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Autoimmune Responses to the Brain After Stroke Are Associated With Worse Outcome Kyra J. Becker, MD; Angela J. Kalil, BA; Pat Tanzi, BSN, RNCCRC; Dannielle K. Zierath, BS; Anna V. Savos, BS; J. Michael Gee, MS; Jessica Hadwin, BS; Kelly T. Carter, BS; Dean Shibata, MD; Kevin C. Cain, PhD Downloaded from http://stroke.ahajournals.org/ by guest on June 17, 2017 Background and Purpose—Immune responses to brain antigens occur after stroke, and experimental studies show that the likelihood of developing a detrimental autoimmune response to these antigens is increased by systemic inflammation at the time of stroke. The aim of this study was to determine if patients who developed infection in the poststroke period would be similarly predisposed to develop autoimmune responses to central nervous system antigens. Methods—We enrolled 114 patients within 72 hours of ischemic stroke. Clinical and demographic data were obtained, and cellular immune responses to a panel of central nervous system antigens were assessed during the initial week and again at Day 90. Outcome was assessed using the modified Rankin Scale. Results—Patients who developed an infection, especially pneumonia, in the 15 days after stroke were more likely to evidence a Th1(⫹) response to myelin basic protein and glial fibrillary acidic protein (P⫽0.019 and P⫽0.039, respectively) at 90 days after stroke. Further, more robust Th1 responses to myelin basic protein at 90 days were associated with a decreased likelihood of good outcome, even after adjusting for baseline stroke severity and patient age (OR, 0.477; 95% CI, 0.244 to 0.935; P⫽0.031). Conclusions—This study demonstrates that immune responses to brain antigens occur after stroke. Although these responses are likely to be an epiphenomenon of ischemic brain injury, the response to myelin basic protein appears to have clinical consequences. The potential role of postischemic autoimmune-mediated brain injury deserves further investigation. (Stroke. 2011;42:2763-2769.) Key Words: autoimmune 䡲 infection 䡲 MBP 䡲 outcome 䡲 stroke A fter stroke, there is a breakdown of the blood– brain barrier that allows for an encounter of central nervous system (CNS) antigens by the systemic immune system; this encounter can occur in the injured brain as well as in the periphery.1–5 Given that brain antigens are generally sequestered from the systemic immune system, the possibility for developing an autoimmune response to these antigens thus exists. In fact, studies have shown increased titers of circulating antibodies to neurofilaments and a portion of the N-methyl-D-aspartic acid receptor in individuals with a history of stroke.6,7 Cellular immune responses to myelinassociated antigens and other brain antigens are also seen in stroke survivors and are more robust than the responses seen in patients with multiple sclerosis.8 –11 In an animal model of severe focal cerebral ischemia, we showed that the predominant lymphocyte response to the brain antigen myelin basic protein (MBP) is best characterized by antigen-specific secretion of transforming growth factor-1, consistent with a T-cell regulatory response.12 If, however, systemic inflammation was induced during the acute stroke, animals had an increased predisposition to develop an antigen-specific inflammatory response characterized by lymphocyte secretion of interferon-␥, consistent with a Th1 response.12 Furthermore, those animals with a Th1(⫹) response to MBP experienced worse 1-month outcomes.12,13 Based on these observations, we hypothesized that patients who become infected in the immediate poststroke period would be more likely to develop a Th1(⫹) response to brain antigens and that this response would be associated with a worse long-term outcome. Materials and Methods Research Subjects Patients with ischemic stroke admitted to Harborview Medical Center from September 2005 through May 2009 who were at least 18 years of age were enrolled within 72 hours of symptom onset. Individuals with ongoing therapy for malignancy, known history of HIV, hepatitis B or C, history of brain tumor, anemia (hematocrit Received March 7, 2011; final revision received March 31, 2011; accepted April 25, 2011. From the Departments of Neurology (K.J.B., A.J.K., P.T., D.K.Z., A.V.S., J.M.G.), Radiology (D.S.), and Biostatistics (K.C.C.), University of Washington School of Medicine, Harborview Medical Center, Seattle, WA; and the University of Washington Medical Center (J.H., K.T.C.), Seattle, WA. The online-only Data Supplement is available at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.111.619593/-/DC1. Correspondence to Kyra J. Becker, MD, University of Washington School of Medicine, Box 359775, Harborview Medical Center, 325 9th Avenue, Seattle, WA 98104-2499. E-mail [email protected] © 2011 American Heart Association, Inc. Stroke is available at http://stroke.ahajournals.org DOI: 10.1161/STROKEAHA.111.619593 2763 2764 Stroke October 2011 ⬍35 on admission), and those taking immunomodulatory drugs were excluded. Blood was drawn as soon as possible after stroke onset and at 72 hours, 7 days, and 90 days after stroke onset. Blood was also drawn from 40 volunteers to determine normative data for cellular immune responses. The study was approved by the Institutional Review Board; all patients or their surrogates as well as control subjects provided informed consent. Clinical Data Downloaded from http://stroke.ahajournals.org/ by guest on June 17, 2017 Clinical and demographic data were collected on all patients. Stroke severity was determined by the National Institutes of Health Stroke Scale (NIHSS) score and outcome by the modified Rankin Scale.14,15 Details about therapeutic interventions for the treatment of stroke were collected and there was active ascertainment of infection data. Infection was defined as clinical symptoms of an infection (fever and/or pyuria for urinary tract infection and fever and/or productive cough and radiographic evidence of consolidation for pneumonia [PNA]) and positive culture data (for both PNA and urinary tract infection). Antibiotic therapy was used as appropriate to treat infections; prophylactic antibiotics were not used. Stroke etiology was determined using the Trial of ORG 10172 in Acute Stroke Treatment criteria.16 Total infarct volume on initial diffusionweighted MRI was calculated by the ABC/2 method.17 Lymphocyte Responses Mononuclear cells were isolated over a Ficoll gradient and frozen in liquid nitrogen until use. Enzyme linked immunoSPOT assays were done to detect the antigen specific secretion of interferon-␥ and transforming growth factor-1 (R&D Systems). Cells were cultured for 24 hours in 96-well plates (MultiScreen-IP; Millipore) at a concentration of 1⫻106 per mL in media alone or with human MBP (25 g/mL; Sigma-Aldrich), human proteolipid protein (PLP; 5 g/mL; ABD Serotec), human neuron-specific enolase (NSE; 5 g/mL; Sigma-Aldrich), bovine S100 (5 g/mL; Fitzgerald), or human glial fibrillary acidic protein (GFAP 5 g/mL; CalbiochemEMD BioSci) and incubated for 24 hours. The response to tetanus toxin (TT 5 g/mL; Sigma-Aldrich) was also assessed as a control. Experiments were performed in triplicate; spots were counted using a semiautomated system (MetaMorph). The degree of Th1 response to each antigen is measured as the ratio of the relative increase in the number of cells secreting interferon-␥ to the relative increase in the number of cells secreting transforming growth factor- to a given antigen. The median response to each antigen during the first week after stroke was determined for each patient. For the 90-day responses, those that were ⬎75th percentile seen in control subjects were considered to represent a Th1(⫹) response. Laboratory Studies Leukocyte counts were determined by the clinical hematology laboratory. The concentrations of high-sensitivity C-reactive protein and glucose were determined by the hospital laboratory using standard methods. Additional plasma was immediately frozen at ⫺80°C; the concentrations of circulating cytokines (interleukin [IL]-2, IL-6, tumor necrosis factor [TNF]-␣) were later measured with a cytometric bead-based system (Fluorokine MAP; R&D Systems). The sensitivity of the assays for IL-2, IL-6, and TNF-␣ was 2.23 pg/mL, 1.11 pg/mL, and 1.50 pg/mL, respectively. Values below the limit of detection are referred to as not detected and assigned the lowest limit of detection for statistical testing. Statistics Descriptive data are presented as median and interquartile range for continuous variables and percentages for categorical variables; group comparisons were performed using the Kruskal-Wallis H test, the Mann-Whitney U, test or the 2 test statistic as appropriate. Logistic regression was used to estimate the OR and 95% CI for the effect of a Th1 immune response to each antigen (as a continuous measure) at Day 90 on neurological outcome at that time point. Given the relatively severe strokes seen in this study, good outcome was defined as independent ambulation (modified Rankin Scale ⱕ3). Logistic regression was also used to estimate the OR and 95% CI for variables that predict the probability of having a Th1(⫹) immune response to a given antigen at 90 days. Significance was set at Pⱕ0.05. Results A total of 114 patients were enrolled in CASIS; none had a history of autoimmune disease. The median age was 57 years (range, 44 to 67 years) and the median NIHSS score was 11 (range, 4 to 19). Patients were divided into tertiles based on initial stroke severity (the highest NIHSS score in the first 72 hours); group characteristics are presented in Table 1. The degree of the Th1 response to the panel of brain antigens (as well as to TT) in the first week after stroke onset is depicted in Figure A; these responses are similar among all tertiles of stroke severity. Four patients died in the hospital as a direct result of their stroke/withdrawal of support. Among the survivors, the Th1 response to PLP and MBP at 90 days was higher in patients with more severe strokes (Figure B). In comparison to the healthy volunteers, the Th1 response to TT was lower in patients with stroke during the first week after symptom onset (0.80 [0.49 to 1.79] versus 1.26 [0.92 to 1.62]; P⫽0.049), but this difference was no longer significant at Day 90 (0.88 [0.48 to 1.80] versus 1.26 [0.92 to 1.62]; P⫽0.181). In patients with less severe strokes (NIHSS ⱕ5), however, the response to TT was less than in the control group early after stroke as well as at 90 days after stroke. The responses of each tertile in comparison to the controls are presented in Table 2. The effect of the immune response to each antigen (as a continuous measure) on outcome at Day 90 is presented in Table 3. Patients with more robust Th1 responses to MBP are less likely to experience a good outcome (modified Rankin Scale ⱕ3). Furthermore, this effect is independent of initial stroke severity and patient age. There are similar but less robust effects of the immune response to PLP and GFAP on outcome. In this study, we defined a Th1(⫹) response to an antigen as a response that was ⬎75% percentile of the control response to that antigen. Patients with more severe strokes (NIHSS ⱖ17) were more likely to develop Th1(⫹) responses to PLP (P⫽0.026) and MBP (P⬍0.001) than patients with less severe strokes. The primary hypothesis for this study was that infection in the poststroke period would increase the likelihood of an individual developing a Th1(⫹) response to brain antigens. Table 4 shows the proportion of patients with and without infection who had evidence of a Th1(⫹) response to each antigen at 90 days. The median response to PLP at 90 days was also higher among patients who developed PNA in the first 15 days after stroke than those who were not infected (1.56 [1.23 to 2.08] versus 0.90 [0.48 to 1.30]; P⫽0.025). Stroke severity was much worse among patients who developed an infection compared to those who did not (NIHSS⫽21 [12 to 26] versus 8 [3 to 16]; P⬍0.001). Patients with PNA had more severe strokes (NIHSS⫽25 [21 to 30]) than patients with urinary tract infection (NIHSS⫽18 [9 to 24]) and patients without infection (NIHSS⫽8 [3 to 16]; P⬍0.001). The effect of infection on immunologic outcome is lost after controlling for stroke severity. Becker et al Table 1. Poststroke Autoimmune Responses 2765 Comparison of Patients With Stroke by Initial Stroke Severity* NIHSS ⱕ5 (N⫽39) NIHSS 6 –16 (N⫽36) NIHSS ⱖ17 (N⫽39) P 60 (46–70) NS Baseline demographics Age, y 54 (40–66) 56 (43–68) Female 11/39 (28%) 16/36 (44%) 12/39 (31%) NS HTN 19/39 (49%) 22/36 (61%) 20/39 (51%) NS DM 7/39 (18%) 8/36 (22%) 13/39 (33%) NS CHD 7/39 (18%) 10/36 (28%) 10/39 (26%) NS CABG 6/39 (15%) 7/36 (19%) 6/39 (15%) NS HLD 25/39 (64%) 26/36 (72%) 30/39 (77%) NS AF 5/39 (13%) 6/36 (17%) 5/39 (13%) NS 13/39 (33%) 11/36 (31%) 19/39 (49%) NS Cardioembolic 9/39 (23%) 10/36 (28%) 12/39 (31%) Lacunar 8/39 (20%) 3/36 (8%) Smoker Stroke etiology Downloaded from http://stroke.ahajournals.org/ by guest on June 17, 2017 Atherosclerosis Other known cause Unknown 0 NS 0.009 3/39 (8%) 4/36 (11%) 10/39 (26%) 0.062 11/39 (28%) 9/36 (25%) 10/39 (26%) NS 8/39 (20%) 10/36 (28%) 7/39 (18%) NS Stroke characteristics and treatment Infarct volume, mL IV tPA 0.8 (0.3–10.1) 4/39 (10%) 11.6 (1.8–32.8) 144.0 (51.1–292.7) ⬍0.001 12/36 (33%) 12/39 (31%) 0.037 Endovascular therapy 0/39 7/36 (19%) 8/39 (20%) 0.011 Hemicraniectomy 0/39 1/36 (3%) 8/39 (20%) 0.001 37.8 (37.4–38.6) 0.003 Complications Highest temperature in first 72 h 37.3 (37.2–37.8) 37.6 (37.2–37.9) Infection by day 15 4/39 (10%) 4/36 (11%) PNA by day 15 21/38 (55%) ⬍0.001 12/38 (32%) ⬍0.001 0/39 0/36 hsCRP per mg/L† 2.4 (1.5–6.2) 10.6 (4.6–40.0) 66.6 (19.2–112.2) ⬍0.001 ⬍0.001 Concentrations of inflammatory markers IL-6 pg/mL† 1.0 (ND-1.9) 2.2 (0.8–5.8) 9.5 (4.2–25.6) TNF-␣ pg/mL† 1.2 (ND-2.8) 1.9 (0.7–3.0) 2.1 (ND-3.7) NS IL-2 pg/mL† ND (ND-ND) ND (ND-ND) ND (ND-2.6) 0.005 Laboratory variables Glucose, mg/dL† 119 (107–143) 123 (107–151) 143 (129–171) Cortisol, g/dL† 9.8 (7.6–13.8) 13.9 (10.9–16.7) 20.2 (13.8–27.4) 19.0 (13.0–28.2) 20.0 (11.5–40.5) 22.5 (16.5–32.0) NS 8.1 (6.0–10.1) 10.3 (8.7–13.8) 12.1 (10.5–15.0) ⬍0.001 ACTH, pg/mL† WBCs, thousands/mL† 0.002 ⬍0.001 PMNs, thousands/mL† 3.9 (3.0–5.2) 5.9 (4.5–7.6) 8.1 (6.3–9.7) ⬍0.001 Lymphs, thousands/mL‡ 1.7 (1.3–2.1) 1.4 (1.2–1.7) 1.2 (0.9–1.5) 0.002 NIHSS indicates National Institutes of Health Stroke Scale; HTN, hypertension; DM, diabetes mellitus; CHD, coronary heart disease; CABG, coronary artery bypass grafting; HLD, hyperlipidemia; AF, atrial fibrillation; IV tPA, intravenous tissue plasminogen activator; PNA, pneumonia; hsCRP, high-sensitivity C-reactive protein; IL, interleukin; TNF, tumor necrosis factor; ACTH, adrenocorticotrophic hormone; WBCs, white blood cells; PMNs, polymorphonuclear cells; lymphs, lymphocytes; ND, not detected; NS, nonsignificant. *Data are presented as either the median and interquartile range or the proportion. Statistics are by Kruskal-Wallis H test or 2 as appropriate. †The highest value in the first 72 h. ‡Lowest value in the first 72 h. Univariate predictors of developing a Th1 response ⬎75th percentile of the control population at Day 90 for each of the antigens studied (a Th1[⫹] response) are shown in Supplemental Table I (http://stroke.ahajournals.org). Stroke severity was one of the most important predictors of developing a Th1(⫹) response to most antigens and was more predictive than infarct volume. For each 1-point increase in the NIHSS score, there was nearly a 10% increase in the odds of developing a Th1(⫹) response to MBP. There were trends toward increased risk of developing Th1(⫹) response to TT and GFAP among patients undergoing hemicraniectomy; these trends no longer existed after controlling for stroke 2766 Stroke October 2011 Downloaded from http://stroke.ahajournals.org/ by guest on June 17, 2017 Figure. Median Th1 responses (Y-axis) to TT and brain antigens in the first week after stroke onset (A) do not differ based on tertile of initial stroke severity (X-axis). By Day 90, the responses to PLP and MBP are more robust in patients with severe strokes (B). Statistics are by Kruskal-Wallis H; *P⬍0.05, **P⬍0.001. TT indicates tetanus toxin; PLP, proteolipid protein; MBP, myelin basic protein; NSE, neuron specific enolase; GFAP, glial fibrillary acidic protein. severity. Curiously, higher numbers of lymphocytes were associated with a decreased risk of developing a Th1(⫹) response to PLP and GFAP. Not surprisingly, infection (and fever) in the first 15 days after stroke onset was associated with increased risk of developing a Th1(⫹) response to TT, PLP, MBP, and GFAP. Finally, Gram-positive infections were more likely to be associated with a subsequent Th1(⫹) response (likely because the preponderance of Gram-positive infections were PNAs).18 Discussion After stroke, lymphocytes infiltrate the ischemic brain allowing for contact with CNS antigens from numerous different cell types (neurons, astrocytes, oligodendrocytes), which are normally sequestered from the peripheral immune system.1–3 Furthermore, there is an increase in the concentration of antigens such as MBP, NSE, S-100, and GFAP in the systemic circulation after stroke, which allows for lymphocyte encounter with these antigens in peripheral lymphoid Table 2. Differences Between the Th1 Response to Each Antigen for Each Tertile of Stroke Severity and Controls After Stroke Onset and at Day 90* Control Subjects (N⫽39) Tertile 1 NIHSS ⱕ5 (N⫽38) P Tertile 2 NIHSS 6 –16 (N⫽36) P Tertile 3 NIHSS ⱖ17 (N⫽36) P TT 1.26 (0.92–1.62) PLP 0.85 (0.63–1.85) 0.87 (0.47–1.33) 0.020 1.04 (0.73–1.45) NS 0.70 (0.45–2.09) 0.073 1.05 (0.60–2.17) NS 0.95 (0.58–1.40) NS 1.08 (0.56–1.67) MBP 1.02 (0.73–1.47) 1.04 (0.71–1.73) NS NS 0.88 (0.63–1.50) NS 1.04 (0.70–1.66) NSE 0.92 (0.55–1.38) NS 1.12 (0.70–1.92) NS 0.86 (0.44–1.56) NS 0.98 (0.72–1.55) NS GFAP S-100B 1.11 (0.51–1.46) 1.00 (0.55–1.69) NS 0.80 (0.45–1.31) NS 1.12 (0.78–1.57) NS 1.00 (0.60–1.42) 0.98 (0.71–1.60) NS 0.76 (0.50–1.46) NS 1.20 (0.82–1.47) NS TT 1.26 (0.92–1.62) 0.78 (0.35–1.88) 0.033 0.83 (0.52–1.49) 0.092 1.38 (0.52–1.97) NS PLP 0.85 (0.63–1.85) 0.89 (0.65–1.30) NS 0.78 (0.43–1.55) NS 1.40 (0.80–2.31) 0.115 MBP 1.02 (0.73–1.47) 0.81 (0.54–1.06) 0.060 0.88 (0.57–1.42) NS 1.68 (0.71–2.60) 0.094 NSE 0.92 (0.55–1.38) 0.68 (0.57–1.19) NS 0.93 (0.51–1.41) NS 1.00 (0.72–1.74) NS GFAP 1.11 (0.51–1.46) 0.78 (0.40–1.05) 0.062 0.98 (0.48–1.44) NS 0.96 (0.53–2.13) NS S-100B 1.00 (0.60–1.42) 0.86 (0.53–1.54) NS 0.77 (0.45–1.97) NS 1.65 (0.69–3.43) 0.079 Within first week Day 90 NIHSS indicates National Institutes of Health Stroke Scale; TT, tetanus toxin; PLP, proteolipid protein; MBP, myelin basic protein; NSE, neuron-specific enolase; GFAP, glial acidic fibrillary acidic protein; NS, signifies P⫾0.200. *Data are displayed as median (interquartile range); statistics are by Mann-Whitney U test (stroke patients vs control subjects). Becker et al Table 3. The Effect of the Immune Response to Each Antigen at 90 Days (as a Continuous Variable) on 90-Day Outcome* 90-Day Outcome OR P At 90 d TT (N⫽80) PLP (N⫽79) MBP (N⫽80) NSE Downloaded from http://stroke.ahajournals.org/ by guest on June 17, 2017 (N⫽80) GFAP (N⫽77) Unadjusted 1.015 (0.866–1.190) NS Adjusted for NIHSS 1.029 (0.876–1.208) NS Adjusted for NIHSS/age 1.023 (0.868–1.205) NS Unadjusted 0.644 (0.426–0.973) 0.037 Adjusted for NIHSS 0.702 (0.460–1.071) 0.100 Adjusted for NIHSS/age 0.677 (0.438–1.045) 0.078 Unadjusted 0.437 (0.251–0.763) 0.004 Adjusted for NIHSS 0.469 (0.240–0.919) 0.027 Adjusted for NIHSS/age 0.477 (0.244–0.935) 0.031 Unadjusted 0.833 (0.505–1.373) NS Adjusted for NIHSS 1.000 (0.556–1.801) NS Adjusted for NIHSS/age 1.060 (0.590–1.904) NS Unadjusted 0.669 (0.442–1.012) 0.057 Adjusted for NIHSS 0.783 (0.497–1.232) NS Unadjusted 1.001 (0.775–1.294) NS Adjusted for NIHSS 1.171 (0.829–1.654) NS Adjusted for NIHSS/age NS Adjusted for NIHSS/age S-100 (N⫽73) mRS indicates modified Rankin Scale; TT, tetanus toxin; PLP, proteolipid protein; MBP, myelin basic protein; NSE, neuron-specific enolase; GFAP, glial fibrillary acidic protein; NIHSS, National Institutes of Health Stroke Scale; NS signifies P⫾0.200; OR, odds ratio. *The ORs are either unadjusted or adjusted for baseline NIHSS score or baseline NIHSS score and age. organs.5,19 Given this lymphocyte contact with novel CNS antigens, irrespective of site, it is thus possible for an (auto)immune response to occur to these antigens. Indeed, antibodies and cellular immune responses to brain antigens are documented in stroke survivors.6 –11 Our observation that Th1(⫹) responses to CNS antigens like MBP occur after stroke is thus not novel, but the finding that these responses may have pathological consequences is. In experimental studies of severe stroke, we showed that Th1(⫹) responses to MBP were uncommon under usual Table 4. Proportion of Patients With a Th1(ⴙ) Response (Response >75th Percentile of the Control Population) to a Given Antigen Based on Infection History* Any Infection by Day 15? Yes 2767 circumstances; the tendency to develop a Th1(⫹) response to MBP, however, could be increased by induction of a systemic inflammatory response with lipopolysaccharide, a component of the Gram-negative bacterial cell wall, at the time of stroke.12,13 The systemic inflammatory response induced by lipopolysaccharide was intended to mimic the response that occurs during infection. Based on our animal data, we hypothesized that patients who became infected in the immediate poststroke period would be more likely to develop Th1(⫹) responses to brain antigens. The findings from this study support our hypothesis that infection would increase the likelihood of developing a Th1(⫹) response to brain antigens given that a greater proportion of patients with poststroke infection (especially PNA) developed Th1(⫹) responses to the myelin-associated antigens (MBP and PLP) and the glial antigen GFAP. The fact that infections with Gram-positive organisms were associated with increased risk for developing Th1(⫹) responses in this study likely relates to the fact that patients with urinary tract infections, which were caused primarily by Gram-negative pathogens, had less severe strokes than patients with PNA, which were predominantly related to Gram-positive organisms.18 Potentially the most important observation in this study is that the more robust the immune response to MBP (and to a lesser extent, PLP and GFAP) at 90 days, the greater the chances of a poor outcome at this time point. A similar association between a Th1(⫹) response to MBP and poor outcome after stroke was demonstrated in animal studies.12,13,20 MBP is a myelin-associated protein found in oligodendrocytes within the CNS, and PLP is the most abundant protein in myelin. Both MBP and PLP are implicated as autoantigens in multiple sclerosis and can induce experimental autoimmune encephalomyelitis.21 We also observed a trend toward worse outcome in patients with more robust responses to GFAP. Both humoral responses and cellular immune responses to GFAP have been described in patients with dementia.22–24 Immune responses to neuronspecific enolase, S-100, and TT, on the other hand, were not predictive of outcome. Given that severe strokes and infections are both associated with the development of more robust immune responses to TT, PLP, MBP, and GFAP, it is possible to argue that the responses mRS ⱕ3 Response to Poststroke Autoimmune Responses PNA by Day 15? No P Yes No P TT 11/22 (50%) 14/59 (24%) 0.023 5/10 (50%) 14/59 (24%) 0.085 PLP 6/22 (27%) 10/58 (17%) NS 4/10 (40%) 10/58 (17%) 0.100 MBP 9/22 (41%) 14/59 (24%) 0.127 6/10 (60%) 14/59 (24%) 0.019 NSE 5/23 (22%) 14/58 (24%) NS 4/11 (36%) 14/58 (24%) NS GFAP 8/22 (36%) 11/56 (20%) 0.122 5/10 (50%) 11/56 (20%) 0.039 S-100B 8/21 (38%) 18/53 (34%) NS 3/9 (33%) 18/53 (34%) NS PNA indicates pneumonia; TT, tetanus toxoid; PLP, proteolipid protein; MBP, myelin basic protein; NSE, neuron-specific enolase; GFAP, glial fibrillary acidic protein; NS signifies Pⱖ0.200. *Statistics are by 2 and compare patients with infection (any vs no infection or PNA vs no infection) with those without infection. 2768 Stroke October 2011 Downloaded from http://stroke.ahajournals.org/ by guest on June 17, 2017 to these antigens are merely a marker for patients with severe stroke and hence worse outcome. The fact that more robust immune responses to TT were not associated with worse outcome, however, tends to dispute this argument as does the fact that the predictive value of the immune response on outcome, at least for MBP, was largely unaltered after controlling for stroke severity. Furthermore, the fact that the responses to NSE and S-100 were not substantially related to stroke severity, infection, or outcome suggests that the observed responses to PLP, MBP, and GFAP are more than just a nonspecific inflammation in patients with severe stroke. Molecular mimicry and bystander activation are concepts that have been proposed to explain how infection can induce autoimmunity.25 For molecular mimicry, it is believed that the immune response to the pathogen cross-reacts with a self-antigen; for bystander activation, it is believed that the infection/inflammation leads to the expression of the necessary costimulatory molecules (“danger signals”) that activate lymphocytes leading to an immune response to antigens in the vicinity of the “danger signals.” Our previous experimental work showed that administration of lipopolysaccharide at the time of stroke led to upregulation of B7.1, an important costimulatory molecule, in the brain.12 There are also abundant data showing that lipopolysaccharide and other inflammatory stimuli lead to upregulation of B7.1 in monocytes and dendritic tissue in the periphery.26,27 It is thus plausible that an infection like PNA provokes enough of an inflammatory response to upregulate the expression of costimulatory molecules in either peripheral lymphoid organs or in the brain to create a microenvironment that promotes bystander activation of lymphocytes encountering CNS antigens. The finding that increasing lymphocyte numbers were associated with a decreased likelihood of developing a Th1(⫹) response to PLP and GFAP is counterintuitive but may be explained by the profound effect of stroke severity on lymphocyte numbers (Table 1) and the fact that stroke severity is such a potent predictor of the Th1(⫹) response to these antigens. Limitations of this study include the relatively small sample size and the fact that we assessed responses to only a small number of potential antigens. Furthermore, our control population was not fully characterized and matched to the patients with stroke; for the most part, the control subjects were largely young and healthy with no history of stroke. Larger studies will need to be done to confirm our results and determine whether stroke severity or infection is more important in driving the autoimmune responses to CNS antigens and to establish normative values for larger cohorts of healthy volunteers. These studies could also assess the response to portions of MBP and PLP with defined antigenicity and evaluate the Th17 response as well as the Th1 response to these antigens. Finally, new approaches to identifying novel autoantigens could also be considered in future studies. In summary, this study again confirms that cellular immune responses to brain antigens occur after stroke. More importantly, we showed that the response to some of these antigens, MBP in particular, predicts long-term outcome. The risk of developing a Th1(⫹) response to MBP is increased by PNA in the first 15 days after stroke, consistent with observations from our animal model. The development of a detrimental autoimmune response to the brain after stroke may help to explain why infection is an independent predictor of poor outcome after stroke. Sources of Funding This research was funded by National Institute of Neurological Disorders and Stroke 5R01NS049197. Disclosures None. References 1. 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Downloaded from http://stroke.ahajournals.org/ by guest on June 17, 2017 Autoimmune Responses to the Brain After Stroke Are Associated With Worse Outcome Kyra J. Becker, Angela J. Kalil, Pat Tanzi, Dannielle K. Zierath, Anna V. Savos, J. Michael Gee, Jessica Hadwin, Kelly T. Carter, Dean Shibata and Kevin C. Cain Downloaded from http://stroke.ahajournals.org/ by guest on June 17, 2017 Stroke. 2011;42:2763-2769; originally published online July 28, 2011; doi: 10.1161/STROKEAHA.111.619593 Stroke is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2011 American Heart Association, Inc. All rights reserved. Print ISSN: 0039-2499. Online ISSN: 1524-4628 The online version of this article, along with updated information and services, is located on the World Wide Web at: http://stroke.ahajournals.org/content/42/10/2763 Data Supplement (unedited) at: http://stroke.ahajournals.org/content/suppl/2011/07/29/STROKEAHA.111.619593.DC1 Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Stroke can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Stroke is online at: http://stroke.ahajournals.org//subscriptions/ SUPPLEMENTAL MATERIAL Supplemental Table. Predictors of a TH1(+) response to each antigen at 90 days after stroke. Laboratory data represent the most extreme value for the first 72 hours after stroke onset. Results are expressed as the odds ratio (OR) and 95% confidence interval (CI). TT antigen: PLP OR P 1.008 NS MBP OR P 1.006 NS NSE OR P 1.022 NS GFAP OR P 1.002 NS S100 OR P 1.017 NS OR P 1.012 NS baseline demographics age (per year) gender (female) NIHSS score (0.972-1.046) 0.526 (0.181-1.527) 1.056 (1.003-1.112) NS 0.039 (0.965-1.049) 0.223 (0.047-1.065) 1.070 (1.008-1.136) 0.060 0.025 (0.983-1.062) 0.605 (0.206-1.773) 1.099 (1.038-1.164) NS 0.001 (0.964-1.043) 0.649 (0.206-2.043) 1.023 (0.968-1.080) NS NS (0.977-1.059) 0.483 (0.142-1.643) 1.069 (1.011-1.131) NS 0.020 (0.974-1.051) 1.059 (0.387-2.897) 1.052 (0.998-1.109) NS 0.059 stroke etiology cardioembolic 1.286 (0.460-3.594) 0.347 lacunar (0.040-3.048) atherosclerosis other known cause 1.136 (0.260-4.961) 0.667 (0.227-1.955) 1.591 unknown (0.533-4.751) NS NS NS NS NS 0.789 (0.226-2.762) 0.644 (0.072-5.769) 1.163 (0.218-6.221) 1.533 (0.486-4.840) 0.824 (0.206-3.303) NS NS NS NS NS 1.148 (0.398-3.310) 1.010 (0.182-5.615) 3.750 (0.907-15.497) 0.570 (0.184-1.769) 0.662 (0.193-2.274) NS NS 0.068 NS NS 0.945 (0.295-3.027) 2.719 (0.551-13.412) 0.924 (0.175-4.876) 0.750 (0.237-2.372) 0.914 (0.261-3.202) NS NS NS NS NS 1.354 (0.437-4.195) 1.271 (0.226-7.153) 0.354 (0.041-3.032) 1.051 (0.345-3.205) 0.944 (0.267-3.337) NS NS NS NS NS 1.425 (0.510-3.987) 0.917 (0.156-5.375) 0.583 (0.109-3.121) 0.600 (0.201-1.788) 1.596 (0.516-4.941) NS NS NS NS NS stroke characteristics and treatment infarct volume (per 10 cc) 1.038 (0.998-1.080) 2.062 IV tPA (0.732-5.815) hemicraniectomy 4.417 (0.965-20.213) 0.066 0.171 0.056 1.037 (0.994-1.081) 1.364 (0.411-4.523) 2.723 (0.577-12.858) 0.089 NS NS 1.038 (0.998-1.080) 0.729 (0.232-2.294) 1.590 (0.347-7.277) 0.063 NS NS 1.000 (0.957-1.046) 0.706 (0.205-2.430) 1.098 (0.203-5.950) NS NS NS 1.034 (0.993-1.077) 0.960 (0.297-3.097) 3.667 (0.819-16.415) 0.101 NS 0.089 1.029 (0.990-1.071) 2.787 (0.981-7.918) 2.000 (0.456-8.763) 0.150 0.054 NS inflammatory cytokine concentrations (highest by 72 hours) IL-6 (per pg/mL) hsCRP (per 10 mg/L) TNF-α (per pg/mL) IL-2 (per pg/mL) 1.010 (0.993-1.028) 1.070 (0.975-1.016) 1.226 (0.970-1.549) 3.396 (0.895-12.884) NS 0.153 0.089 0.072 1.003 (0.985-1.021) 1.064 (0.963-1.177) 1.042 (0.791-1.374) 1.235 (0.706-2.158) NS NS NS NS 1.002 (0.986-1.019) 1.097 (0.998-1.207) 1.100 (0.870-1.392) 1.090 (0.627-1.893) NS 0.056 NS NS 1.000 (0.982-1.019) 0.973 (0.872-1.085) 1.010 (0.773-1.319) 0.914 (0.436-1.914) NS NS NS NS 1.017 (0.996-1.039) 1.063 (0.965-1.170) 1.066 (0.835-1.362) 1.335 (0.741-2.403) 0.109 NS NS NS 0.997 (0.979-1.015) 1.026 (0.990-1.132) 1.063 (0.810-1.395) 2.245 (0.670-7.421) NS NS NS 0.185 laboratory values (highest by 72 hours) blood glucose (per 10 mg/dL) WBCs (per thou/mL) PMNs (per thou/mL) lymphs* (per thou/mL) 0.957 (0.870-1.051) 1.014 (0.906-1.135) 1.088 (0.925-1.279) 0.566 (0.215-1.489) NS NS NS NS 1.014 (0.923-1.114) 0.981 (0.856-1.124) 1.098 (0.913-1.322) 0.188 (0.043-0.813) NS NS NS 0.025 1.045 (0.962-1.134) 1.070 (0.955-1.198) 1.157 (0.979-1.368) 0.729 (0.286-1.859) NS NS 0.087 NS 1.014 (0.928-1.109) 0.975 (0.860-1.106) 0.963 (0.805-1.152) 0.933 (0.367-2.375) NS NS NS NS 1.040 (0.954-1.133) 1.018 (0.900-1.152) 1.164 (0.969-1.399) 0.220 (0.059-0.825) NS NS 0.105 0.025 0.958 (0.871-1.053) 0.975 (0.869-1.094) 0.996 (0.845-1.174) 0.726 (0.295-1.789) NS NS NS NS complications highest temp in 1st 72 hrs (per OC) 2.394 (1.111-5.158) st highest temp in 1 week 1.347 (per C) (0.787-2.307) O infection by day 15 PNA by day 15 gram positive infection gram negative infection 3.214 (1.149-8.992) 2.550 (0.666-9.769) 5.056 (1.323-19.313) 1.895 (0.580-6.193) 0.026 NS 0.026 0.172 0.018 NS 3.124 (1.306-7.469) 1.481 (0.798-2.749) 1.800 (0.565-5.739) 3.222 (0.787-13.193) 2.714 (0.685-10.759) 0.619 (0.124-3.094) 0.010 NS NS 0.104 0.155 NS 1.678 (0.798-3.527) 1.534 (0.881-2.670) 2.225 (0.786-6.297) 4.765 (1.202-18.893) 3.741 (1.013-13.817) 1.011 (0.282-3.618) 0.172 0.130 0.132 0.026 0.048 NS 1.068 (0.529-2.154) 1.334 (0.729-2.44) 0.873 (0.274-2.782) 2.095 (0.541-8.120) 2.095 (0.541-8.120) 0.443 (0.091-2.169) NS NS NS NS NS NS 3.263 (1.390-7.661) 1.823 (0.986-3.371) 2.338 (0.786-6.956) 3.857 (0.978-15.208) 4.985 (1.315-18.891) 1.307 (0.358-4.774) 0.007 0.056 0.127 0.054 0.018 NS 1.294 (0.633-2.645) 0.952 (0.567-1.598) 1.197 (0.419-3.413) 0.913 (0.209-3.996) 2.048 (0.534-7.859) 0.691 (0.193-2.467) NS NS NS NS NS NS TT=tetanus toxoid, PLP=proteolipid protein, MBP=myelin basic protein, NSE=neuron specific enolase, GFAP=glial fibrillary acidic protein, NIHSS=NIH stroke scale, IV tPA=intravenous tissue plasminogen activator, IL=interleukin, TNF=tumor necrosis factor, WBCs=white blood cells, PMNs=polymorphonuclear cells, lymphs=lymphocytes, temp=temperature, PNA=pneumonia. *denotes lowest value by 72 hours. NS signifies P≥0.020.