Horst Ulrich Beuscher

The Anti-Inflammatory Effect of Low-Dose Radiation Therapy Involves a Diminished CCL20 Chemokine Expression and Granulocyte/Endothelial Cell Adhesion

Background and Purpose:Low-dose radiotherapy (LD-RT) is known to exert an anti-inflammatory effect, however, the underlying molecular mechanisms are not fully understood. The manipulation of polymorphonuclear neutrophil (PMN) function and/or recruitment may be one mechanism. Chemokines contribute to this process by creating a chemotactic gradient and by activating integrins. This study aimed to characterize the effect of LD-RT on CCL20 chemokine production and PMN/endothelial cell (EC) adhesion.Material and Methods:The EC line EA.hy.926 was irradiated with doses ranging from 0 to 3 Gy and was co-cultured with PMNs from healthy donors either by direct cell contact or separated by transwell membrane chambers. CXCL8, CCL18, CCL20 chemokine and tumor necrosis factor-(TNF-)α cytokine levels in supernatants were determined by ELISA and adhesion assays were performed. The functional impact of the cytokines transforming growth factor-(TGF-)β1 and TNF-α and of the intercellular adhesion molecule-(ICAM-)1 on CCL20 expression was analyzed by using neutralizing antibodies.Results:As compared to CXCL8 and CCL18, CCL20 chemokine secretion was found to be exclusively induced by a direct cell-cell contact between PMNs and EA.hy.926 ECs in a TNF-α-dependent, but ICAM-1-independent manner. Furthermore, irradiation with doses between 0.5 and 1 Gy resulted in a significant reduction of CCL20 release which was dependent on TGF-β1 (p < 0.01). The decrease of CCL20 paralleled with a significant reduction in PMN/EA.hy.926 EC adhesion (p < 0.001).Conclusion:The modulation of CCL20 chemokine expression and PMN/EC adhesion adds a further facet to the plethora of mechanisms contributing to the anti-inflammatory efficacy of LD-RT.Hintergrund und Ziel:Eine niedrigdosierte Strahlentherapie (LD-RT) kennzeichnet eine entzündungshemmende Wirksamkeit, die zugrundeliegenden molekularen Mechanismen sind jedoch noch unvollständig aufgeklärt. Die Beeinflussung der Funktion oder Rekrutierung von polymorphkernigen neutrophilen Granulozyten (PMNs) könnte einen Mechanismus darstellen. Chemokine tragen zu diesem Prozess durch die Ausbildung eines chemotaktischen Gradienten und die Aktivierung von Integrinen bei. In dieser Studie wurde der Effekt der LD-RT auf die Expression des Chemokins CCL20 und die Adhäsion von PMNs an Endothelzellen (ECs) untersucht.Material und Methodik:Die EC-Linie EA.hy.926 wurde mit Dosen von 0–3 Gy bestrahlt und mit PMNs gesunder Spender, entweder in direktem Zell-Zell-Kontakt oder durch Transwell-Membran-Kammern getrennt, kultiviert. Die Konzentrationen der Chemokine CXCL8, CCL18, CCL20 und von Tumor-Nekrose-Faktor-(TNF-)α im Kulturüberstand wurden in einem ELISA bestimmt. Ein funktioneller Einfluss der Zytokine „transforming growth factor“-(TGF-)β1 und TNF-α sowie des „intercellular adhesion molecule“-(ICAM-)1 auf die CCL20-Expression wurde mittels neutralisierender Antikörper untersucht.Ergebnisse:Im Vergleich zu CXCL8 und CCL18 wurde die Sekretion des Chemokins CCL20 ausschließlich durch einen direkten Zell-Zell-Kontakt zwischen PMNs und EA.hy.926-ECs in einem TNF-α-abhängigen, jedoch nicht durch ICAM-1 vermittelten Prozess induziert. Eine Bestrahlung mit Dosen zwischen 0,5 und 1 Gy führte zu einer signifikanten TGF-β1-vermittelten Minderung der CCL20-Sekretion (p < 0,01). Neben der Reduktion von CCL20 konnte eine gleichzeitige signifikante Verminderung der Adhäsion von PMNs an EA.hy.926-ECs beobachtet werden (p < 0,001).Schlussfolgerung:Die Modulation der CCL20-Chemokin-Expression und der PMN/EC-Adhäsion stellt eine neuartige Facette der Mechanismen dar, die zur entzündungshemmenden Wirksamkeit der LD-RT beitragen.

Constitutive Expression of Macrophage-Inflammatory Protein 2 (MIP-2) mRNA in Bone Marrow Gives Rise to Peripheral Neutrophils with Preformed MIP-2 Protein

Macrophage-inflammatory protein 2 (MIP-2) is a major CXC chemokine involved in the migration of polymorphonuclear neutrophils (PMNs) to sites of inflammation. Although cell culture experiments have identified different cell types that can produce MIP-2, the cellular sources in vivo are not clearly defined. By using immunohistochemical staining and analysis of chemokine mRNA expression, the present study aimed to localize cells producing MIP-2 in tissues of normal mice and mice challenged with Yersinia enterocolitica. The results showed a constitutive expression of MIP-2 mRNA in bone marrow (BM) of normal mice, but not in other organs such as spleen, lung, or liver. MIP-2 protein was found in all organs tested but it was exclusively associated with PMNs that stained positive with the cell surface marker Gr-1. Bacterial infection caused a 5-fold increase in the number of MIP-2-positive PMNs recruited to spleens concomitant with a strong increase of splenic MIP-2 mRNA. This correlated well with a 3-fold loss of MIP-2-producing cells in BM. Because MIP-2 mRNA expression in PMNs was increased after stimulation with TNF, the results indicate that newly recruited PMNs can supplement their MIP-2 content through TNF-stimulated transcription. Together, the data imply a constitutive production of MIP-2 by a subset of PMNs in BM and argue for the possibility of a rapid mobilization of MIP-2 through its storage in circulating PMNs.

Expression and regulation of CCL18 in synovial fluid neutrophils of patients with rheumatoid arthritis

Rheumatoid arthritis (RA) is characterized by the recruitment of leukocytes and the accumulation of inflammatory mediators within the synovial compartment. Release of the chemokine CCL18 has been widely attributed to antigen-presenting cells, including macrophages and dendritic cells. This study investigates the production of CCL18 in polymorphonuclear neutrophils (PMN), the predominant cell type recruited into synovial fluid (SF). Microarray analysis, semiquantitative and quantitative reverse transcriptase polymerase chain reaction identified SF PMN from patients with RA as a novel source for CCL18 in diseased joints. Highly upregulated expression of other chemokine genes was observed for CCL3, CXCL8 and CXCL10, whereas CCL21 was downregulated. The chemokine receptor genes were differentially expressed, with upregulation of CXCR4, CCRL2 and CCR5 and downregulation of CXCR1 and CXCR2. In cell culture experiments, expression of CCL18 mRNA in blood PMN was induced by tumor necrosis factor α, whereas synthesis of CCL18 protein required additional stimulation with a combination of IL-10 and vitamin D3. In comparison, recruited SF PMN from patients with RA were sensitized for CCL18 production, because IL-10 alone was sufficient to induce CCL18 release. These results suggest a release of the T cell-attracting CCL18 by PMN when recruited to diseased joints. However, its production is tightly regulated at the levels of mRNA expression and protein synthesis.

Interferon-γ Inhibits the Efficacy of Interleukin 1 to Generate a Th2-Cell Biased Immune Response Induced by Leishmania major

Splenic adherent cells from L. major-infected resistant and susceptible mice were restimulated in vitro and analyzed for the expression of IL-1 activity. Three weeks or later after infection, cells from parasite infected susceptible BALB/c mice produced substantially more IL-1 activity than those from non-infected controls or from L. major-infected resistant C57BL/6 animals. More than 95% of the IL-1 bioactivity was mediated by IL-1 alpha, as determined by blocking experiments with an anti-IL-1 alpha antiserum. The strain-specific differences in IL-1 production correlated with different accumulation of IL-1 producing adherent cells in the spleens of infected animals, but also with different IL-1 producing capacity on a per cell basis. When adherent cells were mixed with syngeneic IFN-gamma producing CD4+ T lymphocytes from L. major-infected C57BL mice or from animals that had been pretreated with anti-CD4 monoclonal antibody prior to infection, the level of detectable IL-1 decreased depending on the number of T cells added. This inhibition could be blocked completely with an anti-IFN-gamma antibody. No such effect was seen, when CD4+ cells were used that were derived from parasite-infected BALB/c mice and did not produce IFN-gamma. In contrast to L. major, L. donovani antigen not only failed to induce IL-1 production, but also dose-dependently suppressed the IL-1 activity elaborated by L. major antigen. We conclude from these data that IFN-gamma effectively inhibits the efficacy to IL-1 to generate to Th2-cell biased immune response induced by L. major. A T cell independent and as yet unknown mechanism to inhibit the IL-1 response is used by L. donovani.

Suppression of TNF by V antigen of Yersinia spp. involves activated T cells.

The 39‐kDa V antigen (Vag) of pathogenic Yersinia species has been described to be a potent suppressor of TNF production. The underlying cellular and molecular mechanisms, however, are completely undefined. Here we show that Vag does not act directly on macrophages, the primary source of TNF, but rather requires help of activated T cells for TNF suppression. Suppression of TNF strictly required the presence of T cell stimuli, i.  e. concanavalin A or immobilized anti‐CD3 antibody. In controls, suppression of TNF was completely blocked by anti‐recombinant polyhistidine V antigen (rVagHis) IgG. As determined by transwell chamber experiments, suppression of TNF by rVagHis did not depend on cell‐to‐cell contact, indicating that it is mediated by an as yet unknown soluble factor. This is the first report to show a suppressive effect of rVagHis on TNF production in tissue culture. The results demonstrate the importance of activated T cells for suppression of TNF expression by rVagHis in vitro.

Tumor necrosis factor-α expression induced by anti-YopB antibodies coincides with protection against Yersinia enterocolitica infection in mice

Abstract Previous studies have suggested that virulence of pathogenic Yersiniae is associated with a suppression of the local cytokine response. In this context, the plasmid-encoded 41-kDa Yersinia outer protein B (YopB) has been implicated with the lack of tumor necrosis factor-α (TNF-α) expression in Peyers patches (PP), following oral infection of mice with the enteropathogenic Yersinia enterocolitica. The present study was performed to further evaluate the relationships between YopB-induced suppression of TNF-α and bacterial survival in host tissue. Results are presented to show the ability of purified YopB to suppress the release of TNF-α by macrophages, the effect of which was neutralized by monospecific anti-YopB antiserum. In mice orally infected with Y. enterocolitica, anti-YopB treatment on days 3 and 5 postinfection, significantly decreased the recovery of live bacteria from PP. This observation correlated with a strong increase in TNF-α expression, as determined by reverse transcription-polymerase chain reaction and measuring the levels of TNF activity in homogenates of PP. Moreover, treatment of mice with a combination of anti-YopB and anti-TNF-α antiserum, completely abrogated the beneficial effect of the anti-YopB antiserum. In controls, expression of other proinflammatory cytokines such as interleukin-1 remained unaffected by either treatment. Therefore, the results indicate that endogenous TNF-α is required for eradication of Y. enterocolitica from host tissue, and further imply that YopB significantly contributes to suppression of the local TNF-α response in PP.

Lipopolysaccharides as Complement Inhibitors by Complex Formation with the Purified Third Complement Component (C3)

Lipopolysaccharides (LPS) from different bacteria in smooth or rough form (Y. enterocolitica, Y. pseudotuberculosis, E. coli, S. typhimurium, S. marcescens) strongly inhibited hemolytic C3 in incubation mixtures with purified C3. LPS from a core deficient mutant was still reactive, whereas lipid A no longer affected C3 activity. The physical state of LPS was critical for its effect on C3. Strand-like LPS structures formed by Ca++-induced aggregation of solubilized LPS, as shown by electron microscopy, demonstrated the highest reactivity with C3. Inhibition of hemolytic C3 was found to be due to complex formation between LPS and C3 by a hydrophobic reaction. The binding capacity of 1 microgram LPS-R and LPS-S was as high as 125 ng C3 and 56 ng C3, respectively. The C3b fragment required different reaction conditions for maximal binding. The strong binding capacity of LPS for the complement component C3 raises the possibility that LPS act as inhibitors of complement by interruption of the reaction cascade at local infectious sites with gram-negative bacteria.

Formation of EAC{ie} and EAC{ie} with Macrophage Culture Supernatant Containing the Secreted Complement Components C1 to C3

Culture supernatants of thioglycollate-elicited guinea pig peritoneal macrophages contained hemolytic C1, C4, C2 and C3, whereas hemolytic C5, C6, C7, C8 or C9 were not detected. Activity of C1, C2 and C3 increased up to a 48 h culture period, whereas C4 activity already declined in 2 day old cultures. After secretion, the hemolytic activity of C1 was least stable in culture supernatant. Sensitized sheep erythrocytes (EA) when incubated with culture supernatant initiated activation and functional cooperation of secreted C1 to C3 as indicated by formation of EAC142 and EA1423 intermediates. Decay and regeneration with purified C2 was shown for EAC142 and deposition of C3 fragments on EAC1423 was demonstrated with anti-C3. On an average, supernatants of 2 day old macrophage cultures were most suitable for formation of EAC142 and EAC1423 . The rate of EAC142 and EAC1423 formation, and also of C2 and C3 inactivation, during incubation of EA with culture supernatant was slow; addition of purified C1 to culture supernatant, however, greatly enhanced the same reactions of EA with supernatant which indicated that C1 was the rate limiting factor. Local secretion of hemolytic C1, C4, C2 and C3 by macrophages may have an important role in antimicrobial defense mechanisms due to the well-known functional cooperation between macrophages and activated C3.

Ergebnisse mit den IgM-FTA-Abs- und IgM-TPHA-Testen bei der Untersuchung von seropositiven Luetikerseren

Abstract The IgM fraction was separated by gel filtration chromatography from patient sera which were positive in the FTA-abs-test as well as in the TPHA-test. After absorption the isolated IgM fraction was tested for specific IgM antibodies against Treponema pallidum in the IgM-FTA-abs- and also in the IgM-TPHA-test. The following results were obtained: 1. Both IgM tests often showed positive reactions even with negative results in the cardio-lipin complement fixation assay (CFA) (Tables 1 and 2). 2. In most positive cases specific IgM was detectable with both IgM tests in the same sample (Table 3). 3. The CFA titers were low ( 1:20) where the IgM tests gave positive results (Table 1 and 3). Clinical symptoms of active syphilis were only reported for cases with high CFA titer and positive IgM tests in the same sample. Therefore only these latter results of diagnostic tests can be clearly interpreted to indicate a requirement for specific therapy against syphilis.