Polarized cytokine release from airway epithelium differentially influences macrophage phenotype.

Epithelial cells and macrophages represent two major cell populations in the lung. They reside in physical proximity and are influenced by inhaled substances, microbial- and host-derived factors, as well as by crosstalk between each other. Here, we report the first systematic study to compare the effects of apical and basolateral secretomes from primary human small airway epithelial cells (SAEC) on human macrophages. We exposed monocyte-derived macrophages (MDMs) to the secretome supernatants (SN) from the apical and basolateral chamber of SAEC culture in an air-liquid interface (ALI) setting and analyzed expression of macrophage surface markers. We found that the apical SN increased the expression of CD11c and CD16, whereas basolateral SN increased the expression of CD163 and CD300e, consistent with apical and basolateral epithelial secretions inducing an M1-biased and M2-biased macrophage polarization, respectively. Conversely, in the presence of Nontypeable Haemophilus influenzae (NTHi), apical SN from NTHi-exposed SAEC induced CD36, CD163 and CD300e and supressed CD11c expression suggesting a switch towards an M2-biased macrophage polarization. Analysis of SN from polarized epithelium revealed a number of factors with differential expression in the apical and basolateral secretome. Functional neutralization of IL6, IL8 or IL1α in the apical secretome led to a decrease in expression of 'M2-like' surface markers, supporting the concept of epithelial-derived secreted factors influencing macrophage phenotype. In conclusion, we show, for the first time to our knowledge, that SN from polarized epithelium, depending on the side of secretion, apical or basolateral, can elicit a differential influence on the macrophages polarization phenotype.

Antibody-dependent and antibody-independent uptake of HBsAg across human leucocyte subsets is similar between individuals with chronic hepatitis B virus infection and healthy donors.

Maintaining detectable levels of antibodies to hepatitis B surface antigen (HBsAg) in serum after HBsAg sero-conversion is the key clinical endpoint indicative of recovery from infection with hepatitis B virus (HBV). As HBV-infected hepatocytes secrete HBsAg subviral particles in vast excess over HBV virions, detectable hepatitis B surface antibody (anti-HBs) titres imply complete elimination of HBV virions as well as HBsAg particles. Although intrahepatic phagocytes, for example Kupffer cells, are thought to mediate clearance of HBsAg via antibody (Ab)-dependent and Ab-independent mechanisms, the relative contributions of circulating phagocytic cell types to HBsAg elimination are poorly characterized. Understanding the role of various immune cell subsets in the clearance of HBsAg is important because Ab-dependent or Ab-independent phagocytic HBsAg uptake may modulate presentation of HBsAg-derived epitopes to antigen-specific T cells and hence plays a critical role in adaptive immunity against HBV. This study aims to characterize phagocytic leucocyte subsets capable of internalizing HBsAg immune complexes (HBsAg:IC) or un-complexed HBsAg particles in whole blood directly ex vivo. The data show that uptake of HBsAg:IC occurs most prominently in monocytes, B cells, dendritic cells and in neutrophils. In contrast, B cells, and to a lesser degree also monocytes, seem to be effective phagocytes for un-complexed HBsAg. Importantly, a similar pattern of phagocytic HBsAg uptake was observed in blood from chronic hepatitis B (CHB) patients compared to healthy controls, suggesting that phagocytosis-related cellular functions are not altered in the context of CHB.

[Adjuvant autologous tumour cell vaccination in patients with renal cell carcinoma. Overall survival analysis with a follow-up period in excess of more than 10 years]. / Adjuvante autologe Tumorvakzine beim Nierenzellkarzinom. Gesamtüberlebensanalyse mit einem Nachbeobachtungszeitraum von mehr als 10 Jahren.

BACKGROUND: Organ-confined renal cell carcinoma (RCC) is associated with tumour progression after surgical therapy in approximately 30% of cases. However, of all recently available adjuvant treatment options, only the autologous tumour cell lysate vaccination therapy (Reniale) has been able to demonstrate a significant positive impact on progression-free survival in a phase III trial. Nevertheless, this therapeutic option has not yet been established as a standard adjuvant treatment. MATERIALS AND METHODS: Between August 1993 and December 1996, a total of 1,267 patients who underwent radical tumour nephrectomy at 84 German centres received Reniale outside a controlled trial. Of these patients, 692 presented at stage pT2-3, pNx-2, M0 (based on the 4th version of TNM classification). These patients were matched with a cohort of 861 patients not receiving any adjuvant treatment who underwent surgical therapy for RCC in a 15-year period in the Carl-Thiem-Klinikum in Cottbus, Germany. Matching criteria included age, gender, pT stage, pN stage, grading, histological cell type, and UICC stage. This resulted in 495 matched pairs (study group n=990) that were comparable regarding demographic and tumour-specific criteria. Statistical analyses included univariate and multivariate analyses of overall survival (OS). Median follow-up time of all patients still alive at the end of the trial (n=667) was 11 years. RESULTS: In the vaccine group, OS after 5 and 10 years was 80.6% and 68.9%, respectively, whereas control patients had an OS of 79.2% and 62.1%, respectively (p=0.066). The 5-year OS of patients with pT3 RCC was 71.3% after vaccination therapy and 65.4% for control patients. After 10 years, 53.6% of the patients in the vaccine group and 36.2% in the control group were still alive (p=0.022). Median survival of patients with pT3 RCC was 81 months (SD 7.8) in the control group. This period was not achieved in the vaccine group. Multivariate Cox analysis revealed a significant positive impact of Reniale on OS among the whole study group [hazard ratio (HR) 1.28, p=0.030]. The analysis of patient subgroups showed a significant positive influence of Reniale for patients presenting with pT3 tumours (HR 1.67, p=0.001). CONCLUSION: Adjuvant postsurgical treatment with Reniale in patients presenting with stage pT3 RCC results in a significant enhancement of OS and should be considered especially in this group of patients. Further clinical trials integrating the recent TNM classification and comprising different risk constellations should follow in order to ultimately assess the value of adjuvant treatment with vaccination immunotherapy.

Pre-operative renal arterial embolisation does not provide survival benefit in patients with radical nephrectomy for renal cell carcinoma.

Currently, there is no widespread use of percutaneous renal artery embolisation (PRAE) as a pre-operative treatment in the management of renal cell carcinoma (RCC). There is also a scarcity of studies concerning the potential benefits of this procedure. All patients with RCC who underwent pre-operative PRAE before nephrectomy (n = 227) and all patients solely undergoing surgery (n = 607) at our institution from 1992 to 2006 were included. Information on techniques used, perioperative transfusion requirements, pathological and clinical variables, acute toxicity and complications were obtained from a retrospective review of medical records. Propensity modelling techniques were used to compare cancer-specific survival (CSS) and overall survival (OS) in both groups. Propensity scores were calculated from a logistic matching model including age, gender, clinical tumour size, grading, pN stage, cM stage, pT stage, histology and microvascular invasion. This resulted in 189 matches. The mean follow-up of the entire group of matched patients was 81 months. The 5-year actuarial CSS and OS for the total group of matched patients was 80.8% and 73.9%, respectively. CSS and OS did not show any significant differences between the matched treatment groups. There were no statistical differences in surgical complications between all patients treated with pre-operative PRAE (n = 227) and all patients without PRAE (n = 607), except for blood transfusion (61% vs 24%; p<0.01). Symptoms of post-embolization syndrome, including lumbar pain, fever, nausea, hypertension and macroscopic haematuria, were reported by 202 patients (89%), in most cases being mild and self-limited. There is no conclusive evidence that pre-operative PRAE provides survival benefits in the management of surgically resected RCC.

[Value of the postoperative Störkel score. Predict disease-free survival of patients with surgically resected renal cell carcinoma]. / Die Wertigkeit des Störkel-Scores. Postoperative Vorhersage des tumorspezifischen Uberlebens von Patienten mit Nierenzellkarzinom.

OBJECTIVE: To determine the value of clinical and pathological parameters defining the Störkel score in order to predict outcomes of patients with surgically treated renal cell carcinoma (RCC). MATERIAL AND METHODS: A total of 834 consecutive patients having radical or partial nephrectomy were retrospectively reviewed. For each patient with RCC, the prognostic Störkel score was calculated according to the following variables: Robson stage, Thoenes nuclear grading, histological type, pattern of growth, and age. Based on the Störkel score, patients were divided into groups: those with good prognosis (GP), intermediate prognosis (IP), and poor prognosis (PP). Cancer-specific survival (CSS) and overall survival (OS) were estimated using the Kaplan-Meier method. The accuracy of prediction of CSS and OS with the Störkel score was analyzed using Kaplan-Meier analysis, proportional hazards regression, and graphic representation [(Kaplan-Meier curves, area under the curve (AUC)]. In 564 patients who were still alive, the median follow-up was 79 months (mean 84.8 months). RESULTS: In the GP, IP, and PP groups, CSS after 8 years was 86.7%, 75.6%, and 13.7%, respectively (p<0.001). In the multiple analysis, only the Robson stage and Thoenes nuclear grading independently predicted CSS. Accordingly, the prognostic accuracy of the Störkel score (CSS prediction: AUC=0.744, 95% CI=0.70-0.79) was not better than with a reduced model that included the Robson stage and grading only (CSS prediction: AUC=0.765, 95%CI=0.72-0.81). CONCLUSIONS: Of all parameters included in the Störkel score, only the Robson stage and nuclear grading are significant prognostic factors. Hence, we recommend an accordant modification of the score with additional variables.

Expression of interleukin-27 by human trophoblast cells.

Cytokines produced at the fetal-maternal interface play a key role in regulating maternal tolerance to the fetus and successful pregnancy. Previously, we showed that EBV-induced gene 3 (EBI3), an interleukin (IL)-12 p40 homologue, was expressed at very high levels by syncytiotrophoblasts and extravillous trophoblasts throughout human pregnancy. EBI3 was recently shown to associate with a novel ligand, p28, to form a new heterodimeric cytokine with important immunoregulatory functions, IL-27. In this study, we investigated whether EBI3 expression by trophoblast cells is associated with that of p28 to form IL-27. We found that genes encoding IL-27 (EBI3 and p28) and its receptor (IL-27R and gp130) were expressed in the placenta at various stages of pregnancy. Co-immunoprecipitation experiments performed from placental lysates, and ELISA of culture supernatants from placental explants, showed that IL-27 heterodimer was produced and released from placental cells. In situ studies of placentae of first, second and third trimester of pregnancy, and of choriocarcinomas, demonstrated that syncytiotrophoblast cells co-expressed EBI3 and p28. Similarly, extravillous trophoblast cells invading the decidua were found to co-express both subunits of IL-27. These data suggest that IL-27 may be part of the cytokine network regulating local immune responses and angiogenesis during human pregnancy.

Occupational stress and psychiatric illness in the military: investigation of the relationship between occupational stress and mental illness among military mental health patients.

OBJECTIVE: This was a pilot study aimed at gathering preliminary data on the relationship between occupational stress and mental illness among military personnel. The primary goal of this study was to determine to what extent military mental health patients report suffering from significant occupational stress. METHODS: Eighty-five active duty military mental health outpatients at the Wilford Hall U.S. Air Force Medical Center mental health clinic answered a 65-item survey that included items on the perception of occupational stress and reported life changes. Participation in this cross-sectional study was anonymous, voluntary, and random. The 85 participants represent 83% of those surveyed and approximately 10% of the clinic's total population of military mental health outpatients. The survey incorporated the 43-item Schedule of Recent Experiences (SRE). By adding the weighted values assigned to the 43 items, each respondent was given an SRE score, which is a measure of overall stress that has been shown to be predictive of future illnesses. RESULTS: A majority (60%) reported suffering from significant work stress. A majority (52%) reported that work stress was causing them significant emotional distress. Almost half (42.5%) reported that work stress was a significant contributor to the onset of their mental illness. The average SRE score for all respondents was 266, reflecting increased risk for future illnesses. Generic work stressors were endorsed more frequently than military-specific stressors. CONCLUSIONS: The results reveal that this population of military mental health clinic outpatients perceived that work stress had a strong negative effect on their emotional health. These results raise the possibility that work stress could be a significant occupational health hazard in the U.S. military, a possibility that warrants further investigation. By gathering additional data on the relationship between work stress and emotional health in the military, interventions can be planned to mitigate the effect of stress caused by the military work environment on the mental health of military personnel.

Signal transducer gp130: biochemical characterization of the three membrane-proximal extracellular domains and evaluation of their oligomerization potential.

Glycoprotein 130 (gp130) is a type I transmembrane protein and serves as the common signal-transducing receptor subunit of the interleukin-6-type cytokines. Whereas the membrane-distal half of the gp130 extracellular part confers ligand binding and has been subject to intense investigation, the structural and functional features of its membrane-proximal half are poorly understood. On the basis of predictions of tertiary structure, the membrane-proximal part consists of three fibronectin-type-III-like domains D4, D5 and D6. Here we describe the bacterial expression of the polypeptides predicted to comprise each of these three domains. The recombinant proteins were refolded from solubilized inclusion bodies in vitro, purified to homogeneity and characterized by means of size-exclusion chromatography and CD spectroscopy. For the first time the prediction of three individual membrane-proximal protein domains for gp130 has been verified experimentally. The three domains do not show intermediate-affinity or high-affinity interactions between each other. Mapping of a neutralizing gp130 monoclonal antibody against D4 suggested a particular functional role of this domain for gp130 activation, because above that an intrinsic tendency for low-affinity oligomerization was demonstrated for D4.

Two different epitopes of the signal transducer gp130 sequentially cooperate on IL-6-induced receptor activation.

Cytokines are key mediators for the regulation of hemopoiesis and the coordination of immune responses. They exert their various functions through activation of specific cell surface receptors, thereby initiating intracellular signal transduction cascades which lead to defined cellular responses. As the common signal-transducing receptor subunit of at least seven different cytokines, gp130 is an important member of the family of hemopoietic cytokine receptors which are characterized by the presence of at least one cytokine-binding module. Mutants of gp130 that either lack the Ig-like domain D1 (DeltaD1) or contain a distinct mutation (F191E) within the cytokine-binding module have been shown to be severely impaired with respect to IL-6 induced signal transduction. After cotransfection of COS-7 cells with a combination of both inactive gp130 mutants, signal transduction in response to IL-6 is restored. Whereas cells transfected with DeltaD1 do not bind IL-6/sIL-6R complexes, cells transfected with the F191E mutant bind IL-6/sIL-6R with low affinity. Combination of DeltaD1 and F191E, however, leads to high-affinity ligand binding. These data suggest that two different gp130 epitopes, one on each receptor chain, sequentially cooperate in asymmetrical binding of IL-6/IL-6R in a tetrameric signaling complex. On the basis of our data, a model for the mechanism of IL-6-induced gp130 activation is proposed.

Monoclonal antibodies against the human interleukin-11 receptor alpha-chain (IL-11Ralpha) and their use in studies of human mononuclear cells.

A panel of 14 hybridoma cell lines secreting monoclonal antibodies against the human interleukin-11 receptor alpha chain (hIL-11Ralpha) was obtained using two different approaches. Two antibodies were raised against peptides of the N- and C-terminal sequences, respectively, of the extracellular part of the hIL-11Ralpha. Another group of 12 antibodies was generated against a hybrid protein consisting of the extracellular part of the hIL-11Ralpha fused to mature full-length human IL-2. All these antibodies recognized native hIL-11Ralpha and most also recognized the denatured receptor on immunoblots after SDS-PAGE. Four different epitopes were identified on the extracellular part of the hIL-11Ralpha. One epitope, defined by the E27 antibody, is located at the N-terminus and the other three epitopes are clustered in the membrane-proximal, C-terminal region. The antibodies defining epitopes I and II recognized membrane-bound hIL-11Ralpha expressed in gp130/hIL-11Ralpha-co-transfected Ba/F3 cells. The E27 antibody cross-reacted with murine IL-11Ralpha, in agreement with the fact that the N-terminal region is highly conserved between species. The other 13 antibodies all recognized a region between amino acids 319 and 363, which is the membrane-proximal part of the hIL-11Ralpha. This region, which is less conserved between mouse and human, is shown here to be an immunodominant region. Anti-IL-11Ralpha monoclonal antibodies, which have not been described previously enabled us to explore the expression and tissue distribution of IL-11Ralpha on human peripheral blood mononuclear cells and cell lines. The antibodies provide powerful tools for the study of the regulation and function of the receptor.

Different epitopes are required for gp130 activation by interleukin-6, oncostatin M and leukemia inhibitory factor.

Gp130 is the common signal transducing receptor subunit of interleukin (IL)-6, IL-11, leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor and cardiotrophin-1. IL-6 and IL-11 induce gp130 homodimerization whereas the others lead to the formation of heterodimers with LIFR or OSMR. Binding epitopes for IL-6 and IL-11 are located in the immunoglobulin-like domain and the cytokine binding module (CBM). Here we show that a gp130 mutant lacking domain 1, although unresponsive to IL-6 and IL-11, can still activate signal transducer and activator of transcription (STAT) transcription factors in response to LIF or OSM. Moreover, point mutations in the CBM of gp130 (F191E and V252D) that severely impair signal transduction in response to IL-6 and IL-11 differentially interfere with gp130 activation in response to LIF and OSM. Thus, epitopes involved in gp130 homodimerization are distinct from those leading to the formation of gp130/LIFR or gp130/OSMR heterodimers. These findings may serve as the base for rational design of gp130 antagonists that specifically interfere with bioactivity of distinct IL-6-type cytokines.

Importance of the membrane-proximal extracellular domains for activation of the signal transducer glycoprotein 130.

The transmembrane glycoprotein gp130 is the common signal transducing receptor subunit of the IL-6-type cytokines. The gp130 extracellular part is predicted to consist of six individual domains. Whereas the role of the three membrane-distal domains (D1-D3) in binding of IL-6 and IL-11 is well established, the function of the membrane-proximal domains (D4-D6) is unclear. Mapping of a neutralizing mAb to the membrane-proximal part of gp130 suggests a functional role of D4-D6 in receptor activation. Individual deletion of these three domains differentially interferes with ligand binding of the soluble and membrane-bound receptors. All deletion mutants do not signal in response to IL-6 and IL-11. The deletion mutants Delta4 and, to a lesser extent, Delta6 are still activated by agonistic monoclonal gp130 Abs, whereas the deletion mutant Delta5 does not respond. Because membrane-bound Delta5 binds IL-6/soluble IL-6R as does wild-type gp130, but does not transduce a signal in response to various stimuli, this domain plays a prominent role in coupling of ligand binding and signal transduction. Replacement of the fifth domain of gp130 by the corresponding domain of the homologous G-CSF receptor leads to constitutive activation of the chimera upon overexpression in COS-7 cells. In HepG2 cells this mutant responds to IL-6 comparable to wild-type gp130. Our findings suggest a functional role of the membrane-proximal domains of gp130 in receptor activation. Thus, within the hematopoietic receptor family the mechanism of receptor activation critically depends on the architecture of the receptor ectodomain.

A fusion protein of interleukin-11 and soluble interleukin-11 receptor acts as a superagonist on cells expressing gp130.

Interleukin-11 is a hematopoietic cytokine that signals via the signal transducer gp130. Although gp130 is ubiquitously expressed, interleukine-11 responsiveness is restricted to cells that express the interleukine-11 receptor alpha-subunit. The interleukine-11 receptor alpha-subunit can be functionally replaced by its soluble form indicating that the transmembrane and cytoplasmic parts are not required for signal transduction. Here, we show that a recombinant fusion protein of a fragment of the human interleukine-11 receptor alpha-subunit ectodomain linked to human interleukine-11 acts as a superagonist on cells expressing gp130 but lacking the membrane-bound interleukine-11 receptor alpha-subunit. It induces acute phase protein synthesis in hepatoma cells and efficiently promotes proliferation of Ba/F3 cells stably, transfected with gp130. In these bioassays, the fusion protein of a fragment of the human interleukine-11 receptor alpha-subunit ectodomain linked to human interleukine-11 is 50 times more potent than the combination of interleukine-11 and the soluble interleukine-11 receptor alpha-subunit. Thus, our findings support the concept that covalent fusion of two soluble proteins required for receptor activation dramatically increases their bioactivity.

Psychiatric illness and the workplace: perspectives for occupational medicine in the military.

In inpatient psychiatric wards and outpatient mental health clinics throughout the military, psychiatrists and other mental health professionals are often faced with patients suffering from emotional distress attributed to occupational stress. There has been scant research into how the routine military work environment affects the mental health status of military employees. This paper provides a review of the occupational medicine literature on the relationship between the work environment and employee mental health. There is a growing recognition that stress resulting from the workplace can provoke psychiatric illness, but the research is limited at this time. The data existing on the work force in general are examined, and the relationship of these findings to the military work environment is discussed. This review suggests that a comprehensive examination of the relationship between the military work environment and the mental health of military employees is needed. By gathering these data, interventions can be planned to mitigate the effect of stress caused by the military work environment on the mental health of its members.

The signal transducer gp130: solution structure of the carboxy-terminal domain of the cytokine receptor homology region.

The transmembrane glycoprotein gp130 is the common signal transducing receptor subunit of the interleukin-6-type cytokines. It is a member of the cytokine-receptor superfamily predicted to consist of six domains in its extracellular part. The second and third domain constitute the cytokine-binding module defined by a set of four conserved cysteines and a WSXWS motif, respectively. The three-dimensional structure of the carboxy-terminal domain of this region was determined by multidimensional NMR. The domain consists of seven beta-strands constituting a fibronectin type III-like topology. The structure reveals that the WSDWS motif of gp130 is part of an extended tryptophan/arginine zipper which modulates the conformation of the CD loop.

Activation of the signal transducer glycoprotein 130 by both IL-6 and IL-11 requires two distinct binding epitopes.

The coordination and regulation of immune responses are primarily mediated by cytokines that bind to specific cell surface receptors. Glycoprotein 130 (gp130) belongs to the family of class I cytokine receptors and is the common signal-transducing receptor subunit shared by the so-called IL-6 type cytokines (IL-6, IL-11, ciliary neurotrophic factor, leukemia inhibitory factor, oncostatin M, and cardiotrophin-1). The inflammatory cytokines IL-6 and IL-11 induce gp130 homodimerization after binding to their specific alpha receptors, which leads to the activation of the Janus kinase/STAT signal transduction pathway. A molecular model of IL-6/IL-6R/gp130, which is based on the structure of the growth hormone/growth hormone receptor complex, allowed the selection of several amino acids located in the cytokine-binding module of gp130 for mutagenesis. The mutants were analyzed with regard to IL-6- or IL-11-induced STAT activation and ligand binding. It was found that Y190 and F191 are essential for the interaction of gp130 with IL-6 as well as IL-11, suggesting a common mode of recognition of helical cytokines by class I cytokine receptors. Furthermore, the requirement of the gp130 N-terminal Ig-like domain for ligand binding and signal transduction was demonstrated by the use of deletion mutants. Thus, besides the observed analogy to the growth hormone/growth hormone receptor complex, there is a substantial difference in the mechanism of receptor engagement by cytokines that signal via gp130.

The signal transducer gp130--bacterial expression, refolding and properties of the carboxy-terminal domain of the cytokine-binding module.

Gp130 is the signal transducing receptor subunit of the so-called interleukin-6-type cytokines. This transmembrane protein is a member of the cytokine-receptor superfamily predicted to consist of six fibronectin-type-III-like domains in its extracellular part. The second and the third domain constitute the so-called cytokine-binding module. Domain 2 is characterized by a set of four conserved Cys residues, domain 3 by a conserved WSXWS motif. As a first approach to a more detailed characterization of the cytokine-binding domains of human gp130, we have expressed in Escherichia coli two forms of domain 3 differing in length. Both proteins were purified and refolded in a single step applying size-exclusion chromatography. According to the rotational correlation times deduced from fluorescence anisotropy decay, they do not form aggregates. CD and fluorescence spectroscopy were used to study thermal unfolding and denaturation by guanidinium hydrochloride. It was shown that N- and C-terminal extension by residues of the adjacent hinge regions substantially increase the thermal stability of the domain, which is conceivable from a molecular model. These results are the basis for further structural investigation by NMR spectroscopy.