Siegbert Rossol

Proinflammatory cytokines in serum of patients with acute cerebral ischemia : kinetics of secretion and relation to the extent of brain damage and outcome of disease

The release of the proinflammatory cytokines IL-1 beta, IL-6, TNF-alpha and soluble TNF-receptors p55 and p75 in peripheral blood was serially determined in 19 patients with acute cerebral ischemia. Only patients admitted within 4 h following onset of symptoms were studied. In contrast to serum levels of IL-1 beta, TNF-alpha and TNF-receptors, which did not exhibit a significant response, IL-6 showed a significant increase of serum levels already within the first hours following onset of disease and reached a plateau at 10 h until day 3 and returned to baseline by day 7. The increase of levels of this cytokine was significantly (P < 0.05) correlated with increasing volumes of brain lesion and was also significantly (P < 0.005) associated with poor functional and neurological outcome. The increase of levels of IL-6 despite a considerable dilution in peripheral blood shown in this preliminary study suggests an early inflammatory response in ischemic brain lesion.

Inflammatory cytokines in subarachnoid haemorrhage: association with abnormal blood flow velocities in basal cerebral arteries

Subarachnoidal release of inflammatory cytokines (interleukin (IL)-1β, IL-6, and tumour necrosis factor (TNF)-α) was characterised in 35 patients with subarachnoid haemorrhage (SAH) and control subjects and compared with development of complicating haemodynamic abnormalities in basal cerebral arteries and clinical outcome. Serial analysis allowed the observation of a subacute response profile of these key mediators of inflammation in the subarachnoidal space. This compartmentalised inflammatory host response was closely associated in time and extent with development of increased blood flow velocities in the basal cerebral vessels as recorded by transcranial Doppler sonography. Moreover, intrathecal secretion of inflammatory cytokines was significantly increased in patients with poor clinical outcome. Together, these findings suggest a role of excessive compartmentalised inflammatory host response in pathogenesis of cerebrovascular complications after SAH.

Interleukin-12 induction of Th1 cytokines is important for viral clearance in chronic hepatitis B.

Interleukin-12, a cytokine with an important role against intracellular pathogens, promotes Th1 cell development, cellmediated cytotoxicity, and interferon-gamma production. We investigated the immunoregulatory role of IL-12 in 72 chronic hepatitis B virus (HBV) carriers, 33 of whom were monitored longitudinally during interferon-alpha treatment. Serum levels of IL-12 heterodimer, IL-12 p40 subunit, IL-4, and Th1 cytokines were determined by specific ELISAs, and hepatitis B core antigen-specific T cell response by a proliferation assay. Chronic HBV carriers had higher serum levels of IL-12 and IL-12 p40 in comparison with controls (P < 0.01), suggesting that IL-12 production is not impaired. The longitudinal analysis revealed a further substantial increase (> 2.5x baseline level) of bioactive IL-12 and Th1 cytokines in patients who cleared HBV and seroconverted to anti- hepatitis B e, unlike the 23 nonresponders with persistent HBV replication (P < 0.01). The IL-12 peak followed the peak of hepatocytolysis by 9.8+/-2.8 wk and occurred either before or simultaneously with hepatitis B e seroconversion. Hepatitis B core antigen-specific T cell proliferation closely correlated with hepatocytolysis and increased significantly in all patients (8 responders and 15 nonresponders) who developed hepatitis flare, irrespective of the virological outcome. These results provide in vivo evidence that IL-12 may have an important role for viral clearance in chronic HBV infection.

Endothelin-1 in Subarachnoid Hemorrhage An Acute-Phase Reactant Produced by Cerebrospinal Fluid Leukocytes

Background and Purpose The most potent vasoconstrictor known, endothelin-1, is currently considered to mediate cerebral vasospasm in subarachnoid hemorrhage (SAH), which can cause delayed cerebral ischemia. In our study, we performed clinical and in vitro experiments to investigate the origin and the mechanisms of the secretion of endothelin-1 in SAH. Methods Endothelin-1 and markers of inflammatory host response (interleukin [IL]-1&bgr;, IL-6, and tumor necrosis factor-&agr;) were comparatively quantified in the cerebrospinal fluid (CSF) of SAH patients and control subjects, and concentrations were related to clinical characteristics. Furthermore, mononuclear leukocytes isolated from the CSF of SAH patients and control subjects were analyzed regarding their mRNA expression of endothelin-1 and inflammatory cytokines. Finally, complementary in vitro experiments were performed to investigate whether coincubation of blood and CSF can trigger leukocytic mRNA expression and release of these factors. Results Activated mononuclear leukocytes in the CSF of SAH patients synthesize and release endothelin-1 in parallel with known acute-phase reactants (IL-1&bgr;, IL-6, and tumor necrosis factor-&agr;). Complementary in vitro experiments not only further confirmed this leukocytic origin of endothelin-1 but also showed that aging and subsequent hemolysis of blood is sufficient to induce such endothelin-1 production. Conclusions The demonstration that endothelin-1 is produced by activated CSF mononuclear leukocytes suggests that subarachnoid inflammation may represent a therapeutic target to prevent vasospasm and delayed cerebral ischemia after SAH.

Butyrate modulates intestinal epithelial cell‐mediated neutrophil migration

Butyrate, a short‐chain fatty acid released by colonic bacteria and administered therapeutically in inflammatory bowel diseases, exerts immunomodulatory properties. The aim of the study was to determine the functional consequences of butyrate exposure on the proinflammatory responsiveness of human intestinal epithelial cells (IEC). IL‐8 promoter activity in IEC pretreated with butyrate then exposed to proinflammatory stimuli was assayed by transfection of luciferase constructs. IL‐8 secretion was determined by ELISA and neutrophil migration by flow cytometry. Receptor mRNA was assessed by reverse transcriptase–polymerase chain reaction (RT‐PCR). Butyrate modulated proinflammatory IL‐8 secretion differentially in Caco‐2 and HT‐29 cells on the transcriptional level. Pointing to the potentially underlying mechanism of increased IL‐1β‐stimulated IL‐8 secretion in HT‐29 cells, butyrate up‐regulated IL‐1RI mRNA but not IL‐1RII. Butyrate pretreatment of IEC lines stimulated by IL‐1β modulated neutrophil migration significantly: reduction towards Caco‐2 and enhancement towards HT‐29/p cells. Pharmacological inhibition of protein tyrosine phosphatases or treatment with mesalamine or sulphasalazine diminished IL‐1β‐stimulated IL‐8 secretion by butyrate‐exposed HT‐29 cells substantially. Immunomodulatory effects of butyrate on IEC are functionally relevant for neutrophil migration. Pharmacological inhibition of enhanced IL‐1β‐mediated IL‐8 secretion in a subpopulation of IEC may improve the clinical efficacy of butyrate.

Enhanced production of IL-18 in butyrate-treated intestinal epithelium by stimulation of the proximal promoter region

Expression of IL‐18 in intestinal epithelial cells (IEC) has been implicated in Th1 cell‐mediated chronic intestinal inflammation and anti‐tumor immunity. However, physiological regulatory factors have not been identified. Besides their effects on proliferation and restitution, immunomodulatory functions have been attributed to short chain fatty acids (SCFA). We investigated the effect of SCFA (butyrate, propionate, acetate) on expression of IL‐18 in IEC in vitro and in vivo. Expression of IL‐18 mRNA and protein in human carcinoma‐derived HT‐29 and Caco‐2 cells was analyzed by reverse transcription‐PCR and Western blot. Transcriptional regulation of IL‐18 gene expression was determined by transient transfection of wild‐type and mutated IL‐18 promoter. Further, in vivo expression of IL‐18 in the intestine from butyrate‐treated and untreated mice was assessed by immunohistochemistry. IL‐18 mRNA and the IL‐18 protein were expressed in IEC, while IL‐18 secretion was not observed. Butyrate and acetate increased intracellular IL‐18 content in a time‐ and dose‐dependent fashion. In contrast to proinflammatory stimuli butyrate potently activated the IL‐18 promoter, indicating that IL‐18 is regulated at the transcriptional level by SCFA. Furthermore, a 108‐bp sequence in the proximal region was identified to be essential for IL‐18 promoter activation by butyrate. As proof of principle butyrate effects were confirmed in vivo by demonstration of increased IL‐18 protein expression in IEC from butyrate‐treated mice. In conclusion, SCFAup‐regulate IL‐18 protein expression in IEC, suggesting a potential regulatory contribution of these luminal constituents to T cell mediated inflammatory and neoplastic intestinal conditions.

Gene expression of TNF-receptors in peripheral blood mononuclear cells of patients with alcoholic cirrhosis

BACKGROUND/AIMS Elevated concentrations of tumor necrosis factor receptors have been detected in alcoholic cirrhosis, but it remains unknown whether or not peripheral blood mononuclear cells are a source of tumor necrosis factor receptors and reflect the clinical disease activity of patients with advanced alcoholic liver disease. METHODS Twenty-two abstinent patients in different stages of alcohol-induced cirrhosis according to the criteria of the Child-Pugh classification (Child-Pugh stage A: 4, Child-Pugh stage B: 10, Child-Pugh stage C: 8) were compared with four healthy individuals. Semi-quantitative reverse transcriptase-polymerase chain reaction was used for the measurement of the expression of tumor necrosis factor-alpha, soluble tumor necrosis factor receptors-p55, -p75, interleukin-10 and inducible nitric oxide synthase in unstimulated peripheral blood mononuclear cells. RESULTS Unstimulated peripheral blood mononuclear cells of patients with alcoholic cirrhosis demonstrate a stage-dependent enhanced RNA expression of tumor necrosis factor-alpha (healthy controls 0/4, Child-Pugh stage A 2/4, stage B 10/10, stage C 8/8; p<0.01). The mRNA expression of TNF-receptors-p55/-p75 is significantly higher in patients with severe alcoholic cirrhosis (Child-Pugh stage B or C patients) than healthy controls (p<0.05), while peripheral blood mononuclear cells from patients with Child-Pugh stage A show a similiar pattern of gene expression to healthy controls. No significant up-regulation of interleukin-10 was found. Inducible nitric oxide synthase was detectable in Child-Pugh stage C (p<0.05). CONCLUSIONS Unstimulated peripheral blood mononuclear cells of patients with severe alcoholic cirrhosis (Child-Pugh stage B and C) demonstrate a systemic leukocyte activation and gene expression of tumor necrosis factor-alpha and tumor necrosis factor receptors-p55/-p75, which is correlated with the activity of the disease. Our data confirm previous studies that reported a correlation between plasma levels of pro-inflammatory cytokines and the severity of alcoholic cirrhosis. The role of interleukin-10 and inducible nitric oxide synthase in the pathogenesis of alcoholic cirrhosis remains to be fully elucidated.

Proinflammatory cytokines: indicators of infection in high-risk patients.

Proinflammatory cytokines play an eminent role in pathophysiology of infection and inflammation. Their actual clinical importance is, however, uncertain. In this study, we tested the hypothesis that inflammatory cytokines could be useful in detection of infections in high-risk patients. We prospectively studied the diagnostic value of determination of concentrations of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and the 55- and 75-kd soluble TNF receptors (sTNFR-p55 and sTNFR-p75) in detection of nosocomial infections in 52 patients with acute ischemic stroke, as an exemplary high-risk group, and compared these findings to those of conventional inflammatory indicators of inflammation (C-reactive protein and leukocyte count). After 1 week of hospitalization, 27% of the patients had minor or moderately severe nosocomial infections. This subpopulation exhibited significantly increased concentrations of IL-6 and sTNFR-p55 but not of IL-1beta, TNF-alpha, or sTNFR-p75. As expected, levels of C-reactive protein and leukocytes were increased in infected patients. The sensitivity and specificity for detection of nosocomial infections at day 7 of hospitalization was highest for IL-6, followed by C-reactive protein and the leukocyte count. The data suggest that the proinflammatory cytokine IL-6, in addition to its considerable pathophysiologic importance in systemic inflammation, may be valuable in detection of infections in high-risk patients.

Increase in Vδ1+γδ T cells in the peripheral blood and bone marrow as a selective feature of HIV‐1 but not other virus infections

Dysregulation of T‐cell receptor (TCR) αβ bearing lymphocytes and an increase in Vδ1+γδ T cells are typical features of HIV‐1 infection. However, the role of γδ T cells remains unclear. Therefore, peripheral blood mononuclear cells (PBMC) of 103 HIV‐1‐infected patients were investigated with respect to expression of Vδ1. These results were compared to the Vδ1 expression of bone marrow mononuclear cells (BMMC). In contrast to healthy controls, Vδ1+ cells dominated among both PBMC and BMMC in HIV‐1‐infected patients. Analysis of the coexpression of CD25, CD8, HLA‐DR and CD45RO revealed a high prevalence of Vδ1/CD45RO and Vδ1/HLA‐DR double‐positive PBMC only in HIV‐1‐infected patients but not in healthy donors. Furthermore, analysis of the γδ TCR repertoire in patients infected with hepatitis B virus, hepatitis C virus, herpes simplex virus (HSV)‐1 and HSV‐2 showed that the selective enhancement of Vδ1+ cells was restricted to HIV infection and not observed in other virus diseases. Our data provide further support for the involvement of γδ T cells in immunosuppression and progression of HIV infection.

Gene expression of interleukin 18 in unstimulated peripheral blood mononuclear cells of patients with alcoholic cirrhosis

BACKGROUND Most patients with alcohol induced cirrhosis (AC) and chronic endotoxinaemia are not suffering from clinically evident systemic inflammatory reactions. This may be due to altered gene expression of cytokines, possibly related to endotoxin (for example, tolerance and sensitisation). Interleukin 18 (IL-18; interleukin γ inducing factor) modulates local cytokine production in response to endotoxin (lipopolysaccharide (LPS)). AIM To investigate the systemic immune response of patients with AC and to see if unstimulated peripheral blood mononuclear cells (PBMC) from patients with AC are activated and contribute to gene expression of IL-18. METHODS Plasma levels of endotoxin (LPS) and serum levels of IL-18 were measured by enzyme linked immunoassay and the amoebocyte lysate test in 74 abstinent patients with different stages of AC (Child-Pugh stage A, n=18; B, n=22; C, n=34) and compared with healthy controls (n=43). Gene expression of IL-18 was assessed by semiquantitative reverse transcription-polymerase chain reaction in freshly isolated unstimulated PBMC of a subgroup of 14 patients with AC compared with five healthy controls. RESULTS Gene expression of IL-18 specific mRNA in unstimulated PBMC was significantly enhanced in patients with advanced AC (Child-Pugh stage C) and correlated with plasma LPS and serum CD14 levels (Spearman rank correlation factors r=0.76 andr=0.72). Serum concentrations of IL-18 were also elevated compared with healthy controls (p<0.001) but correlation with serum levels of CD14 and plasma levels of LPS was much weaker compared with mRNA data (Spearman rank correlation factorsr=0.47 andr=0.26). CONCLUSIONS Our in vivo data suggest a presensitisation of “unstimulated” PBMC in the circulation of patients with AC by endotoxin. The term “unstimulated” may be inadequate in patients with AC. Further investigations are needed to define the exact mechanisms and localisation of sensitisation of PBMC in vivo.