Elisabeth Buchberger

Efficient phagocytosis of periodontopathogens by neutrophils requires plasma factors, platelets and TLR2

Summary.  Background: Periodontitis represents a chronic infection of supportive dental tissues by distinct gram‐negative bacteria. It is characterized by chronic and local inflammation as well as transient bacteremia with frequently occurring infections at distant sites. Objectives: The present work aimed to clarify the role of platelets and plasma factors in neutrophil interactions with the periodontopathogens A. actinomycetemcomitans and P. gingivalis. Methods: Phagocytosis, cell–cell interactions and activation of platelets and neutrophils in response to periodontopathogens were analyzed by flow cytometry, confocal microscopy and bacteria survival assay. Plasma factors, platelet signaling pathways and receptors involved in platelet‐neutrophil‐bacteria interactions were determined. The role of platelet and neutrophil TLR2 in phagocytosis was further evaluated in a murine TLR2 knockout model. Results: In the presence of plasma neutrophil‐mediated clearance of periodontopathogens is doubled due to opsonisation of bacteria. Platelets, which become activated by periodontopathogens, further enhance clearance of bacteria by 20%, via direct interaction with neutrophils. Plasma factors (e.g. CD14) are required for platelet activation, which is mainly TLR2 dependent and results in PI3K/Akt activation. In a murine TLR2 knockout model we prove that platelet TLR2 is important for formation of platelet–neutrophil aggregates and enhanced phagocytosis of periodontopathogens. In contrast, neutrophil TLR2 is not involved in platelet–neutrophil aggregate formation but is required for efficient phagocytosis. Conclusions: These data indicate that efficient elimination of periodontopathogens by neutrophils involves a complex interplay of plasma factors as well as platelets and requires functional TLR2. By enhancing neutrophil activation platelets contribute to immune defense but may also foster inflammation.

Intermediate Monocytes but Not TIE2-Expressing Monocytes Are a Sensitive Diagnostic Indicator for Colorectal Cancer

We have conducted the first study to determine the diagnostic potential of the CD14++CD16+ intermediate monocytes as compared to the pro-angiogenic subset of CD14++CD16+TIE2+ TIE2-expressing monocytes (TEMs) in cancer. These monocyte populations were investigated by flow cytometry in healthy volunteers (N = 32) and in colorectal carcinoma patients with localized (N = 24) or metastatic (N = 37) disease. We further determined blood levels of cytokines associated with monocyte regulation. The results revealed the intermediate monocyte subset to be significantly elevated in colorectal cancer patients and to show the highest frequencies in localized disease. Multivariate regression analysis identified intermediate monocytes as a significant independent variable in cancer prediction. With a cut-off value at 0.37% (intermediate monocytes of total leukocytes) the diagnostic sensitivity and specificity ranged at 69% and 81%, respectively. In contrast, TEM levels were elevated in localized cancer but did not differ significantly between groups and none of the cytokines correlated with monocyte subpopulations. Of interest, in vitro analyses supported the observation that intermediate monocytes were more potently induced by primary as opposed to metastatic cancer cells which may relate to the immunosuppressive milieu established in the advanced stage of metastatic disease. In conclusion, intermediate monocytes as compared to TIE2-expressing monocytes are a more sensitive diagnostic indicator of colorectal cancer.

Periodontopathogens induce soluble P-selectin release by endothelial cells and platelets.

INTRODUCTION Soluble P-selectin plays a pivotal role in inflammation and the development of thrombotic and cardiovascular disease. Accordingly, elevated levels of soluble P-selectin are found in periodontitis and (other forms of) inflammatory diseases. However, the cellular source of soluble P-selectin in periodontitis and the effects of periodontopathogens on P-selectin release are unknown. MATERIAL AND METHODS Soluble P-selectin was determined in 26 patients with periodontitis and 19 controls. Furthermore, human endothelial cells and platelets were investigated for their ability to elicit soluble and surface P-selectin in response to periodontopathogens A. actinomycetemcomitans Y4 and P. gingivalis. Moreover surface E-selectin and ICAM-1 expression as well as NFκB translocation in response to these bacteria were determined on endothelial cells as well as the formation of platelet-leukocyte complexes. RESULTS Plasma levels of soluble P-selectin are significantly elevated in periodontitis and correlate with severity of disease and bacterial infection. Stimulation of endothelial cells with periodontopathogens results in rapid surface expression of P-selectin but does not induce NFκB translocation and subsequent de novo synthesis of P-selectin, E-selectin or ICAM-1. In platelets, bacterial stimulation leads to surface expression of P-selectin and fosters the formation of platelet-leukocyte aggregates within minutes. P-selectin is rapidly shed from the surface of platelets and endothelial cells and results in increased levels of soluble P-selectin. CONCLUSIONS Periodontopathogens are able to directly cause activation of endothelial cells and platelets within minutes. Given that transient periodontitis-associated bacteremia commonly occurs after tooth brushing or chewing, our data suggest that reduction of periodontopathogens might result in potential cardiovascular benefits.

Neutralizing Type I IFN Antibodies Trigger an IFN-Like Response in Endothelial Cells

Neutralizing Abs to type I IFNs are of therapeutic significance, i.e., are currently evaluated for the treatment of autoimmune diseases with pathogenic IFN-α production such as for systemic lupus erythematosus. Unexpectedly, we observed that several neutralizing Abs reportedly known to counteract IFN-α or IFN-β activity triggered an “IFN-like” response in quiescent primary human endothelial cells leading to activation of the transcription factor IFN-stimulated gene factor 3 and the expression of IFN-responsive genes. Furthermore, these Abs were found to enhance rather than inhibit type I IFN signals, and the effect was also detectable for distinct other cell types such as PBMCs. The stimulatory capacity of anti-IFN-α/β Abs was mediated by the constitutive autocrine production of “subthreshold” IFN levels, involved the type I IFNR and was dependent on the Fc Ab domain, as Fab or F(ab′)2 fragments potently inhibited IFN activity. We thus propose that a combined effect of IFN recognition by the Ab paratope and the concomitant engagement of the Fc domain may trigger an IFN signal via the respective type I IFNR, which accounts for the observed IFN-like response to the neutralizing Abs. With respect to clinical applications, the finding may be of importance for the design of recombinant Abs vs Fab or F(ab′)2 fragments to efficiently counteract IFN activity without undesirable activating effects.

Monocytes with angiogenic potential are selectively induced by liver resection and accumulate near the site of liver regeneration

BackgroundMonocytes reportedly contribute to liver regeneration. Three subsets have been identified to date: classical, intermediate, non-classical monocytes. The intermediate population and a subtype expressing TIE2 (TEMs) were suggested to promote angiogenesis. In a clinical setting, we investigated which monocyte subsets are regulated after liver resection and correlate with postoperative liver function.MethodsIn 38 patients monocyte subsets were evaluated in blood and subhepatic wound fluid by flow cytometry before and 1-3 days after resection of colorectal liver metastases. The monocyte-regulating cytokines macrophage colony stimulating factor (M-CSF), transforming growth factor beta 1 (TGFβ1), and angiopoietin 2 (ANG-2) were measured in patient plasma by ELISA. C-reactive protein (CRP) and liver function parameters were retrieved from routine hospital analyses.ResultsOn post-operative day (POD) 1 blood monocytes shifted to significantly elevated levels of intermediate monocytes. In wound fluid, a delayed surge in intermediate monocytes was detected by POD 3. Furthermore, TEMs were highly enriched in wound fluid as compared to circulation. CRP and M-CSF levels were substantially increased in patient blood after surgery and correlated significantly with the frequency of intermediate monocytes. In addition, liver function parameters showed a significant association with intermediate monocyte levels on POD 3.ConclusionsThe reportedly pro-angiogenic subsets of monocytes are selectively increased upon liver resection and accumulate next to the site of liver regeneration. As previously proposed by in vitro experiments, the release of CRP and M-CSF may trigger the induction of intermediate monocytes. The correlation with liver parameters points to a functional involvement of these monocyte populations in liver regeneration which warrants further investigation.

Levosimendan exerts anti-inflammatory effects on cardiac myocytes and endothelial cells in vitro

Levosimendan is a positive inotropic drug for the treatment of acute decompensated heart failure (HF). Clinical trials showed that levosimendan was particularly effective in HF due to myocardial infarction. Myocardial necrosis induces a strong inflammatory response, involving chemoattractants guiding polymorphonuclear neutrophils (PMN) into the infarcted myocardial tissue. Our aim was to examine whether levosimendan exhibits anti-inflammatory effects on human adult cardiac myocytes (HACM) and human heart microvascular endothelial cells (HHMEC). Cardiac myocytes and endothelial cells were stimulated with interleukin-1β (IL)-1β (200 U/ml) and treated with levosimendan (0.1-10 µM) for 2-48 hours. IL-1β strongly induced expression of IL-6 and IL-8 in HACM and E-selectin and intercellular adhesion molecule-1 (ICAM-1) in HHMEC and human umbilical vein endothelial cells (HUVEC). Treatment with levosimendan strongly attenuated IL-1β-induced expression of IL-6 and IL-8 in HACM as well as E-selectin and ICAM-1 in ECs. Levosimendan treatment further reduced adhesion of PMN to activated endothelial cells under both static and flow conditions by approximately 50 %. Incubation with 5-hydroxydecanoic acid, a selective blocker of mitochondrial ATP-dependent potassium channels, partly abolished the above seen anti-inflammatory effects. Additionally, levosimendan strongly diminished IL-1β-induced reactive oxygen species and nuclear factor-κB (NF-κB) activity through inhibition of S536 phosphorylation. In conclusion, levosimendan exhibits anti-inflammatory effects on cardiac myocytes and endothelial cells in vitro. These findings could explain, at least in part, the beneficial effects of levosimendan after myocardial infarction.

The VEGF rise in blood of bevacizumab patients is not based on tumor escape but a host-blockade of VEGF clearance

Vascular endothelial growth factor (VEGF) has become a major target in cancer treatment as it promotes tumor angiogenesis. Therapy with anti-VEGF antibody bevacizumab reportedly induces high levels of circulating VEGF which may potentially contribute to resistance. Based on animal or computational models, mechanisms of VEGF induction by bevacizumab have been proposed but not verified in the clinical setting. Hence, we evaluated sixty patients with colorectal cancer metastases for changes in plasma VEGF during neoadjuvant/conversion and adjuvant chemotherapy with or without bevacizumab. VEGF expression was assessed in tissue sections of liver metastases. The VEGF source was investigated with in vitro cultures of tumor, endothelial cells, fibroblasts and platelets, and potential protein stabilization due to anti-VEGF therapy was addressed. A VEGF rise was observed in blood of bevacizumab patients but not in chemotherapy controls, and VEGF was found to be largely complexed by the antibody. A comparable VEGF increase occurred in the presence (neoadjuvant) and absence of the tumor (adjuvant). Accordingly, VEGF expression in tumor tissue was not determined by bevacizumab treatment. Investigations with isolated cell types did not reveal VEGF production in response to bevacizumab. However, antibody addition to endothelial cultures led to a dose-dependent blockade of VEGF internalization and hence stabilized VEGF in the supernatant. In conclusion, the VEGF rise in cancer patients treated with bevacizumab is not originating from the tumor. The accumulation of primarily host-derived VEGF in circulation can be explained by antibody interference with receptor-mediated endocytosis and protein degradation. Thus, the VEGF increase in response to bevacizumab therapy should not be regarded as a tumor escape mechanism.

Engraftment of retrovirally transduced Bet v 1-GFP expressing bone marrow cells leads to allergen-specific tolerance

Molecular chimerism is a promising strategy to induce tolerance to disease-causing antigens expressed on genetically modified haematopoietic stem cells. The approach was employed successfully in models of autoimmunity and organ transplantation. Recently, we demonstrated that molecular chimerism induces robust and lasting tolerance towards the major grass pollen allergen Phl p 5. Since allergens are a group of antigens differing widely in their function, origin and structure we further examined the effectiveness of molecular chimerism using the Phl p 5-unrelated major birch pollen allergen Bet v 1, co-expressed with the reporter GFP. Besides, inhibition of CD26 was used to promote engraftment of modified stem cells. Retrovirus VSV-Betv1-GFP was generated to transduce 5-FU-mobilized BALB/c hematopoietic cells to express membrane-bound Bet v 1 (VSV-GFP virus was used as control). Myeloablated BALB/c mice received Betv1-GFP or GFP expressing bone marrow cells, pre-treated with a CD26 inhibitor. Chimerism was followed by flow cytometry. Tolerance was assessed by measuring allergen-specific isotype levels in sera, RBL assays and T-cell proliferation assays. Mice transplanted with transduced BMC developed multi-lineage molecular chimerism which remained stable long-term (>8 months). After repeated immunizations with Bet v 1 and Phl p 5 serum levels of Bet v 1-specific antibodies (IgE, IgG1, IgG2a, IgG3 and IgA) remained undetectable in Betv1-GFP chimeras while high levels of Phl p 5-specific antibodies developed. Likewise, basophil degranulation was induced in response to Phl p 5 but not to Bet v 1 and specific non-responsiveness to Bet v 1 was observed in proliferation assays. These data demonstrate successful tolerization towards Bet v 1 by molecular chimerism. Stable long-term chimerism was achieved under inhibition of CD26. These results provide evidence for the broad applicability of molecular chimerism as tolerance strategy in allergy.

Overexpression of the Transcriptional Repressor Complex BCL-6/BCoR Leads to Nuclear Aggregates Distinct from Classical Aggresomes

Nuclear inclusions of aggregated proteins have primarily been characterized for molecules with aberrant poly-glutamine repeats and for mutated or structurally altered proteins. They were termed “nuclear aggresomes” and misfolding was shown to promote association with molecular chaperones and proteasomes. Here, we report that two components of a transcriptional repressor complex (BCL-6 and BCoR) of wildtype amino acid sequence can independently or jointly induce the formation of nuclear aggregates when overexpressed. The observation that the majority of cells rapidly downregulate BCL-6/BCoR levels, supports the notion that expression of these proteins is under tight control. The inclusions occur when BCL-6/BCoR expression exceeds 150-fold of endogenous levels. They preferentially develop in the nucleus by a gradual increase in aggregate size to form large, spheroid structures which are not associated with heat shock proteins or marked by ubiquitin. In contrast, we find the close association of BCL-6/BCoR inclusions with PML bodies and a reduction in aggregation upon the concomitant overexpression of histone deacetylases or heat shock protein 70. In summary, our data offer a perspective on nuclear aggregates distinct from classical “nuclear aggresomes”: Large complexes of spheroid structure can evolve in the nucleus without being marked by the cellular machinery for protein refolding and degradation. However, nuclear proteostasis can be restored by balancing the levels of chaperones.

Contamination with recombinant IFN accounts for the unexpected stimulatory properties of commonly used IFN-blocking antibodies.

The cellular response to IFN is an essential part of immune reactions and has been subject to investigations for over 50 years 1. The analyses on IFN function frequently involve the use of neutralizing antibodies to block responses and to document the dependence on IFN signals. In this context, we have previously described an unusual “IFN-like” response initiated by blocking antibodies to type I IFN in primary human endothelial cells (EC) or mononuclear blood cells. In the absence of exogenously added recombinant IFN (rIFN), the exposure of EC to increasing concentrations of IFN-blocking mAb resulted in the dose-dependent induction of IFN response genes at the mRNA and protein level 2. The effect was observed for four different mAb directed against human IFN-α or -β and was dependent on the type I IFN receptor. We concluded that an intrinsic feature of the IFN-blocking antibodies was responsible for the observed “IFN-like” activation of EC; a model was proposed of antibody binding to surface Fc-receptors with sequestration of autocrine IFN and subsequent release to nearby IFN receptors, which would result in the observed “IFN-like” signal. We have now obtained evidence that refutes this hypothesis showing that the “IFN-like” activity associated with IFN-blocking mAb is indeed a discrete component that can be separated from the antibody moiety by sequential cycles of antibody immunoprecipitation (Supporting Information Fig. 1 and Supporting Information Methodology). Furthermore, when the standard two-step procedure for antibody purification as performed by the manufacturer (based on ammonium sulfate precipitation and ion exchange chromatography) was extended by a third step of hydrophobic interaction chromatography, the “IFN-like” activity was lost and the neutralizing capacity of the respective antibodies prevailed (Fig. 1A–C). Figure 1 The “IFN-like” activity in antibody preparations can be eliminated by additional antibody purification (three-step process) and is inhibited by polyclonal anti-IFN-α antiserum. The neutralizing anti-IFN-α mAb MMHA-2 and ... Having established that the “IFN-like” activity was attributable to a discrete contaminant of the applied anti-IFN antibody preparations, the possible contamination with microbial products was first examined. Since the majority of pathogen-associated signals leading to the IFN pathway are mediated by the TLR family 3, 4 we screened for hallmarks of TLR activity. However, we did not observe the induction of the transcription factor NF-κB or the pro-inflammatory activation of EC, strongly arguing against TLR involvement (Supporting Information Fig. 2). We then obtained an indication towards contamination with type I IFN from competition studies showing that the contaminant in mAb preparations was neutralized by rabbit (data not shown) or sheep polyclonal anti-human IFN-α antiserum (Fig. 1D). Polyclonal anti-IFN-β antiserum or control antiserum obtained prior to immunization did not affect the “IFN-like” activity (data not shown). The co-purification (and cross-reactivity) of mouse IFN upon mAb isolation from mouse ascites was a potential source of contamination, which was addressed by cytopathic effect inhibition assays on mouse versus human target cells. There was a significantly higher impact on the human target cells, thus arguing for the presence of human rather than mouse IFN-α (Supporting Information Fig. 3A). However, the question remained as to why the contaminating human IFN-α was not neutralized by the investigated anti-IFN-α-blocking mAb (e.g. MMHA-2). When comparing the neutralizing capacity towards various rIFN-α subtypes, the three-step purified mAb failed to inhibit individual family members (IFN-α subtypes 8, 14, and 16) while the sheep polyclonal antiserum potently repressed all IFN-α subtypes (Supporting Information Fig. 3C). This finding supported the notion that a distinct human IFN-α subtype not neutralized by the respective monoclonal was present in the antibody preparation. In accordance, we found that the purified mAb could not block the “IFN-like” activity present in the contaminated mAb preparation (Supporting Information Fig. 3B). Of note, rIFN-α8 and rIFN-α14 had been produced by PBL prior to the preparation of the contaminated antibody MMHA-2. By applying two anti-human IFN-α ELISA tests (not mouse cross-reactive) with distinct sensitivity towards rIFN-α8 and rIFN-α14 evidence was gained for a predominant antibody contamination by human rIFN-α14 (Supporting Information Table 1). However, a combination of contaminating IFN cannot be excluded. Table 1 Level of detectable contamination with rIFN-α in various antibody preparationsa) Thus, the source of contamination could be traced to the sequential production of rIFN and anti-IFN-blocking antibodies with common equipment. Despite a time window of several months between productions, despite the regular two-step purification procedure, and despite standard equipment cleansing, the contamination of antibody preparations with functional type I IFN was substantial. The importance of our observation was further demonstrated by the frequent occurrence of detectable IFN activity in a considerable number of tested antibodies (Table 1). Apart from various mouse monoclonals against human IFN-α and IFN-β (MMHA-2, MMHA-3, MMHA-9, MMHA-13, MMHB-3, MMHB-12), rat anti-mouse antibodies directed against IFN-α (RMMA-1) or IFN-γ (RMMG-1) also presented with significant amounts of human rIFN. While most of these monoclonals originated from PBL and were supplied by PBL or associated distributors in the contaminated form, further examples for contaminated antibodies were found for an alternative supplier. Two anti-pig IFN mAb (K9, F17) similarly showed contamination with rIFN-α (Table 1). Based on the diverse specificity of contaminated antibodies we propose that unspecific co-purification rather than specific antibody binding accounts for the presence of contaminants. While most of the affected antibodies showed IFN-α contamination, the subtype present may vary. The range of detectable IFN activity varied considerably (by a factor of 1000). The highest levels of anti-viral activity as recorded for the anti-IFN-α mAb clone MMHA-2 equalled a concentration of 800 U/mL of human rIFN-α when applying the antibody at a dilution of 50 μg/mL (common for in vitro experiments). For example, stimulation of target cells with 1000 U/mL of biological or rIFN-α2a in the presence of 50 μg/mL of contaminated blocking mAb MMHA-2 would be expected to result in the complete neutralization of the α2a subtype, while exposing the cells to 800 U/mL of non-neutralized rIFN-α14. The net inhibitory effect on the target cells would be minor leading to the false interpretation of results, especially for an experimental setup where the involvement and concentration of type I IFN is the unknown parameter under investigation. Thus, the information given in this report may be of help in interpreting previously conducted experiments with the listed antibodies. With respect to PBL products, all mAb preparations have been carefully evaluated, and contaminated antibodies were found to date back to the last 2–8 years. More stringent purification and equipment cleaning procedures as well as routine testing for contaminating activity have been put in place at PBL in part due to these experiments. With respect to K9, F17, the company producing these antibodies was informed and has, in the meantime, provided the respective clones to PBL for antibody production. Hence, all products identified in this report to have previously been affected by contamination are now being supplied to the research community in a purified form; however, it is easy to envision that reagent providers who prepare multiple cytokines and mAb could face similar issues as those noted here.