk, hazard ratio (HR) (95% CI) was 1.45 (1.07-1.97). Higher inflammatory biomarker concentrations were independently associated with death but not ESRD or NFCVE in the total population. For each 1 standard deviation (SD) increase in log IL-6, VWF and CRP, the HR for all-cause mortality were 1.35 (1.10-1.70), 1.26 (1.05-1.51) and 1.34 (1.12-1.61), respectively. CRP retained its independent association (HR 1.47 (1.15-1.87)) with death in the stroke population. CONCLUSION: Previous stroke is an important determinant of mortality. However, the adverse combination of stroke and ND-CKD does not seem to be driven by higher levels of inflammation detected after the stroke event. Biomarkers of inflammation were associated with worse outcome in both stroke and non-stroke ND-CKD patients. TRIAL REGISTRATION: 15/NW/0818 .

SCIL-STROKE (Subcutaneous Interleukin-1 Receptor Antagonist in Ischemic Stroke): A Randomized Controlled Phase 2 Trial.

BACKGROUND AND PURPOSE: The proinflammatory cytokine IL-1 (interleukin-1) has a deleterious role in cerebral ischemia, which is attenuated by IL-1 receptor antagonist (IL-1Ra). IL-1 induces peripheral inflammatory mediators, such as interleukin-6, which are associated with worse prognosis after ischemic stroke. We investigated whether subcutaneous IL-1Ra reduces the peripheral inflammatory response in acute ischemic stroke. METHODS: SCIL-STROKE (Subcutaneous Interleukin-1 Receptor Antagonist in Ischemic Stroke) was a single-center, double-blind, randomized, placebo-controlled phase 2 trial of subcutaneous IL-1Ra (100 mg administered twice daily for 3 days) in patients presenting within 5 hours of ischemic stroke onset. Randomization was stratified for baseline National Institutes of Health Stroke Scale score and thrombolysis. Measurement of plasma interleukin-6 and other peripheral inflammatory markers was undertaken at 5 time points. The primary outcome was difference in concentration of log(interleukin-6) as area under the curve to day 3. Secondary outcomes included exploratory effect of IL-1Ra on 3-month outcome with the modified Rankin Scale. RESULTS: We recruited 80 patients (mean age, 72 years; median National Institutes of Health Stroke Scale, 12) of whom 73% received intravenous thrombolysis with alteplase. IL-1Ra significantly reduced plasma interleukin-6 (P<0.001) and plasma C-reactive protein (P<0.001). IL-1Ra was well tolerated with no safety concerns. Allocation to IL-1Ra was not associated with a favorable outcome on modified Rankin Scale: odds ratio (95% confidence interval)=0.67 (0.29-1.52), P=0.34. Exploratory mediation analysis suggested that IL-1Ra improved clinical outcome by reducing inflammation, but there was a statistically significant, alternative mechanism countering this benefit. CONCLUSIONS: IL-1Ra reduced plasma inflammatory markers which are known to be associated with worse clinical outcome in ischemic stroke. Subcutaneous IL-1Ra is safe and well tolerated. Further experimental studies are required to investigate efficacy and possible interactions of IL-1Ra with thrombolysis. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: ISRCTN74236229.

Reduction of inflammation after administration of interleukin-1 receptor antagonist following aneurysmal subarachnoid hemorrhage: results of the Subcutaneous Interleukin-1Ra in SAH (SCIL-SAH) study.

OBJECTIVE Aneurysmal subarachnoid hemorrhage (aSAH) is a devastating cerebrovascular event with long-term morbidity and mortality. Patients who survive the initial bleeding are likely to suffer further early brain injury arising from a plethora of pathological processes. These may result in a worsening of outcome or death in approximately 25% of patients and may contribute to longer-term cognitive dysfunction in survivors. Inflammation, mediated by the cytokine interleukin-1 (IL-1), is an important contributor to cerebral ischemia after diverse forms of brain injury, including aSAH. Its effects are attenuated by its naturally occurring antagonist, IL-1 receptor antagonist (IL-1Ra [anakinra]). The authors hypothesized that administration of additional subcutaneous IL-1Ra would reduce inflammation and associated plasma markers associated with poor outcome following aSAH. METHODS This was a randomized, open-label, single-blinded study of 100 mg subcutaneous IL-1Ra, administered twice daily in patients with aSAH, starting within 3 days of ictus and continuing until 21 days postictus or discharge from the neurosurgical center, whichever was earlier. Blood samples were taken at admission (baseline) and at Days 3-8, 14, and 21 postictus for measurement of inflammatory markers. The primary outcome was difference in plasma IL-6 measured as area under the curve between Days 3 and 8, corrected for baseline value. Secondary outcome measures included similar area under the curve analyses for other inflammatory markers, plasma pharmacokinetics for IL-1Ra, and clinical outcome at 6 months. RESULTS Interleukin-1Ra significantly reduced levels of IL-6 and C-reactive protein (p < 0.001). Fibrinogen levels were also reduced in the active arm of the study (p < 0.002). Subcutaneous IL-1Ra was safe, well tolerated, and had a predictable plasma pharmacokinetic profile. Although the study was not powered to investigate clinical effect, scores of the Glasgow Outcome Scale-extended at 6 months were better in the active group; however, this outcome did not reach statistical significance. CONCLUSIONS Subcutaneous IL-1Ra is safe and well tolerated in aSAH. It is effective in reducing peripheral inflammation. These data support a Phase III study investigating the effect of IL-1Ra on outcome following aSAH. Clinical trial registration no.: EudraCT: 2011-001855-35 ( www.clinicaltrialsregister.eu ).

The effect of intravenous interleukin-1 receptor antagonist on inflammatory mediators in cerebrospinal fluid after subarachnoid haemorrhage: a phase II randomised controlled trial.

BACKGROUND: Interleukin-1 (IL-1) is a key mediator of ischaemic brain injury induced by stroke and subarachnoid haemorrhage (SAH). IL-1 receptor antagonist (IL-1Ra) limits brain injury in experimental stroke and reduces plasma inflammatory mediators associated with poor outcome in ischaemic stroke patients. Intravenous (IV) IL-1Ra crosses the blood-brain barrier (BBB) in patients with SAH, to achieve cerebrospinal fluid (CSF) concentrations that are neuroprotective in rats. METHODS: A small phase II, double-blind, randomised controlled study was carried out across two UK neurosurgical centres with the aim of recruiting 32 patients. Adult patients with aneurysmal SAH, requiring external ventricular drainage (EVD) within 72 hours of ictus, were eligible. Patients were randomised to receive IL-1Ra (500 mg bolus, then a 10 mg/kg/hr infusion for 24 hours) or placebo. Serial samples of CSF and plasma were taken and analysed for inflammatory mediators, with change in CSF IL-6 between 6 and 24 hours as the primary outcome measure. RESULTS: Six patients received IL-1Ra and seven received placebo. Concentrations of IL-6 in CSF and plasma were reduced by one standard deviation in the IL-1Ra group compared to the placebo group, between 6 and 24 hours, as predicted by the power calculation. This did not reach statistical significance (P = 0.08 and P = 0.06, respectively), since recruitment did not reach the target figure of 32. No adverse or serious adverse events reported were attributable to IL-1Ra. CONCLUSIONS: IL-1Ra appears safe in SAH patients. The concentration of IL-6 was lowered to the degree expected, in both CSF and plasma for patients treated with IL-1Ra.

Overcoming matrix matching problems in multiplex cytokine assays.

Failure to match assay matrices with samples in immunoassays can result in incorrect sample values being reported. For multiplex assays this presents particular problems, due to the need to find a matrix suitable for all the analytes. Here, we describe strategies adopted to overcome matrix problems identified in establishing a cytokine multiplex assay in human plasma. Standard analytes were diluted in plasma samples to identify representative plasma for assay development. Horse sera were screened to evaluate potential interference before using to adjust a matrix to match plasma samples. Suitability of the matrix match was confirmed by evaluating recovery of known concentrations of analytes from plasma. Individual plasmas modified the assay signal for some analytes to a variable extent, particularly for IL-1α and IL-1ß. Addition of horse serum to assay buffer improved matching to plasma samples, although endogenous MCP-1 activity was apparent in one sample. Matching of plasma and assay matrices allowed recoveries within 10% to 20% of the expected values, unless the samples contained atypical interfering activity. Attention to choice of samples and diluent used for assay development is particularly important for measurement of sample analytes in cytokine multiplex assays.

Cerebrospinal fluid and plasma cytokines after subarachnoid haemorrhage: CSF interleukin-6 may be an early marker of infection.

BACKGROUND: Cytokines and cytokine receptor concentrations increase in plasma and cerebrospinal fluid (CSF) of patients following subarachnoid haemorrhage (SAH). The relationship between plasma and CSF cytokines, and factors affecting this, are not clear. METHODS: To help define the relationship, paired plasma and cerebrospinal fluid (CSF) samples were collected from patients subject to ventriculostomy. Concentrations of key inflammatory cytokines, interleukin (IL)-1ß, IL-1 receptor antagonist (IL-1Ra), IL-1 receptor 2, IL-6, IL-8, IL-10, tumour necrosis factor (TNF)-α, and TNF receptors (TNF-R) 1 and 2 were determined by immunoassay of CSF and plasma from 21 patients, where samples were available at three or more time points. RESULTS: Plasma concentrations of IL-1ß, IL-1Ra, IL-10, TNF-α and TNF-R1 were similar to those in CSF. Plasma TNF-R2 and IL-1R2 concentrations were higher than in CSF. Concentrations of IL-8 and IL-6 in CSF were approximately10 to 1,000-fold higher than in plasma. There was a weak correlation between CSF and plasma IL-8 concentrations (r = 0.26), but no correlation for IL-6. Differences between the central and peripheral pattern of IL-6 were associated with episodes of ventriculostomy-related infection (VRI). A VRI was associated with CSF IL-6 >10,000 pg/mL (P = 0.0002), although peripheral infection was not significantly associated with plasma IL-6. CONCLUSIONS: These data suggest that plasma cytokine concentrations cannot be used to identify relative changes in the CSF, but that measurement of CSF IL-6 could provide a useful marker of VRI.

Interleukin-1 receptor antagonist reverses stroke-associated peripheral immune suppression.

INTRODUCTION: Infections are common following stroke and adversely affect outcome. Cellular immune suppression associated with acute stroke may increase susceptibility to infection. Cytokines are important contributors to both stroke pathology and the response to infection. Since interleukin (IL)-1 blockade is a candidate treatment for cerebral ischemia, we examined whether administration of interleukin-1 receptor antagonist (IL-1Ra) to patients with acute stroke affected innate cellular immune responses in a phase II placebo-controlled trial. METHODS: Venous blood samples were taken prior to treatment initiation, at 24h and 5 to 7d. Blood was also drawn from stroke-free controls. Lipopolysaccharide (LPS) stimulation of whole-blood cultures assessed the potential of leukocytes to produce cytokines. RESULTS: Induction of tumor necrosis factor (TNF)-α, IL-1ß, IL-6, IL-8 and IL-10 by LPS was significantly reduced in patients at admission, compared to controls. At 24h, cytokine induction remained suppressed in the placebo group. In contrast, for patients treated with IL-1Ra, induction of TNF-α, IL-6 and IL-10 was similar to controls and IL-1ß induction was significantly greater than in the placebo group. At 5 to 7d, TNF-α and IL-1ß induction remained suppressed only in the placebo group (p<0.05). Plasma cortisol concentrations, elevated at admission in patients compared to controls, were substantially reduced at 24h in the patients receiving IL-1Ra (p<0.05) and inversely correlated (p<0.001) with either TNF-α (r=-0.71) or IL-1ß induction (r=-0.67) at admission. CONCLUSION: Treatment with IL-1Ra reverses peripheral innate immune suppression in the acute phase of stroke, which is associated with attenuated cortisol production. The mechanisms underlying these observations, including the potential impact of IL-1Ra on stroke severity and the clinical significance of immune suppression, require further evaluation in larger studies.

Reconstituting National Institute for Biological Standards and Control (NIBSC) chemokines.

We observed significant discrepancies between immunoassay results when using different internally prepared reference preparations for interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) from the National Institute for Biological Standards and Control (NIBSC). To evaluate the reasons for this we prepared the chemokines using diluents that incorporated protein at different steps. This showed that even brief addition of water to these preparations, in the absence of additional protein, resulted in loss of immunoreactivity in assays. The data obtained emphasise the importance of adding protein at an early stage of preparation to avoid loss of material and potential loss of activity.

Clinical outcome following acute ischaemic stroke relates to both activation and autoregulatory inhibition of cytokine production.

BACKGROUND: As critical mediators of local and systemic inflammatory responses, cytokines are produced in the brain following ischaemic stroke. Some have been detected in the circulation of stroke patients, but their role and source is unclear. Focusing primarily on interleukin(IL)-1-related mechanisms, we serially measured plasma inflammatory markers, and the production of cytokines by whole blood, from 36 patients recruited within 12 h and followed up to 1 year after acute ischaemic stroke (AIS). RESULTS: Admission plasma IL-1 receptor antagonist (IL-1ra) concentration was elevated, relative to age-, sex-, and atherosclerosis-matched controls. IL-1beta, soluble IL-1 receptor type II, tumour necrosis factor (TNF)-alpha, TNF-RII, IL-10 and leptin concentrations did not significantly differ from controls, but peak soluble TNF receptor type I (sTNF-RI) in the first week correlated strongly with computed tomography infarct volume at 5-7 days, mRS and BI at 3 and 12 months. Neopterin was raised in patients at 5-7 d, relative to controls, and in subjects with significant atherosclerosis. Spontaneous IL-1beta, TNF-alpha and IL-6 gene and protein expression by blood cells was minimal, and induction of these cytokines by lipopolysaccharide (LPS) was significantly lower in patients than in controls during the first week. Minimum LPS-induced cytokine production correlated strongly with mRS and BI, and also with plasma cortisol. CONCLUSION: Absence of spontaneous whole blood gene activation or cytokine production suggests that peripheral blood cells are not the source of cytokines measured in plasma after AIS. Increased plasma IL-1ra within 12 h of AIS onset, the relationship between sTNF-RI and stroke severity, and suppressed cytokine induction suggests early activation of endogenous immunosuppressive mechanisms after AIS.

Comparison of 'real-time' and immunometric RT-PCR: RNA interference of reverse transcriptase-PCR.

We measured interleukin-6 (IL-6) mRNA in whole blood, using an immunometric reverse transcriptase-polymerase chain reaction (IRT-PCR) method and 'real-time' RT-PCR using the Roche LightCycler. Both methods showed similar precision, in terms of inter- and intra-assay variation, where a lower total RNA concentration was used at a relatively high ratio of (IL-6) mRNA to total RNA. However, the 'real-time' RT-PCR method showed a greater loss of accuracy at higher concentrations of total RNA.

Simple, quantitative measurement of cytokine gene expression using an immunometric reverse transcriptase-polymerase chain reaction.

We have developed a novel system to quantify mRNA, using a coupled reverse transcriptase-polymerase chain reaction (RT-PCR) with RNA standards and an immunometric optical readout. Biotinylated RT-PCR products were captured onto avidin-coated microplates and hybridised to a dinitrophenol (DNP)-labelled oligonucleotide probe. Enzyme-linked anti-DNP antibodies were used to detect the product via a colorimetric enzyme assay. Unknown mRNA samples were quantified by interpolating the signal on a standard curve generated from dilutions of RNA (cRNA) standards for interleukin-6 (IL-6), tumour necrosis factor-alpha (TNFalpha), IL-1beta and actin mRNA. The assay for IL-6 mRNA was able to discriminate between samples with a two- to fourfold difference in concentration, had an intra-assay coefficient of variation (CV) of 17% and an inter-assay CV of 24%. The method was used to quantify IL-6 mRNA, relative to expression of IL-6 protein and biological activity, in human blood that had been activated by endotoxin. The IL-6 mRNA could be detected 1 h after endotoxin addition and peaked at 4 h. This paralleled the increase in protein production that followed approximately 1 h later.