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Stroke-induced Immunodeficiency Promotes Spontaneous Bacterial Infections and Is Mediated by Sympathetic Activation Reversal by Poststroke T Helper Cell Type 1–like Immunostimulation

Stroke-induced Immunodeficiency Promotes Spontaneous Bacterial Infections and Is Mediated by... Infections are a leading cause of death in stroke patients. In a mouse model of focal cerebral ischemia, we tested the hypothesis that a stroke-induced immunodeficiency increases the sus- ceptibility to bacterial infections. 3 d after ischemia, all animals developed spontaneous septicemia and pneumonia. Stroke induced an extensive apoptotic loss of lymphocytes and a shift from T helper cell (Th)1 to Th2 cytokine production. Adoptive transfer of T and natural killer cells from wild-type mice, but not from interferon (IFN)-–deficient mice, or administration of IFN- at day 1 after stroke greatly decreased the bacterial burden. Importantly, the defective IFN- response and the occurrence of bacterial infections were prevented by blocking the sympathetic nervous system but not the hypothalamo-pituitary-adrenal axis. Furthermore, admin- istration of the -adrenoreceptor blocker propranolol drastically reduced mortality after stroke. These data suggest that a catecholamine-mediated defect in early lymphocyte activation is the key factor in the impaired antibacterial immune response after stroke. Key words: T lymphocytes • natural killer cells • interferon  • pneumonia • brain ischemia Introduction Infectious complications, predominantly chest and urinary tions in stroke patients (10). The high incidence of infec- tract infections, have been reported to occur in 23–65% of tions in these patients is likely to be a result of an impaired all stroke patients within the first few days after stroke (1–5). immune function. Immunodepression has been reported in Although early mortality is due to direct complications other potentially life-threatening conditions, such as myo- from large strokes, pneumonia is the leading cause of death cardial infarction, polytrauma, or major surgery, resulting in the postacute phase of stroke, regardless of hospitalization in an increased risk of infectious complications (11–13). In (6–9). The high frequency of antibiotic treatment on spe- trauma patients, the presence of brain injury was identified cialized stroke units underlines the clinical problem of infec- as an independent risk factor for infectious complications, possibly due to a central nervous shutdown of the immune defense (14, 15). As early as 1974, an immunosuppressive K. Prass and C. Meisel contributed equally to this work. state was associated with stroke (16). However, the mecha- Address correspondence to Christian Meisel, Wellcome Trust Centre nisms leading to an increased susceptibility to infections after for Human Genetics, University of Oxford, Roosevelt Drive, OX3 7BN Oxford, United Kingdom. Phone: 44-1865-287538; Fax: 44-1865- stroke are still poorly understood. Here, we demonstrate 287533; email: chr.meisel@charite.de; or Ulrich Dirnagl, Department of Neurology, Charité Hospital, Humboldt University, Schumannstraße 20- 21, D-10098 Berlin, Germany. Phone: 49-30-450-560184; Fax: 49-30- Abbreviations used in this paper: 6-OHDA, 6-hydroxydopamine HBr; HE, 450-560915; email: ulrich.dirnagl@charite.de hematoxylin/eosin; HPA, hypothalamic-pituitary-adrenal axis; MCA, C. Meisel’s present address is Wellcome Trust Centre for Human middle cerebral artery; MCAO, middle cerebral artery occlusion; SNS, Genetics, University of Oxford, Roosevelt Drive, OX3 7BN Oxford, sympathetic nervous system; TUNEL, in situ terminal deoxynucleotide United Kingdom. transferase-mediated dUTP nick-end labeling. 725 J. Exp. Med.  The Rockefeller University Press • 0022-1007/2003/09/725/12 $8.00 Volume 198, Number 5, September 1, 2003 725–736 http://www.jem.org/cgi/doi/10.1084/jem.20021098 The Journal of Experimental Medicine that focal cerebral ischemia induces a rapid and long-lasting Biosciences) were used: CD3 (145-2C11; T cells), CD4 (L3T4), CD8 (Ly-2), CD45R (RA3-6B2; B cells), anti–pan-NK cell inhibition of cell-mediated immunity resulting in sponta- marker (DX5), CD11b (M1/70; monocytes/macrophages, gran- neous systemic bacterial infection, and that early IFN- ulocytes), and CD11c (HL3; dendritic cells). In blood and spleen production by T and NK cells is crucial in controlling bac- samples, RBCs were lysed with BD Lysis Solution (Becton Dick- terial infections. Our finding that -adrenoreceptor block- inson) before analysis. The degree of apoptotic cell death in thy- ade enhances cellular immune responses and prevents bac- mus and spleen single cell suspensions was quantified using fluo- terial infections, provides strong evidence that an activation rescein-labeled annexin V (Qbiogene). Cell phenotyping and of the sympathetic nervous system (SNS) is the main im- identification of apoptotic cells was performed by four-color flow munosuppressive mechanism leading to a high incidence of cytometry on a FACSCalibur™ using CELLQuest™ Software infections after stroke. (BD Biosciences). Analysis of Ex Vivo Cytokine Production. Whole blood was di- luted 1:5 in heparinized RPMI 1640 and incubated at 37C and Materials and Methods 5% CO . For analysis of TNF- synthesis, samples were stimu- tm1Mom Animals. SV129/J mice (BGVV), B6.129P2-Tcrb ( lated with 100 ng/ml LPS (endotoxin) from Escherichia coli 0127:B8 tm1Mom T cell–deficient), B6.129P2-Tcrd ( T cell–deficient), (Sigma-Aldrich) for 4 h. For analysis of IFN- and IL-4 produc- tm1Ts B6.129S7-INF (IFN-–deficient), and C57BL/6J wild-type tion, blood samples were stimulated with 100 g/ml Con A mice (The Jackson Laboratory) were housed in the animal care (Sigma-Aldrich) for 24 h. For analysis of IFN- production in facility at the Department of Neurology (Charité Hospital, Ber- spleen cells, 10 cells/ml were stimulated with 10 g/ml Con A lin, Germany) until killed. for 24 h. Concentrations of cytokines in culture supernatants Experimental Model of Stroke. We used gender-mixed 7-wk- were determined using commercially available kits and according old or older SV129/J or, where indicated, C57BL/6J mice weigh- to the manufacturer’s instructions (BD Biosciences). Each sample ing 18–22 g. The surgical procedure of middle cerebral artery was assayed in duplicate. occlusion (MCAO) did not exceed 10 min and was induced as Adoptive Transfer of Spleen Cells. Single cell suspensions were previously described (17). In brief, a monofilament was introduced prepared from spleens of wild-type or knockout mice as de- into the common carotid artery under halothane narcosis, ad- scribed above. Mice that underwent MCAO were injected intra- vanced to the origin of the middle cerebral artery (MCA), and left peritoneally with 5  10 splenocytes 24 h after MCAO. Control there for 60 min until reperfusion. In sham-operated animals, after mice were injected with medium. In some experiments, splenic inserting the filament to the MCA origin, the filament was imme- T, B, and NK cell subsets were depleted using commercially diately withdrawn by 2 mm to avoid ischemia. Occlusion and re- available magnetic bead kits (CD90, CD45R, DX5 microbeads; perfusion were verified by laser Doppler flowmetry (Peri Flux 4001 Miltenyi Biotec). Depletion efficacy was verified as 95% by Master; Perimed). Mice were kept in heated cages for the next 2 h flow cytometry. and body temperature was frequently measured. Animals were Histopathological Analysis of Lung Tissue. After transcardial then returned to their home cages and allowed free access to food perfusion with ethanol-formalin-acetic acid, lungs were removed and water. The infarct volumes were measured histologically as and embedded in paraffin wax. 12-m thick sections were ob- previously described (18). All animal experiments were performed tained by microtome dissection and stained with hematoxylin/ according to institutional and state guidelines. eosin (HE). 20 representative sections of lungs were chosen per Drug Administration. All drugs were injected i.p. and the re- animal (n 4 in each group) and evaluated by two investigators, spective diluents were given to the control animals at the same who were blinded to the treatment groups. The sections were time. RU486 (Sigma-Aldrich) was dissolved in ethanol/sesame graded according to the following criteria: no definite damage oil solution (1:10 vol/vol) at 6 mg/ml and administered (30 mg/ represented no histological changes or minor changes, including kg body weight) 24 h, 5 h, and immediately before MCAO. Pro- unequal distension of alveolar units, mild thickening of the alveo- pranolol (Sigma-Aldrich) was dissolved in 0.9% sodium chloride lar septa, and perivascular and peribronchiolar edema. Definite at 6 mg/ml, and administered (dose 1–30 mg/kg body weight as damage was observed as lung consolidation, thickened alveolar indicated) immediately before as well as 4 and 8 h after MCAO. septae, and the presence of intra-alveolar inflammatory infiltrates. Where indicated, propranolol administration (30 mg/kg body Histochemistry and In Situ Terminal Deoxynucleotide Transferase- weight) was delayed and given 24, 28, and 32 h after MCAO. mediated dUTP Nick-end Labeling (TUNEL) Assay. After trans- 6-hydroxydopamine HBr (6-OHDA; Sigma-Aldrich) was dis- cardial perfusion with ethanol-formalin-acetic acid, thymi were solved in sterile 0.01% ascorbic acid/saline and injected (200 mg/ removed and embedded in paraffin wax. Sections were obtained kg body weight) 3 d before MCAO. IFN- (TEBU) was dis- by microtome dissection and stained with HE. Based on HE solved in phosphate buffered saline at 20 g/ml, and adminis- staining, two representative 12-m sections of thymus were cho- tered i.p. (2 g) at 24 and/or 48 h after MCAO. Control mice sen per animal (n 6) and processed for TUNEL. The Apoptag received heat-denaturated IFN- solution. Kit (Intergen) was used according to the manufacturer’s instruc- Blood Samples, Cell Suspensions, and Cell Count. Mice were tions. Visualization was achieved using the Vectorstain ABC elite killed and blood was collected into heparinized tubes. Single cell kit (Vector Laboratories) in conjunction with 3,3 -diaminobenzi- suspensions from thymus and spleen were prepared by forcing the dine/H O (Sigma-Aldrich). 2 2 tissues through a fine wire mesh. Cells were washed and resus- Bacteriological Analysis. The anesthetized mice were washed pended in 2 ml RPMI 1640 medium containing penicillin, strep- with 70% ethanol under sterile conditions. Blood was collected tomycin, 2 mM glutamine, and 10% FCS (Biochrom KG). by decapitation. The lungs were removed after thoracotomy and Residual RBCs were lysed by hypotonic lysis in ice-cold ammo- homogenized. For determination of CFU, 100 l tissue homo- nium. Cell counts were performed in triplicates. genate or blood was serially diluted, plated onto blood agar plates Flow Cytometry. For flow cytometric analysis the following (Merck), incubated at 37C for 18 h, and bacterial colonies were fluorescently labeled anti–mouse monoclonal antibodies (BD counted. Stroke-induced Immunodepression The Journal of Experimental Medicine Statistical Analysis. Bacteriological data are presented as box Spontaneous Bacteremia and Pneumonia after Focal Cerebral plots and other data are given as mean SD. Data were analyzed Ischemia. 3 d after ischemia, all MCAO animals suffered by Kruskal-Wallis analysis of variance (ANOVA) after pairwise from spontaneous bacterial infections. Bacterial cultures comparison with Dunn’s method or Mann-Whitney U test using from peripheral blood and lungs invariably demonstrated SPSS software (SPSS Inc.). Differences in the group survival were 95% E. coli (Fig. 1 a). Significant bacterial loads were ob- determined using Fisher’s exact test. Values of P 0.05 were served in lungs and blood 24 and 48 h after ischemia, re- considered significant. spectively. Histological examination of lungs revealed typi- cal signs of bacterial pneumonia (19, 20) in all analyzed MCAO-treated animals 72 h after experimental stroke (n Results 4, Fig. 1 b). In contrast, blood and lung cultures from Ischemia was induced in mice by temporary occlusion of sham-operated animals remained sterile at 6, 12, 24, 48 the MCAO. Sham controls received surgery identical to (not depicted), and 72 h (Fig. 1 a). In addition, lung sec- that of MCAO but with regional cerebral blood flow re- tions of sham controls revealed no signs of pneumonia (n 4, maining unaffected. After 60 min of MCAO, animals de- Fig. 1 b). Therefore, susceptibility to infection resulted veloped large infarcts (90 10 mm ) of cortex, striatum, from stroke and not from surgical stress. and hippocampus. Long-lasting Lymphopenia and Impaired Cellular Immune Functions after Cerebral Ischemia. To characterize the un- derlying mechanisms of spontaneous infections after stroke, we analyzed the cellularity and leukocyte populations in lymphoid organs and peripheral blood from MCAO, sham, and control mice using four-color flow cytometry. We found a striking reduction of lymphocyte counts in blood, spleen, and thymus after cerebral ischemia (Fig. 2). The ab- solute numbers of T, B, and NK lymphocytes were de- creased 3–10-fold in blood and 2–3-fold in spleen as early as 12 h after MCAO (Fig. 2, a–f). There was only a mar- ginal decrease in the number of lymphocytes in sham ani- mals, indicating that lymphopenia was mainly caused by ischemia and only partly due to surgical stress. The post- ischemic total lymphocyte count in the blood was signifi- cantly lower even after 14 d (MCAO: 2.7 0.6 vs. con- trol: 4.8 0.5  10 /ml; P 0.05), whereas the number of splenic lymphocytes remained reduced for 42 d (MCAO: 26.9 0.8 vs. control: 46.5 4.7  10 /ml; P 0.05; Fig. 2, a–f). Cellularity in the thymus was also severely reduced (Fig. 2 g) and characterized by a rapid loss of immature CD4 CD8 thymocytes and subsequent re- duction in the number of mature single positive T cells (not depicted). Interestingly, splenic T and B cell counts increased 2 d after stroke, but dropped again at day 5 (Fig. 2, d and e) coinciding with the occurrence of bacteremia. Recently, we have reported that patients with brain sur- gery showed diminished monocytic HLA-DR expression and endotoxin-induced TNF- secretion as signs of sys- temic immunodepression (13, 21). To test whether cerebral ischemia is also associated with alterations in cellular immune functions, we examined the endotoxin-induced TNF- secretion as well as Con A–induced IFN- and IL-4 production ex vivo in whole blood cultures, as parameters of monocyte and T lymphocyte functions, respectively. Secre- tion of endotoxin-induced TNF- was significantly de- Figure 1. Stroke induces bacteremia and pneumonia. (a) Lungs and creased after 12 h and 2 d after MCAO but returned to con- blood samples from sham (n 10) and MCAO-treated mice (n 4–14 trol levels on day 5 (Fig. 2 h). The decrease in the per group) were collected for bacteriological analysis at the indicated time points after surgery. Data are given in CFU/ml (log 10) blood or lung tis- monocytic TNF- release was not due to reduced mono- sue homogenate. (b) In a different set of experiments, lungs from sham- cyte cell numbers, as blood monocyte counts in MCAO operated (n 4) and MCAO mice (n 4) were collected after 72 h for animals did not differ from those in control mice (not histological examination. A representative 12-m section of HE-stained depicted). Furthermore, we found reduced IFN- but in- lung from MCAO but not from sham animals revealed signs (thickening of alveolar walls and neutrophilic infiltrates) of E. coli pneumonia. 160. creased IL-4 production in Con A–stimulated blood cultures Prass et al. The Journal of Experimental Medicine Figure 2. Stroke induces long-lasting lymphopenia and impaired cytokine expression. Spleen, thymus, and peripheral blood samples of untreated SV129/J mice (control) and sham or MCAO mice were collected at different time points (sham, 12 h; MCAO, as indicated) after surgery. Leukocyte counts in blood (a–c), spleen (d–f), and thymus (g) single cell suspensions were determined as described in Materials and Methods. The lymphocyte sub- sets were determined by flow cytometry and their absolute numbers were calculated. (h and i) Aliquots of blood samples were stimulated ex vivo with either LPS for analysis of monocytic TNF- expression or Con A for analysis of IFN- and IL-4 synthesis, as described in Materials and Methods. Cytokines were determined in supernatants by ELISA. The Con A–induced lymphokine expression is given as the ratio of IFN- and IL-4 production calculated for each individual. Data are shown as mean SD. *, results differed from the control group; *, P 0.05; **, P 0.01; ***, P 0.001; #, results differed from the sham-operated group; #, P 0.05; ##, P 0.01. Mann-Whitney U test, n 3–11 per group. resulting in a significant decrease of the IFN-/IL-4 ratio for mice, and only a very modest increase in apoptosis in dou- at least 14 d after MCAO (Fig. 2 i). In contrast, sham-oper- ble positive immature thymocytes was noted (Fig. 3, a and ated animals did not show significant alterations in any of the b). In accordance with these results, light microscopic ex- measured cytokine secretion parameters (Fig. 2, h and i). amination of thymi from MCAO mice, but not sham mice, Importantly, the described alterations of immune parameters showed many apoptotic cells with features of pyknosis or after experimental stroke preceded bacterial infections (com- karyorrhexis (Fig. 3 c, top). Moreover, TUNEL staining pare Figs. 1 a and 2) and, therefore, are more likely to be the demonstrated that the apoptosis occurred predominantly in cause than the result of bacterial infections. the thymic cortex, which is mainly populated by immature Brain Ischemia Causes Rapid and Extensive Apoptosis in double positive thymocytes, and relatively spared the thy- Lymphatic Organs. To test whether lymphopenia was a re- mic medulla (Fig. 3 c, bottom). In addition, thymocyte sult of increased apoptosis, we analyzed spleen and thymus DNA from MCAO animals showed extensive laddering cell suspensions by flow cytometry and found a marked in- (not depicted). crease in apoptotic lymphocytes 12 h after MCAO. In sple- Role of Stress Mediators in Postischemic Immunodepression. nocytes, increased apoptosis affected all lymphocyte subsets, The activation of the hypothalamic-pituitary-adrenal axis as determined by annexin V and cell-type–specific surface (HPA) and the SNS, resulting in a systemic release of adre- marker staining (Fig. 3 a). Similarly, enhanced apoptosis nal steroid hormones and catecholamines, is an essential was found in all thymocyte subsets, mostly affecting the component of the response to major surgery, brain injury, dim immature CD3 CD4 CD8 double positive thymocytes or trauma. As high levels of these stress mediators are (Fig. 3 b). In contrast, there was no significant difference in known to be immunosuppressive (22), we investigated the the degree of apoptosis in splenocytes from sham-operated effects of the -adrenoreceptor antagonist propranolol and Stroke-induced Immunodepression The Journal of Experimental Medicine creased the percentage of apoptotic splenocytes to levels observed in sham-operated mice and prevented the de- crease in peripheral blood lymphocyte counts. The protec- tive effects of both compounds extended to all lymphocyte subsets in spleen and blood (Fig. 4, a and b). Both com- pounds also significantly increased the ex vivo endotoxin- induced TNF- release, but only propranolol normalized the IFN-/IL-4 ratio after Con A stimulation of whole blood cultures (Fig. 4 c). Inhibition of the SNS but Not Steroid Receptor Blockade Pre- vents Systemic Infection. Treatment with propranolol (3 30 mg/kg BW) completely prevented both bacteremia and pneumonia in 8 out of 10 animals (Fig. 4 d). In the remain- ing two mice, bacterial counts in blood and lung were re- 4 6 duced by the order of 10 and 10 , respectively. In contrast, the glucocorticoid receptor antagonist RU486 had no ef- fect on blood or pulmonary bacterial burden. The protec- tive effect of propranolol was dose and time dependent. Propranolol doses (3  10 and 3  30 mg/kg BW starting immediately after MCAO) sufficient to reduce or prevent bacterial dissemination at 72 h after MCAO (Fig. 5 a) also restored the defective IFN- response determined 12 h af- ter experimental stroke (Fig. 5 b), whereas lower doses had no effect on either bacterial load or IFN- production. Im- portantly, delayed administration of propranolol (3  30 mg/kg BW starting 24 h after cerebral ischemia) neither in- creased IFN- production nor decreased bacterial load (Fig. 5), demonstrating that blockade of sympathetic nerves activation early after MCAO is crucial to prevent lympho- cyte dysfunction and, subsequently, bacterial infections. To confirm that the protective effect of propranolol was due to inhibition of sympathetic nerve signaling, MCAO mice were pretreated with 6-OHDA, which selectively enters and destroys sympathetic noradrenergic terminals, thereby depleting tissue catecholamines (23). Like propranolol, chemical sympathectomy by 6-OHDA up-regulated IFN- production and significantly reduced bacterial load in lungs and blood (Fig. 5). Inhibition of the SNS Improves Survival after Experimental Stroke. Mortality in MCAO animals sharply increased from 13% on day 3 to 60% on day 7 after ischemia (n Figure 3. Stroke induces increased apoptosis in lymphoid organs. Spleen and thymus from untreated SV129/J mice (control) and sham or 15). Because animals developed severe hypothermia be- MCAO mice were collected 12 h after surgery. Splenocytes (a) and thy- tween days 3 and 5 ( 33C measured rectally, not de- mocytes (b) were isolated and investigated for apoptosis by annexin V picted) accompanied by bacteremia, sepsis was the likely labeling and flow cytometry. Lymphocyte subpopulations were deter- cause of death. The early mortality in propranolol-treated mined by staining with mAbs against cell-type–specific surface markers. Data are shown as mean SD. *, results differed from the control group; (3  10 mg/kg BW) and diluent-treated animals equally *, P 0.05; **, P 0.01; ***, P 0.001; #, results differed from the amounted to 7-13% until day 3 after stroke. However, sham-operated group; #, P 0.05; ##, P 0.01; ###, P 0.001. only an additional 7% of the propranolol-treated animals Mann-Whitney U test, n 8 per group. (c) Thymic tissue sections from died, whereas 60% of mice treated with diluent were dead sham- and MCAO-treated mice were stained with HE (top) or examined by the fluorescent TUNEL method (bottom). Thymi from MCAO mice by day 7 (P 0.02, diluent vs. propranolol; Fig. 6). showed many apoptotic cells with typical features of nuclear condensation Adoptive Lymphocyte Transfer Prevents Bacterial Infections and fragmentation (HE; 400). Note also the high number of TUNEL after Stroke. Both RU486 and propranolol prevented cells (bright red nuclei) in the thymus cortex from MCAO animals as most of the stroke-induced immunodepressive effects ex- compared with sham mice. 100. cept the changes in the lymphocyte cytokine production, which was only normalized by -adrenoreceptor block- the glucocorticoid receptor inhibitor RU486 on the im- ade. To test whether bacterial infection in MCAO ani- munoinhibitory alterations induced by stroke. Treatment mals was caused by the (functional) loss of lymphocytes, of MCAO animals with either propranolol or RU486 de- we conducted a series of adoptive cell transfer experi- Prass et al. The Journal of Experimental Medicine ments. 24 h after MCAO, mice received either unsepa- rated splenocytes (5  10 ) or subset-depleted spleen cells from normal littermates. Bacterial counts in blood and lungs were determined 48 h later. Transfer of nonsepa- rated spleen cells virtually abolished bacteremia and re- duced pulmonary bacterial load by the order of 10 (Fig. 7 a). This effect was absent upon transfer of spleen cell sus- pensions depleted of all lymphocyte subsets (thus contain- ing mostly monocytes/macrophages). Depletion of B cells only marginally affected the ability to reduce the bacterial load. In contrast, T cell–depleted splenocytes reduced bacteremia only by two orders of magnitude and failed to reduce pulmonary bacterial counts. In addition, NK cell– depleted splenocytes lowered the bacterial load signifi- cantly in the lung but not in peripheral blood (Fig. 7 a). To further investigate the role of specific T cell popula- tions in the antibacterial defense, we performed adoptive transfer studies with spleen cells (5  10 ) from  T cell– or  T cell–deficient mice. Spleen cells from  T cell knockout mice were unable to prevent pulmonary in- fection, whereas they substantially reduced blood bacterial dissemination (Fig. 7 b). In contrast, spleen cells from T cell knockout mice reduced the bacterial burden in the lung but hardly had any effect on bacteremia. These data indicate that T and NK lymphocytes are critical compo- nents of the acute response to bacterial infections after stroke. In particular,  T cells and  T cells are impor- tant for an effective host response to bacterial pneumonia and bacteremia, respectively. IFN- Is Crucial in Controlling Bacterial Infections after Stroke. IFN- is one of the key effector cytokines pro- duced by NK and T cells that enhances microbicidal activ- ity of macrophages and neutrophils. As shown above, blockade of catecholamines restored both IFN- pro- duction and bacterial clearance. Therefore, we examined whether the beneficial effect of lymphocyte transfer in stroke-induced infections is dependent on their ability to produce IFN-. Adoptive transfer of splenocytes from IFN- mice failed to reduce pulmonary and blood bac- terial counts in comparison to spleen cells from wild-type SV129/J mice underwent sham or MCAO surgery (n 9–12 per group). MCAO mice received the -adrenoreceptor antagonist propranolol, the glucocorticoid receptor inhibitor RU486, or only diluent before or after surgery, as described in Materials and Methods. Spleens and blood samples were collected 12 h after surgery. (a) Splenocytes were isolated and inves- tigated for apoptosis by annexin V labeling, staining with mAbs against cell-type–specific surface markers, and flow cytometry. (b) Total leuko- cyte counts in blood samples were determined as described in Materials and Methods. The percentages of different lymphocyte classes were deter- mined by flow cytometry using mAbs against cell-type–specific surface markers and their absolute numbers calculated. (c) For determination of cytokine production, blood samples were processed and cytokine expres- sion was analyzed as described in Fig. 2. Data are shown as mean SD. #, results differed from the sham-operated group; #, P 0.05; ##, P 0.01; §, results differed from the diluent-treated MCAO group; §, P 0.05; §§, P 0.01; §§§, P 0.001. Mann-Whitney U test, n 9–12 per group. (d) In a different set of experiments, lungs and blood samples from sham and inhibitor- or diluent-treated MCAO mice were collected for Figure 4. Effects of -adrenoreceptor and glucocorticoid receptor bacteriological analysis 72 h after surgery. Data are shown as box plots in blockade on stroke-induced immunodepression and bacterial infection. CFU/ml (log 10) blood or lung tissue homogenate. Stroke-induced Immunodepression The Journal of Experimental Medicine Figure 6. Blocking the SNS improves survival after experimental stroke. Mice underwent MCAO and received either diluent or 10 mg/kg BW immediately before, 4, and 8 h after MCAO (n 15 per group). Differences in survival between propranolol-treated versus diluent-treated mice were tested for significance on day 11 after MCAO and are indi- cated by asterisks. *, P 0.05. Fisher’s exact test. Discussion In this study, we provide for the first time experimental proof that a neuroendocrine-mediated systemic immuno- suppression after acute central nervous system lesion results in the development of spontaneous systemic bacterial infec- tions. Our study provides the long-sought explanation for a stroke-induced immunosuppressive state. Our findings may have general implications for the understanding of how se- vere stress and injury may lead to an increased susceptibility to bacterial infections. We demonstrate, in an experimental model, that cerebral ischemia induces long-lasting depres- sion of the cell-mediated immunity (monocyte deactiva- tion, lymphopenia, Th1/Th2 shift) associated with spon- taneous bacteremia and pneumonia. We provide strong Figure 5. Prevention of bacterial infections and restoration of defective IFN- response by SNS inhibitors is dose and time dependent. (a) MCAO evidence that an impaired early NK and T cell response, in mice were treated with diluent or different doses of propranolol (3 particular a reduced IFN- production, is the critical 1–3  30 mg/kg BW) commencing immediately after MCAO or 24 h stroke-induced defect in the antibacterial defense. Thus, ei- (3  30 mg/kg BW) after MCAO, or with 6-OHDA before MCAO as ther adoptive transfer of IFN-–producing lymphocytes described in Materials and Methods and as indicated (n 4 in each group). Lungs and blood samples were collected for bacteriological analy- (i.e., NK and T, but not B cells) or early treatment with re- sis 72 h after MCAO. Data are shown as box plots in CFU/ml (log 10) combinant IFN- inhibited bacteremia and pneumonia. blood or lung tissue homogenate. (b) In a different experiment, spleens of Importantly, the occurrence of life-threatening spontane- untreated mice (control), sham-, or MCAO-operated mice, which re- ous systemic bacterial infections after experimental stroke ceived diluent, propranolol, or 6-OHDA as described above, were col- lected 12 or 36 h (delayed propranolol treatment) after surgery. 10 /ml and the defective IFN- response were only prevented by spleen cells were stimulated ex vivo with Con A for 24 h and IFN- pro- blocking the SNS, but not the HPA, suggesting that cate- duction was analyzed by ELISA, as described in Materials and Methods. cholamine-mediated lymphocyte dysfunction is the key Data are shown as mean SD. #, results differed from the sham-oper- factor in the impaired antibacterial immune response after ated group; #, P 0.05; §, results differed from the MCAO animals; §, P 0.05. Mann-Whitney U test, n 3–6 per group. stroke. We call this phenomenon stroke-induced immuno- deficiency syndrome. It has been long known that activation of the SNS and B6 mice (Fig. 8 a). Moreover, recombinant IFN- reduced the HPA by proinflammatory cytokines in systemic inflam- bacterial dissemination in peripheral blood and lungs when mation results in the release of glucocorticoids and cate- administered 24 h, but not 48 h, after cerebral ischemia cholamines, which in turn inhibit further production of (Fig. 8 b). proinflammatory mediators. More recently, another physi- Prass et al. The Journal of Experimental Medicine Figure 7. Adoptive lymphocyte transfer after experimental stroke pre- vents infection. (a) Adoptive lymphocyte transfer was performed 24 h after MCAO using either unseparated (total) or T, B, and NK cell, or T cell–, Figure 8. IFN- is essential in preventing the bacterial infections. (a) B cell–, or NK cell–depleted spleen cell suspensions from wild-type Adoptive lymphocyte transfer experiments 24 h after MCAO revealed an SV129/J mice. *, results differed from unseparated splenocyte transfer impaired ability of splenocytes from IFN-–deficient mice (IFN- ) to group. Kruskal-Wallis analysis of variance (ANOVA) after pairwise com- prevent bacterial infection in blood and lung when compared with sple- parison with Dunn’s method test, n 4 per group. (b) In a different set of nocytes from wild-type B6 mice (n 8 per group). (b) Administration of experiments, splenocytes from  T cell–deficient (-TCR ),  T 2 g recombinant IFN- 24 h, but not 48 h, after cerebral ischemia re- cell–deficient (-TCR ), or wild-type B6 mice (WT) were trans- duced the bacterial burden in peripheral blood and lungs as measured 72 h ferred into B6 mice 24 h after MCAO. 48 h later, blood and lung samples after MCAO (n 4 per group). For data representation see Fig. 5. were analyzed for bacterial counts. Data are shown as box plots in CFU/ml (log 10) blood or lung tissue homogenate. *, results differed from WT splenocyte transfer group. Kruskal-Wallis analysis of variance (ANOVA) after comparison of pairs with Dunn’s method test, n 8 per group. temic inflammation can inappropriately suppress the immune system and increase the risk of infections. We ological feedback mechanism termed the “cholinergic anti- have recently reported that the intrathecal release of pro- inflammatory pathway” has been uncovered. Activation of inflammatory cytokines is associated with signs of systemic the vagus nerve by inflammatory cytokines during endo- immunodepression and a high incidence of infections in toxemia was found to inhibit macrophage cytokine produc- neurosurgical patients (21). Increased intrathecal levels of tion through release of acetylcholine (24, 25). The rapid proinflammatory cytokines like IL-1, IL-6, and TNF- activation of these pathways in inflammatory conditions have been found in various brain disorders including protects the organism against any adverse effects of an over- trauma, subarachnoidal hemorrhage, and ischemia, which whelming immune response. However, an excessive acti- are commonly associated with HPA and SNS activation vation of inhibitory neuroendocrine pathways without sys- (22, 26–32). Interestingly, diminished ex vivo endotoxin- Stroke-induced Immunodepression The Journal of Experimental Medicine induced TNF- production in rats, after intracerebroven- 13% of the mice die. The mortality in this phase is pre- tricular TNF- or IL-1 infusion, was prevented by hypo- dominantly caused by the brain lesion directly. After day 3, physectomy or -adrenoreceptor blockade (33), suggesting mortality increased dramatically and reached 60% by day that a local increase of proinflammatory cytokines in dam- 7. The results of this study strongly suggest that this second aged brain tissue can trigger a systemic immunoinhibitory phase is a consequence of stroke-induced immunodefi- response via sympathoadrenal activation (13, 21). ciency syndrome–associated severe infections and not di- In this study, we demonstrated that pharmacological in- rectly due to the brain lesion because prevention of in- hibition of the activation of either SNS or HPA prevented fection by antibiotic therapy (unpublished data) or by the stroke-induced lymphocyte apoptosis, lymphopenia, blockade of SNS (Fig. 6) improves survival dramatically and monocytic deactivation. However, poststroke lympho- without affecting infarct size (unpublished data). Although cyte dysfunction (i.e., decrease of IFN- and increase of we have not found a systematic analysis of mortality in IL-4 production) and bacterial infections were only pre- MCAO models in the literature, historic data also suggest a vented by SNS inhibitors (propranolol and 6-OHDA) and two phase time course of survival (45, 47, 51–54). Al- not by the glucocorticoid receptor blocker RU486. Cate- though infectious complications (in particular, bacterial cholamines have been shown to suppress Th1 activities and pneumonia) and their relevance for mortality are well cellular immune responses either directly, inhibiting the known in acute stroke (55, 56), these complications have IFN- synthesis by Th1 cells, or indirectly, by inhibition of not been reported previously in experimental stroke. To Th1-polarizing cytokines like IL-12 and TNF- produced our knowledge, this is the first report of a model with by antigen-presenting cells (34). Instead, catecholamines stress-mediated immunodepression promoting spontaneous appear to promote Th2 cell differentiation and cytokine systemic bacterial infections. This model will be useful to production (34), and trigger the secretion of IL-10 (a po- further investigate the neuroimmunological mechanisms tent inhibitor of Th1 and monocyte functions) by mono- contributing to immunodepression and development of in- cytes (13). Because IFN- stimulates the microbicidal ac- fections after trauma and stress, and to evaluate new thera- tivity of phagocytes, whereas IL-4 inhibits phagocytosis, a peutical approaches to prevent or reverse immunodepres- decreased ratio of IFN-/IL-4 production indicates a com- sion and its infectious complications. promised antibacterial defense (35). A decreased Th1/Th2 In stroke patients, the occurrence of pneumonia is of ratio has been found after major surgery or trauma and as- particular clinical importance. Importantly, the 30-d mor- sociated with lower survival and decreased resistance to in- tality in patients who developed pneumonia is increased up fections after major burn injury (36–39). Our data on stress to sixfold in comparison with stroke patients not suffering mediator blockade underline the importance of functional from pneumonia (55). Because, conservatively estimated, at defects in IFN- production in the control of infectious least 10% of deaths occurring in stroke patients are attribut- complications after stroke. This conclusion is further sup- able to pneumonia, further research to identify patients at ported by the adoptive transfer experiments: prevention of increased risk for pneumonia and alternative approaches for bacterial infections by spleen cell transfer depends not only prevention are needed (55). Additionally, the extent of in- on T and NK cells, but also on their ability to secret IFN-. fectious complications is proportional to the severity of the Moreover, we show that early (24 h) but not late (48 h) initial ischemic deficit (2–4, 55, 56). With respect to stroke administration of recombinant IFN- greatly reduced blood size, our murine model is comparable with large infarctions and pulmonary bacterial burden, suggesting that a rapid in the human MCA region, which may explain the high production of IFN- by lymphocytes is crucial in control- incidence of infections in this model. Although pneumonia ling bacterial infections. Our data support and extend re- is thought to develop in stroke patients most often as a re- cent findings in models of sepsis and pneumonia suggest- sult of aspiration secondary to dysphagia, not all patients ing an important role for early lymphocyte responses in who aspirate develop infections (57, 58). In addition, up to mounting an efficient antibacterial defense (40–42). In mu- a half of patients who develop pneumonia do not aspirate rine models of Gram-negative (Klebsiella pneumoniae) and (59). Thus, although after severe strokes aspiration due to Gram-positive (Nocardia asteroides) pneumonia,  T cells dysphagia is a known risk factor for pneumonia, other fac- are critical for survival (43, 44). In stroke-induced infec- tors have been postulated that might predispose stroke pa- tions,  T cells are essential for pulmonary bacterial clear- tients to pneumonia, e.g., an impaired immune responsive- ance, whereas  T cells are more critical in the peripheral ness (57, 58, 60). Our data strongly support this hypothesis blood. and provide conclusive experimental evidence that aspira- The results of our study may also have important impli- tion alone is not sufficient for the development of bactere- cations for models of stroke as well as sepsis. The high mor- mia and pneumonia, for the following reasons. First, we tality that we have observed in our study is well known in demonstrated that bacterial infections can be prevented by rodent models of stroke (45–47) and seems to be largely propranolol, administration of INF-, as well as by adop- dependent on the duration of the MCAO as well as the tive lymphocyte transfer. These interventions, specifically strain of animals used (48–51). Previously, the high mortal- the latter two, restore the immune function and are un- ity was directly attributed to the central nervous system le- likely to prevent only aspiration. Second, the described sion (45, 46). Interestingly, the time course of the survival changes in immune parameters preceded infections. Third, rate shows two phases. In the first phase (up to day 3) antibiotic treatment prevents infection but does not restore Prass et al. The Journal of Experimental Medicine the altered immune profile (unpublished data). Rather, a 8. Kalra, L., G. Yu, K. Wilson, and P. Roots. 1995. Medical complications during stroke rehabilitation. Stroke. 26:990– defective antibacterial defense after cerebral ischemia is the main factor that determines the occurrence of systemic bac- 9. Viitanen, M., B. Winblad, and K. Asplund. 1987. Autopsy- terial infections. However, we cannot exclude that aspira- verified causes of death after stroke. Acta Med. Scand. 222: tion is a prerequisite for pneumonia in stroke. 401–408. In conclusion, we have characterized for the first time a 10. Ronning, O.M., and B. Guldvog. 1998. Stroke unit versus stroke-induced immunodeficiency that leads to severe bac- general medical wards, II: neurological deficits and activities terial infections, which could have important implications of daily living: a quasi-randomized controlled trial. Stroke. 29: in improving therapy. Prevention of infections in patients 586–590. with cerebral infarctions could reduce morbidity and mor- 11. Livingston, D.H., S.H. Appel, S.R. Wellhausen, G. Sonnen- tality, as chest infections significantly affect the outcome in feld, and H.C. Polk, Jr. 1988. Depressed interferon gamma production and monocyte HLA-DR expression after severe stroke patients (61, 62) and preventive antibiotic treatment injury. Arch. Surg. 123:1309–1312. reduces neurological deficit and mortality in this model of 12. Docke, W.D., F. Randow, U. Syrbe, D. Krausch, K. Asadul- stroke by 60% (unpublished data). In addition to anti- lah, P. Reinke, H.D. Volk, and W. Kox. 1997. Monocyte microbial approaches, immunotherapeutic approaches, such deactivation in septic patients: restoration by IFN-gamma as inhibition of lymphocyte apoptosis (e.g., by caspase in- treatment. Nat. Med. 3:678–681. hibitors) or use of immunostimulatory drugs (e.g., IFN-, 13. Woiciechowsky, C., K. Asadullah, D. Nestler, B. Eberhardt, GM-CSF), may help to prevent or even reverse inappro- C. Platzer, B. Schoning, F. Glockner, W.R. 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Stroke. 31:410–414. 58. Finegold, S.M. 1991. Aspiration pneumonia. Rev. Infect. Dis. Stroke-induced Immunodepression The Journal of Experimental Medicine http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Experimental Medicine Pubmed Central

Stroke-induced Immunodeficiency Promotes Spontaneous Bacterial Infections and Is Mediated by Sympathetic Activation Reversal by Poststroke T Helper Cell Type 1–like Immunostimulation

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Copyright © 2003, The Rockefeller University Press
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0022-1007
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10.1084/jem.20021098
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Abstract

Infections are a leading cause of death in stroke patients. In a mouse model of focal cerebral ischemia, we tested the hypothesis that a stroke-induced immunodeficiency increases the sus- ceptibility to bacterial infections. 3 d after ischemia, all animals developed spontaneous septicemia and pneumonia. Stroke induced an extensive apoptotic loss of lymphocytes and a shift from T helper cell (Th)1 to Th2 cytokine production. Adoptive transfer of T and natural killer cells from wild-type mice, but not from interferon (IFN)-–deficient mice, or administration of IFN- at day 1 after stroke greatly decreased the bacterial burden. Importantly, the defective IFN- response and the occurrence of bacterial infections were prevented by blocking the sympathetic nervous system but not the hypothalamo-pituitary-adrenal axis. Furthermore, admin- istration of the -adrenoreceptor blocker propranolol drastically reduced mortality after stroke. These data suggest that a catecholamine-mediated defect in early lymphocyte activation is the key factor in the impaired antibacterial immune response after stroke. Key words: T lymphocytes • natural killer cells • interferon  • pneumonia • brain ischemia Introduction Infectious complications, predominantly chest and urinary tions in stroke patients (10). The high incidence of infec- tract infections, have been reported to occur in 23–65% of tions in these patients is likely to be a result of an impaired all stroke patients within the first few days after stroke (1–5). immune function. Immunodepression has been reported in Although early mortality is due to direct complications other potentially life-threatening conditions, such as myo- from large strokes, pneumonia is the leading cause of death cardial infarction, polytrauma, or major surgery, resulting in the postacute phase of stroke, regardless of hospitalization in an increased risk of infectious complications (11–13). In (6–9). The high frequency of antibiotic treatment on spe- trauma patients, the presence of brain injury was identified cialized stroke units underlines the clinical problem of infec- as an independent risk factor for infectious complications, possibly due to a central nervous shutdown of the immune defense (14, 15). As early as 1974, an immunosuppressive K. Prass and C. Meisel contributed equally to this work. state was associated with stroke (16). However, the mecha- Address correspondence to Christian Meisel, Wellcome Trust Centre nisms leading to an increased susceptibility to infections after for Human Genetics, University of Oxford, Roosevelt Drive, OX3 7BN Oxford, United Kingdom. Phone: 44-1865-287538; Fax: 44-1865- stroke are still poorly understood. Here, we demonstrate 287533; email: chr.meisel@charite.de; or Ulrich Dirnagl, Department of Neurology, Charité Hospital, Humboldt University, Schumannstraße 20- 21, D-10098 Berlin, Germany. Phone: 49-30-450-560184; Fax: 49-30- Abbreviations used in this paper: 6-OHDA, 6-hydroxydopamine HBr; HE, 450-560915; email: ulrich.dirnagl@charite.de hematoxylin/eosin; HPA, hypothalamic-pituitary-adrenal axis; MCA, C. Meisel’s present address is Wellcome Trust Centre for Human middle cerebral artery; MCAO, middle cerebral artery occlusion; SNS, Genetics, University of Oxford, Roosevelt Drive, OX3 7BN Oxford, sympathetic nervous system; TUNEL, in situ terminal deoxynucleotide United Kingdom. transferase-mediated dUTP nick-end labeling. 725 J. Exp. Med.  The Rockefeller University Press • 0022-1007/2003/09/725/12 $8.00 Volume 198, Number 5, September 1, 2003 725–736 http://www.jem.org/cgi/doi/10.1084/jem.20021098 The Journal of Experimental Medicine that focal cerebral ischemia induces a rapid and long-lasting Biosciences) were used: CD3 (145-2C11; T cells), CD4 (L3T4), CD8 (Ly-2), CD45R (RA3-6B2; B cells), anti–pan-NK cell inhibition of cell-mediated immunity resulting in sponta- marker (DX5), CD11b (M1/70; monocytes/macrophages, gran- neous systemic bacterial infection, and that early IFN- ulocytes), and CD11c (HL3; dendritic cells). In blood and spleen production by T and NK cells is crucial in controlling bac- samples, RBCs were lysed with BD Lysis Solution (Becton Dick- terial infections. Our finding that -adrenoreceptor block- inson) before analysis. The degree of apoptotic cell death in thy- ade enhances cellular immune responses and prevents bac- mus and spleen single cell suspensions was quantified using fluo- terial infections, provides strong evidence that an activation rescein-labeled annexin V (Qbiogene). Cell phenotyping and of the sympathetic nervous system (SNS) is the main im- identification of apoptotic cells was performed by four-color flow munosuppressive mechanism leading to a high incidence of cytometry on a FACSCalibur™ using CELLQuest™ Software infections after stroke. (BD Biosciences). Analysis of Ex Vivo Cytokine Production. Whole blood was di- luted 1:5 in heparinized RPMI 1640 and incubated at 37C and Materials and Methods 5% CO . For analysis of TNF- synthesis, samples were stimu- tm1Mom Animals. SV129/J mice (BGVV), B6.129P2-Tcrb ( lated with 100 ng/ml LPS (endotoxin) from Escherichia coli 0127:B8 tm1Mom T cell–deficient), B6.129P2-Tcrd ( T cell–deficient), (Sigma-Aldrich) for 4 h. For analysis of IFN- and IL-4 produc- tm1Ts B6.129S7-INF (IFN-–deficient), and C57BL/6J wild-type tion, blood samples were stimulated with 100 g/ml Con A mice (The Jackson Laboratory) were housed in the animal care (Sigma-Aldrich) for 24 h. For analysis of IFN- production in facility at the Department of Neurology (Charité Hospital, Ber- spleen cells, 10 cells/ml were stimulated with 10 g/ml Con A lin, Germany) until killed. for 24 h. Concentrations of cytokines in culture supernatants Experimental Model of Stroke. We used gender-mixed 7-wk- were determined using commercially available kits and according old or older SV129/J or, where indicated, C57BL/6J mice weigh- to the manufacturer’s instructions (BD Biosciences). Each sample ing 18–22 g. The surgical procedure of middle cerebral artery was assayed in duplicate. occlusion (MCAO) did not exceed 10 min and was induced as Adoptive Transfer of Spleen Cells. Single cell suspensions were previously described (17). In brief, a monofilament was introduced prepared from spleens of wild-type or knockout mice as de- into the common carotid artery under halothane narcosis, ad- scribed above. Mice that underwent MCAO were injected intra- vanced to the origin of the middle cerebral artery (MCA), and left peritoneally with 5  10 splenocytes 24 h after MCAO. Control there for 60 min until reperfusion. In sham-operated animals, after mice were injected with medium. In some experiments, splenic inserting the filament to the MCA origin, the filament was imme- T, B, and NK cell subsets were depleted using commercially diately withdrawn by 2 mm to avoid ischemia. Occlusion and re- available magnetic bead kits (CD90, CD45R, DX5 microbeads; perfusion were verified by laser Doppler flowmetry (Peri Flux 4001 Miltenyi Biotec). Depletion efficacy was verified as 95% by Master; Perimed). Mice were kept in heated cages for the next 2 h flow cytometry. and body temperature was frequently measured. Animals were Histopathological Analysis of Lung Tissue. After transcardial then returned to their home cages and allowed free access to food perfusion with ethanol-formalin-acetic acid, lungs were removed and water. The infarct volumes were measured histologically as and embedded in paraffin wax. 12-m thick sections were ob- previously described (18). All animal experiments were performed tained by microtome dissection and stained with hematoxylin/ according to institutional and state guidelines. eosin (HE). 20 representative sections of lungs were chosen per Drug Administration. All drugs were injected i.p. and the re- animal (n 4 in each group) and evaluated by two investigators, spective diluents were given to the control animals at the same who were blinded to the treatment groups. The sections were time. RU486 (Sigma-Aldrich) was dissolved in ethanol/sesame graded according to the following criteria: no definite damage oil solution (1:10 vol/vol) at 6 mg/ml and administered (30 mg/ represented no histological changes or minor changes, including kg body weight) 24 h, 5 h, and immediately before MCAO. Pro- unequal distension of alveolar units, mild thickening of the alveo- pranolol (Sigma-Aldrich) was dissolved in 0.9% sodium chloride lar septa, and perivascular and peribronchiolar edema. Definite at 6 mg/ml, and administered (dose 1–30 mg/kg body weight as damage was observed as lung consolidation, thickened alveolar indicated) immediately before as well as 4 and 8 h after MCAO. septae, and the presence of intra-alveolar inflammatory infiltrates. Where indicated, propranolol administration (30 mg/kg body Histochemistry and In Situ Terminal Deoxynucleotide Transferase- weight) was delayed and given 24, 28, and 32 h after MCAO. mediated dUTP Nick-end Labeling (TUNEL) Assay. After trans- 6-hydroxydopamine HBr (6-OHDA; Sigma-Aldrich) was dis- cardial perfusion with ethanol-formalin-acetic acid, thymi were solved in sterile 0.01% ascorbic acid/saline and injected (200 mg/ removed and embedded in paraffin wax. Sections were obtained kg body weight) 3 d before MCAO. IFN- (TEBU) was dis- by microtome dissection and stained with HE. Based on HE solved in phosphate buffered saline at 20 g/ml, and adminis- staining, two representative 12-m sections of thymus were cho- tered i.p. (2 g) at 24 and/or 48 h after MCAO. Control mice sen per animal (n 6) and processed for TUNEL. The Apoptag received heat-denaturated IFN- solution. Kit (Intergen) was used according to the manufacturer’s instruc- Blood Samples, Cell Suspensions, and Cell Count. Mice were tions. Visualization was achieved using the Vectorstain ABC elite killed and blood was collected into heparinized tubes. Single cell kit (Vector Laboratories) in conjunction with 3,3 -diaminobenzi- suspensions from thymus and spleen were prepared by forcing the dine/H O (Sigma-Aldrich). 2 2 tissues through a fine wire mesh. Cells were washed and resus- Bacteriological Analysis. The anesthetized mice were washed pended in 2 ml RPMI 1640 medium containing penicillin, strep- with 70% ethanol under sterile conditions. Blood was collected tomycin, 2 mM glutamine, and 10% FCS (Biochrom KG). by decapitation. The lungs were removed after thoracotomy and Residual RBCs were lysed by hypotonic lysis in ice-cold ammo- homogenized. For determination of CFU, 100 l tissue homo- nium. Cell counts were performed in triplicates. genate or blood was serially diluted, plated onto blood agar plates Flow Cytometry. For flow cytometric analysis the following (Merck), incubated at 37C for 18 h, and bacterial colonies were fluorescently labeled anti–mouse monoclonal antibodies (BD counted. Stroke-induced Immunodepression The Journal of Experimental Medicine Statistical Analysis. Bacteriological data are presented as box Spontaneous Bacteremia and Pneumonia after Focal Cerebral plots and other data are given as mean SD. Data were analyzed Ischemia. 3 d after ischemia, all MCAO animals suffered by Kruskal-Wallis analysis of variance (ANOVA) after pairwise from spontaneous bacterial infections. Bacterial cultures comparison with Dunn’s method or Mann-Whitney U test using from peripheral blood and lungs invariably demonstrated SPSS software (SPSS Inc.). Differences in the group survival were 95% E. coli (Fig. 1 a). Significant bacterial loads were ob- determined using Fisher’s exact test. Values of P 0.05 were served in lungs and blood 24 and 48 h after ischemia, re- considered significant. spectively. Histological examination of lungs revealed typi- cal signs of bacterial pneumonia (19, 20) in all analyzed MCAO-treated animals 72 h after experimental stroke (n Results 4, Fig. 1 b). In contrast, blood and lung cultures from Ischemia was induced in mice by temporary occlusion of sham-operated animals remained sterile at 6, 12, 24, 48 the MCAO. Sham controls received surgery identical to (not depicted), and 72 h (Fig. 1 a). In addition, lung sec- that of MCAO but with regional cerebral blood flow re- tions of sham controls revealed no signs of pneumonia (n 4, maining unaffected. After 60 min of MCAO, animals de- Fig. 1 b). Therefore, susceptibility to infection resulted veloped large infarcts (90 10 mm ) of cortex, striatum, from stroke and not from surgical stress. and hippocampus. Long-lasting Lymphopenia and Impaired Cellular Immune Functions after Cerebral Ischemia. To characterize the un- derlying mechanisms of spontaneous infections after stroke, we analyzed the cellularity and leukocyte populations in lymphoid organs and peripheral blood from MCAO, sham, and control mice using four-color flow cytometry. We found a striking reduction of lymphocyte counts in blood, spleen, and thymus after cerebral ischemia (Fig. 2). The ab- solute numbers of T, B, and NK lymphocytes were de- creased 3–10-fold in blood and 2–3-fold in spleen as early as 12 h after MCAO (Fig. 2, a–f). There was only a mar- ginal decrease in the number of lymphocytes in sham ani- mals, indicating that lymphopenia was mainly caused by ischemia and only partly due to surgical stress. The post- ischemic total lymphocyte count in the blood was signifi- cantly lower even after 14 d (MCAO: 2.7 0.6 vs. con- trol: 4.8 0.5  10 /ml; P 0.05), whereas the number of splenic lymphocytes remained reduced for 42 d (MCAO: 26.9 0.8 vs. control: 46.5 4.7  10 /ml; P 0.05; Fig. 2, a–f). Cellularity in the thymus was also severely reduced (Fig. 2 g) and characterized by a rapid loss of immature CD4 CD8 thymocytes and subsequent re- duction in the number of mature single positive T cells (not depicted). Interestingly, splenic T and B cell counts increased 2 d after stroke, but dropped again at day 5 (Fig. 2, d and e) coinciding with the occurrence of bacteremia. Recently, we have reported that patients with brain sur- gery showed diminished monocytic HLA-DR expression and endotoxin-induced TNF- secretion as signs of sys- temic immunodepression (13, 21). To test whether cerebral ischemia is also associated with alterations in cellular immune functions, we examined the endotoxin-induced TNF- secretion as well as Con A–induced IFN- and IL-4 production ex vivo in whole blood cultures, as parameters of monocyte and T lymphocyte functions, respectively. Secre- tion of endotoxin-induced TNF- was significantly de- Figure 1. Stroke induces bacteremia and pneumonia. (a) Lungs and creased after 12 h and 2 d after MCAO but returned to con- blood samples from sham (n 10) and MCAO-treated mice (n 4–14 trol levels on day 5 (Fig. 2 h). The decrease in the per group) were collected for bacteriological analysis at the indicated time points after surgery. Data are given in CFU/ml (log 10) blood or lung tis- monocytic TNF- release was not due to reduced mono- sue homogenate. (b) In a different set of experiments, lungs from sham- cyte cell numbers, as blood monocyte counts in MCAO operated (n 4) and MCAO mice (n 4) were collected after 72 h for animals did not differ from those in control mice (not histological examination. A representative 12-m section of HE-stained depicted). Furthermore, we found reduced IFN- but in- lung from MCAO but not from sham animals revealed signs (thickening of alveolar walls and neutrophilic infiltrates) of E. coli pneumonia. 160. creased IL-4 production in Con A–stimulated blood cultures Prass et al. The Journal of Experimental Medicine Figure 2. Stroke induces long-lasting lymphopenia and impaired cytokine expression. Spleen, thymus, and peripheral blood samples of untreated SV129/J mice (control) and sham or MCAO mice were collected at different time points (sham, 12 h; MCAO, as indicated) after surgery. Leukocyte counts in blood (a–c), spleen (d–f), and thymus (g) single cell suspensions were determined as described in Materials and Methods. The lymphocyte sub- sets were determined by flow cytometry and their absolute numbers were calculated. (h and i) Aliquots of blood samples were stimulated ex vivo with either LPS for analysis of monocytic TNF- expression or Con A for analysis of IFN- and IL-4 synthesis, as described in Materials and Methods. Cytokines were determined in supernatants by ELISA. The Con A–induced lymphokine expression is given as the ratio of IFN- and IL-4 production calculated for each individual. Data are shown as mean SD. *, results differed from the control group; *, P 0.05; **, P 0.01; ***, P 0.001; #, results differed from the sham-operated group; #, P 0.05; ##, P 0.01. Mann-Whitney U test, n 3–11 per group. resulting in a significant decrease of the IFN-/IL-4 ratio for mice, and only a very modest increase in apoptosis in dou- at least 14 d after MCAO (Fig. 2 i). In contrast, sham-oper- ble positive immature thymocytes was noted (Fig. 3, a and ated animals did not show significant alterations in any of the b). In accordance with these results, light microscopic ex- measured cytokine secretion parameters (Fig. 2, h and i). amination of thymi from MCAO mice, but not sham mice, Importantly, the described alterations of immune parameters showed many apoptotic cells with features of pyknosis or after experimental stroke preceded bacterial infections (com- karyorrhexis (Fig. 3 c, top). Moreover, TUNEL staining pare Figs. 1 a and 2) and, therefore, are more likely to be the demonstrated that the apoptosis occurred predominantly in cause than the result of bacterial infections. the thymic cortex, which is mainly populated by immature Brain Ischemia Causes Rapid and Extensive Apoptosis in double positive thymocytes, and relatively spared the thy- Lymphatic Organs. To test whether lymphopenia was a re- mic medulla (Fig. 3 c, bottom). In addition, thymocyte sult of increased apoptosis, we analyzed spleen and thymus DNA from MCAO animals showed extensive laddering cell suspensions by flow cytometry and found a marked in- (not depicted). crease in apoptotic lymphocytes 12 h after MCAO. In sple- Role of Stress Mediators in Postischemic Immunodepression. nocytes, increased apoptosis affected all lymphocyte subsets, The activation of the hypothalamic-pituitary-adrenal axis as determined by annexin V and cell-type–specific surface (HPA) and the SNS, resulting in a systemic release of adre- marker staining (Fig. 3 a). Similarly, enhanced apoptosis nal steroid hormones and catecholamines, is an essential was found in all thymocyte subsets, mostly affecting the component of the response to major surgery, brain injury, dim immature CD3 CD4 CD8 double positive thymocytes or trauma. As high levels of these stress mediators are (Fig. 3 b). In contrast, there was no significant difference in known to be immunosuppressive (22), we investigated the the degree of apoptosis in splenocytes from sham-operated effects of the -adrenoreceptor antagonist propranolol and Stroke-induced Immunodepression The Journal of Experimental Medicine creased the percentage of apoptotic splenocytes to levels observed in sham-operated mice and prevented the de- crease in peripheral blood lymphocyte counts. The protec- tive effects of both compounds extended to all lymphocyte subsets in spleen and blood (Fig. 4, a and b). Both com- pounds also significantly increased the ex vivo endotoxin- induced TNF- release, but only propranolol normalized the IFN-/IL-4 ratio after Con A stimulation of whole blood cultures (Fig. 4 c). Inhibition of the SNS but Not Steroid Receptor Blockade Pre- vents Systemic Infection. Treatment with propranolol (3 30 mg/kg BW) completely prevented both bacteremia and pneumonia in 8 out of 10 animals (Fig. 4 d). In the remain- ing two mice, bacterial counts in blood and lung were re- 4 6 duced by the order of 10 and 10 , respectively. In contrast, the glucocorticoid receptor antagonist RU486 had no ef- fect on blood or pulmonary bacterial burden. The protec- tive effect of propranolol was dose and time dependent. Propranolol doses (3  10 and 3  30 mg/kg BW starting immediately after MCAO) sufficient to reduce or prevent bacterial dissemination at 72 h after MCAO (Fig. 5 a) also restored the defective IFN- response determined 12 h af- ter experimental stroke (Fig. 5 b), whereas lower doses had no effect on either bacterial load or IFN- production. Im- portantly, delayed administration of propranolol (3  30 mg/kg BW starting 24 h after cerebral ischemia) neither in- creased IFN- production nor decreased bacterial load (Fig. 5), demonstrating that blockade of sympathetic nerves activation early after MCAO is crucial to prevent lympho- cyte dysfunction and, subsequently, bacterial infections. To confirm that the protective effect of propranolol was due to inhibition of sympathetic nerve signaling, MCAO mice were pretreated with 6-OHDA, which selectively enters and destroys sympathetic noradrenergic terminals, thereby depleting tissue catecholamines (23). Like propranolol, chemical sympathectomy by 6-OHDA up-regulated IFN- production and significantly reduced bacterial load in lungs and blood (Fig. 5). Inhibition of the SNS Improves Survival after Experimental Stroke. Mortality in MCAO animals sharply increased from 13% on day 3 to 60% on day 7 after ischemia (n Figure 3. Stroke induces increased apoptosis in lymphoid organs. Spleen and thymus from untreated SV129/J mice (control) and sham or 15). Because animals developed severe hypothermia be- MCAO mice were collected 12 h after surgery. Splenocytes (a) and thy- tween days 3 and 5 ( 33C measured rectally, not de- mocytes (b) were isolated and investigated for apoptosis by annexin V picted) accompanied by bacteremia, sepsis was the likely labeling and flow cytometry. Lymphocyte subpopulations were deter- cause of death. The early mortality in propranolol-treated mined by staining with mAbs against cell-type–specific surface markers. Data are shown as mean SD. *, results differed from the control group; (3  10 mg/kg BW) and diluent-treated animals equally *, P 0.05; **, P 0.01; ***, P 0.001; #, results differed from the amounted to 7-13% until day 3 after stroke. However, sham-operated group; #, P 0.05; ##, P 0.01; ###, P 0.001. only an additional 7% of the propranolol-treated animals Mann-Whitney U test, n 8 per group. (c) Thymic tissue sections from died, whereas 60% of mice treated with diluent were dead sham- and MCAO-treated mice were stained with HE (top) or examined by the fluorescent TUNEL method (bottom). Thymi from MCAO mice by day 7 (P 0.02, diluent vs. propranolol; Fig. 6). showed many apoptotic cells with typical features of nuclear condensation Adoptive Lymphocyte Transfer Prevents Bacterial Infections and fragmentation (HE; 400). Note also the high number of TUNEL after Stroke. Both RU486 and propranolol prevented cells (bright red nuclei) in the thymus cortex from MCAO animals as most of the stroke-induced immunodepressive effects ex- compared with sham mice. 100. cept the changes in the lymphocyte cytokine production, which was only normalized by -adrenoreceptor block- the glucocorticoid receptor inhibitor RU486 on the im- ade. To test whether bacterial infection in MCAO ani- munoinhibitory alterations induced by stroke. Treatment mals was caused by the (functional) loss of lymphocytes, of MCAO animals with either propranolol or RU486 de- we conducted a series of adoptive cell transfer experi- Prass et al. The Journal of Experimental Medicine ments. 24 h after MCAO, mice received either unsepa- rated splenocytes (5  10 ) or subset-depleted spleen cells from normal littermates. Bacterial counts in blood and lungs were determined 48 h later. Transfer of nonsepa- rated spleen cells virtually abolished bacteremia and re- duced pulmonary bacterial load by the order of 10 (Fig. 7 a). This effect was absent upon transfer of spleen cell sus- pensions depleted of all lymphocyte subsets (thus contain- ing mostly monocytes/macrophages). Depletion of B cells only marginally affected the ability to reduce the bacterial load. In contrast, T cell–depleted splenocytes reduced bacteremia only by two orders of magnitude and failed to reduce pulmonary bacterial counts. In addition, NK cell– depleted splenocytes lowered the bacterial load signifi- cantly in the lung but not in peripheral blood (Fig. 7 a). To further investigate the role of specific T cell popula- tions in the antibacterial defense, we performed adoptive transfer studies with spleen cells (5  10 ) from  T cell– or  T cell–deficient mice. Spleen cells from  T cell knockout mice were unable to prevent pulmonary in- fection, whereas they substantially reduced blood bacterial dissemination (Fig. 7 b). In contrast, spleen cells from T cell knockout mice reduced the bacterial burden in the lung but hardly had any effect on bacteremia. These data indicate that T and NK lymphocytes are critical compo- nents of the acute response to bacterial infections after stroke. In particular,  T cells and  T cells are impor- tant for an effective host response to bacterial pneumonia and bacteremia, respectively. IFN- Is Crucial in Controlling Bacterial Infections after Stroke. IFN- is one of the key effector cytokines pro- duced by NK and T cells that enhances microbicidal activ- ity of macrophages and neutrophils. As shown above, blockade of catecholamines restored both IFN- pro- duction and bacterial clearance. Therefore, we examined whether the beneficial effect of lymphocyte transfer in stroke-induced infections is dependent on their ability to produce IFN-. Adoptive transfer of splenocytes from IFN- mice failed to reduce pulmonary and blood bac- terial counts in comparison to spleen cells from wild-type SV129/J mice underwent sham or MCAO surgery (n 9–12 per group). MCAO mice received the -adrenoreceptor antagonist propranolol, the glucocorticoid receptor inhibitor RU486, or only diluent before or after surgery, as described in Materials and Methods. Spleens and blood samples were collected 12 h after surgery. (a) Splenocytes were isolated and inves- tigated for apoptosis by annexin V labeling, staining with mAbs against cell-type–specific surface markers, and flow cytometry. (b) Total leuko- cyte counts in blood samples were determined as described in Materials and Methods. The percentages of different lymphocyte classes were deter- mined by flow cytometry using mAbs against cell-type–specific surface markers and their absolute numbers calculated. (c) For determination of cytokine production, blood samples were processed and cytokine expres- sion was analyzed as described in Fig. 2. Data are shown as mean SD. #, results differed from the sham-operated group; #, P 0.05; ##, P 0.01; §, results differed from the diluent-treated MCAO group; §, P 0.05; §§, P 0.01; §§§, P 0.001. Mann-Whitney U test, n 9–12 per group. (d) In a different set of experiments, lungs and blood samples from sham and inhibitor- or diluent-treated MCAO mice were collected for Figure 4. Effects of -adrenoreceptor and glucocorticoid receptor bacteriological analysis 72 h after surgery. Data are shown as box plots in blockade on stroke-induced immunodepression and bacterial infection. CFU/ml (log 10) blood or lung tissue homogenate. Stroke-induced Immunodepression The Journal of Experimental Medicine Figure 6. Blocking the SNS improves survival after experimental stroke. Mice underwent MCAO and received either diluent or 10 mg/kg BW immediately before, 4, and 8 h after MCAO (n 15 per group). Differences in survival between propranolol-treated versus diluent-treated mice were tested for significance on day 11 after MCAO and are indi- cated by asterisks. *, P 0.05. Fisher’s exact test. Discussion In this study, we provide for the first time experimental proof that a neuroendocrine-mediated systemic immuno- suppression after acute central nervous system lesion results in the development of spontaneous systemic bacterial infec- tions. Our study provides the long-sought explanation for a stroke-induced immunosuppressive state. Our findings may have general implications for the understanding of how se- vere stress and injury may lead to an increased susceptibility to bacterial infections. We demonstrate, in an experimental model, that cerebral ischemia induces long-lasting depres- sion of the cell-mediated immunity (monocyte deactiva- tion, lymphopenia, Th1/Th2 shift) associated with spon- taneous bacteremia and pneumonia. We provide strong Figure 5. Prevention of bacterial infections and restoration of defective IFN- response by SNS inhibitors is dose and time dependent. (a) MCAO evidence that an impaired early NK and T cell response, in mice were treated with diluent or different doses of propranolol (3 particular a reduced IFN- production, is the critical 1–3  30 mg/kg BW) commencing immediately after MCAO or 24 h stroke-induced defect in the antibacterial defense. Thus, ei- (3  30 mg/kg BW) after MCAO, or with 6-OHDA before MCAO as ther adoptive transfer of IFN-–producing lymphocytes described in Materials and Methods and as indicated (n 4 in each group). Lungs and blood samples were collected for bacteriological analy- (i.e., NK and T, but not B cells) or early treatment with re- sis 72 h after MCAO. Data are shown as box plots in CFU/ml (log 10) combinant IFN- inhibited bacteremia and pneumonia. blood or lung tissue homogenate. (b) In a different experiment, spleens of Importantly, the occurrence of life-threatening spontane- untreated mice (control), sham-, or MCAO-operated mice, which re- ous systemic bacterial infections after experimental stroke ceived diluent, propranolol, or 6-OHDA as described above, were col- lected 12 or 36 h (delayed propranolol treatment) after surgery. 10 /ml and the defective IFN- response were only prevented by spleen cells were stimulated ex vivo with Con A for 24 h and IFN- pro- blocking the SNS, but not the HPA, suggesting that cate- duction was analyzed by ELISA, as described in Materials and Methods. cholamine-mediated lymphocyte dysfunction is the key Data are shown as mean SD. #, results differed from the sham-oper- factor in the impaired antibacterial immune response after ated group; #, P 0.05; §, results differed from the MCAO animals; §, P 0.05. Mann-Whitney U test, n 3–6 per group. stroke. We call this phenomenon stroke-induced immuno- deficiency syndrome. It has been long known that activation of the SNS and B6 mice (Fig. 8 a). Moreover, recombinant IFN- reduced the HPA by proinflammatory cytokines in systemic inflam- bacterial dissemination in peripheral blood and lungs when mation results in the release of glucocorticoids and cate- administered 24 h, but not 48 h, after cerebral ischemia cholamines, which in turn inhibit further production of (Fig. 8 b). proinflammatory mediators. More recently, another physi- Prass et al. The Journal of Experimental Medicine Figure 7. Adoptive lymphocyte transfer after experimental stroke pre- vents infection. (a) Adoptive lymphocyte transfer was performed 24 h after MCAO using either unseparated (total) or T, B, and NK cell, or T cell–, Figure 8. IFN- is essential in preventing the bacterial infections. (a) B cell–, or NK cell–depleted spleen cell suspensions from wild-type Adoptive lymphocyte transfer experiments 24 h after MCAO revealed an SV129/J mice. *, results differed from unseparated splenocyte transfer impaired ability of splenocytes from IFN-–deficient mice (IFN- ) to group. Kruskal-Wallis analysis of variance (ANOVA) after pairwise com- prevent bacterial infection in blood and lung when compared with sple- parison with Dunn’s method test, n 4 per group. (b) In a different set of nocytes from wild-type B6 mice (n 8 per group). (b) Administration of experiments, splenocytes from  T cell–deficient (-TCR ),  T 2 g recombinant IFN- 24 h, but not 48 h, after cerebral ischemia re- cell–deficient (-TCR ), or wild-type B6 mice (WT) were trans- duced the bacterial burden in peripheral blood and lungs as measured 72 h ferred into B6 mice 24 h after MCAO. 48 h later, blood and lung samples after MCAO (n 4 per group). For data representation see Fig. 5. were analyzed for bacterial counts. Data are shown as box plots in CFU/ml (log 10) blood or lung tissue homogenate. *, results differed from WT splenocyte transfer group. Kruskal-Wallis analysis of variance (ANOVA) after comparison of pairs with Dunn’s method test, n 8 per group. temic inflammation can inappropriately suppress the immune system and increase the risk of infections. We ological feedback mechanism termed the “cholinergic anti- have recently reported that the intrathecal release of pro- inflammatory pathway” has been uncovered. Activation of inflammatory cytokines is associated with signs of systemic the vagus nerve by inflammatory cytokines during endo- immunodepression and a high incidence of infections in toxemia was found to inhibit macrophage cytokine produc- neurosurgical patients (21). Increased intrathecal levels of tion through release of acetylcholine (24, 25). The rapid proinflammatory cytokines like IL-1, IL-6, and TNF- activation of these pathways in inflammatory conditions have been found in various brain disorders including protects the organism against any adverse effects of an over- trauma, subarachnoidal hemorrhage, and ischemia, which whelming immune response. However, an excessive acti- are commonly associated with HPA and SNS activation vation of inhibitory neuroendocrine pathways without sys- (22, 26–32). Interestingly, diminished ex vivo endotoxin- Stroke-induced Immunodepression The Journal of Experimental Medicine induced TNF- production in rats, after intracerebroven- 13% of the mice die. The mortality in this phase is pre- tricular TNF- or IL-1 infusion, was prevented by hypo- dominantly caused by the brain lesion directly. After day 3, physectomy or -adrenoreceptor blockade (33), suggesting mortality increased dramatically and reached 60% by day that a local increase of proinflammatory cytokines in dam- 7. The results of this study strongly suggest that this second aged brain tissue can trigger a systemic immunoinhibitory phase is a consequence of stroke-induced immunodefi- response via sympathoadrenal activation (13, 21). ciency syndrome–associated severe infections and not di- In this study, we demonstrated that pharmacological in- rectly due to the brain lesion because prevention of in- hibition of the activation of either SNS or HPA prevented fection by antibiotic therapy (unpublished data) or by the stroke-induced lymphocyte apoptosis, lymphopenia, blockade of SNS (Fig. 6) improves survival dramatically and monocytic deactivation. However, poststroke lympho- without affecting infarct size (unpublished data). Although cyte dysfunction (i.e., decrease of IFN- and increase of we have not found a systematic analysis of mortality in IL-4 production) and bacterial infections were only pre- MCAO models in the literature, historic data also suggest a vented by SNS inhibitors (propranolol and 6-OHDA) and two phase time course of survival (45, 47, 51–54). Al- not by the glucocorticoid receptor blocker RU486. Cate- though infectious complications (in particular, bacterial cholamines have been shown to suppress Th1 activities and pneumonia) and their relevance for mortality are well cellular immune responses either directly, inhibiting the known in acute stroke (55, 56), these complications have IFN- synthesis by Th1 cells, or indirectly, by inhibition of not been reported previously in experimental stroke. To Th1-polarizing cytokines like IL-12 and TNF- produced our knowledge, this is the first report of a model with by antigen-presenting cells (34). Instead, catecholamines stress-mediated immunodepression promoting spontaneous appear to promote Th2 cell differentiation and cytokine systemic bacterial infections. This model will be useful to production (34), and trigger the secretion of IL-10 (a po- further investigate the neuroimmunological mechanisms tent inhibitor of Th1 and monocyte functions) by mono- contributing to immunodepression and development of in- cytes (13). Because IFN- stimulates the microbicidal ac- fections after trauma and stress, and to evaluate new thera- tivity of phagocytes, whereas IL-4 inhibits phagocytosis, a peutical approaches to prevent or reverse immunodepres- decreased ratio of IFN-/IL-4 production indicates a com- sion and its infectious complications. promised antibacterial defense (35). A decreased Th1/Th2 In stroke patients, the occurrence of pneumonia is of ratio has been found after major surgery or trauma and as- particular clinical importance. Importantly, the 30-d mor- sociated with lower survival and decreased resistance to in- tality in patients who developed pneumonia is increased up fections after major burn injury (36–39). Our data on stress to sixfold in comparison with stroke patients not suffering mediator blockade underline the importance of functional from pneumonia (55). Because, conservatively estimated, at defects in IFN- production in the control of infectious least 10% of deaths occurring in stroke patients are attribut- complications after stroke. This conclusion is further sup- able to pneumonia, further research to identify patients at ported by the adoptive transfer experiments: prevention of increased risk for pneumonia and alternative approaches for bacterial infections by spleen cell transfer depends not only prevention are needed (55). Additionally, the extent of in- on T and NK cells, but also on their ability to secret IFN-. fectious complications is proportional to the severity of the Moreover, we show that early (24 h) but not late (48 h) initial ischemic deficit (2–4, 55, 56). With respect to stroke administration of recombinant IFN- greatly reduced blood size, our murine model is comparable with large infarctions and pulmonary bacterial burden, suggesting that a rapid in the human MCA region, which may explain the high production of IFN- by lymphocytes is crucial in control- incidence of infections in this model. Although pneumonia ling bacterial infections. Our data support and extend re- is thought to develop in stroke patients most often as a re- cent findings in models of sepsis and pneumonia suggest- sult of aspiration secondary to dysphagia, not all patients ing an important role for early lymphocyte responses in who aspirate develop infections (57, 58). In addition, up to mounting an efficient antibacterial defense (40–42). In mu- a half of patients who develop pneumonia do not aspirate rine models of Gram-negative (Klebsiella pneumoniae) and (59). Thus, although after severe strokes aspiration due to Gram-positive (Nocardia asteroides) pneumonia,  T cells dysphagia is a known risk factor for pneumonia, other fac- are critical for survival (43, 44). In stroke-induced infec- tors have been postulated that might predispose stroke pa- tions,  T cells are essential for pulmonary bacterial clear- tients to pneumonia, e.g., an impaired immune responsive- ance, whereas  T cells are more critical in the peripheral ness (57, 58, 60). Our data strongly support this hypothesis blood. and provide conclusive experimental evidence that aspira- The results of our study may also have important impli- tion alone is not sufficient for the development of bactere- cations for models of stroke as well as sepsis. The high mor- mia and pneumonia, for the following reasons. First, we tality that we have observed in our study is well known in demonstrated that bacterial infections can be prevented by rodent models of stroke (45–47) and seems to be largely propranolol, administration of INF-, as well as by adop- dependent on the duration of the MCAO as well as the tive lymphocyte transfer. These interventions, specifically strain of animals used (48–51). Previously, the high mortal- the latter two, restore the immune function and are un- ity was directly attributed to the central nervous system le- likely to prevent only aspiration. Second, the described sion (45, 46). Interestingly, the time course of the survival changes in immune parameters preceded infections. Third, rate shows two phases. In the first phase (up to day 3) antibiotic treatment prevents infection but does not restore Prass et al. The Journal of Experimental Medicine the altered immune profile (unpublished data). Rather, a 8. 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The Journal of Experimental MedicinePubmed Central

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