• Essential role of CCL2 in clustering of splenic ERTR-9+ macrophages during infection of BALB/c mice by Listeria monocytogenes.

      Jablonska, Jadwiga; Dittmar, Kurt E; Kleinke, Tanja; Buer, Jan; Weiss, Siegfried; Molecular Immunology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany. jja@gbf.de (2007-01)
      Early interactions between pathogens and host cells are often decisive for the subsequent course of infection. Here we investigated early events during infection by Listeria monocytogenes, a ubiquitously occurring facultative intracellular microorganism that exhibits severe pathogenicity, mainly in immunocompromised individuals. We show that the inflammatory chemokine CCL2 is highly up-regulated early after Listeria infection in spleens of BALB/c mice. ERTR-9+ macrophages of the marginal zone were identified as the only infected cells and exclusive producers of CCL2 at the early time point. Consequently, clusters of different cell types were formed around infected ERTR-9+ cells. Metallophilic MOMA-1+ marginal zone macrophages were, however, excluded from the clusters and migrated into the B-cell follicles. Depletion of CCL2 during infection resulted in a different composition of cell clusters in the spleen and increased the mortality rate of treated mice. Interestingly, ERTR-9+ macrophages no longer were part of clusters in such mice but remained at their original location in the marginal zone.
    • High-resolution mass spectrometric analysis of the secretome from mouse lung endothelial progenitor cells.

      Hemmen, Katherina; Reinl, Tobias; Buttler, Kerstin; Behler, Friederike; Dieken, Hauke; Jänsch, Lothar; Wilting, Jörg; Weich, Herbert A; Department of Gene Regulation, HZI, Build. D, Inhoffenstr. 7, 38124, Braunschweig, Germany. katharina.hemmen@ewetel.net (2011-05)
      Recently, we isolated and characterized resident endothelial progenitor cells from the lungs of adult mice. These cells have a high proliferation potential, are not transformed and can differentiate into blood- and lymph-vascular endothelial cells under in vitro and in vivo conditions. Here we studied the secretome of these cells by nanoflow liquid chromatographic mass spectrometry (LC-MS). For analysis, 3-day conditioned serum-free media were used. We found 133 proteins belonging to the categories of membrane-bound or secreted proteins. Thereby, several of the membrane-bound proteins also existed as released variants. Thirty-five proteins from this group are well known as endothelial cell- or angiogenesis-related proteins. The MS analysis of the secretome was supplemented and confirmed by fluorescence activated cell sorting analyses, ELISA measurements and immunocytological studies of selected proteins. The secretome data presented in this study provides a platform for the in-depth analysis of endothelial progenitor cells and characterizes potential cellular markers and signaling components in hem- and lymphangiogenesis.
    • The role of heterodimerization between VEGFR-1 and VEGFR-2 in the regulation of endothelial cell homeostasis.

      Cudmore, Melissa J; Hewett, Peter W; Ahmad, Shakil; Wang, Ke-Qing; Cai, Meng; Al-Ani, Bahjat; Fujisawa, Takeshi; Ma, Bin; Sissaoui, Samir; Ramma, Wenda; et al. (2012)
      VEGF-A activity is tightly regulated by ligand and receptor availability. Here we investigate the physiological function of heterodimers between VEGF receptor-1 (VEGFR-1; Flt-1) and VEGFR-2 (KDR; Flk-1) (VEGFR(1-2)) in endothelial cells with a synthetic ligand that binds specifically to VEGFR(1-2). The dimeric ligand comprises one VEGFR-2-specific monomer (VEGF-E) and a VEGFR-1-specific monomer (PlGF-1). Here we show that VEGFR(1-2) activation mediates VEGFR phosphorylation, endothelial cell migration, sustained in vitro tube formation and vasorelaxation via the nitric oxide pathway. VEGFR(1-2) activation does not mediate proliferation or elicit endothelial tissue factor production, confirming that these functions are controlled by VEGFR-2 homodimers. We further demonstrate that activation of VEGFR(1-2) inhibits VEGF-A-induced prostacyclin release, phosphorylation of ERK1/2 MAP kinase and mobilization of intracellular calcium from primary endothelial cells. These findings indicate that VEGFR-1 subunits modulate VEGF activity predominantly by forming heterodimer receptors with VEGFR-2 subunits and such heterodimers regulate endothelial cell homeostasis.
    • Whole-blood flow-cytometric analysis of antigen-specific CD4 T-cell cytokine profiles distinguishes active tuberculosis from non-active states.

      Sester, Urban; Fousse, Mathias; Dirks, Jan; Mack, Ulrich; Prasse, Antje; Singh, Mahavir; Lalvani, Ajit; Sester, Martina; Department of Internal Medicine IV, Saarland University, Homburg, Germany. (2011)
      T-cell based IFN-γ release assays do not permit distinction of active tuberculosis (TB) from successfully treated disease or latent M. tuberculosis infection. We postulated that IFN-γ and IL-2 cytokine profiles of antigen-specific T cells measured by flow-cytometry ex vivo might correlate with TB disease activity in vivo. Tuberculin (PPD), ESAT-6 and CFP-10 were used as stimuli to determine antigen-specific cytokine profiles in CD4 T cells from 24 patients with active TB and 28 patients with successfully treated TB using flow-cytometry. Moreover, 25 individuals with immunity consistent with latent M. tuberculosis infection and BCG-vaccination, respectively, were recruited. Although the frequency of cytokine secreting PPD reactive CD4 T cells was higher in patients with active TB compared to patients with treated TB (median 0.81% vs. 0.39% of CD4 T cells, p = 0.02), the overlap in frequencies precluded distinction between the groups on an individual basis. When assessing cytokine profiles, PPD specific CD4 T cells secreting both IFN-γ and IL-2 predominated in treated TB, latent infection and BCG-vaccination, whilst in active TB the cytokine profile was shifted towards cells secreting IFN-γ only (p<0.0001). Cytokine profiles of ESAT-6 or CFP-10 reactive CD4 T cells did not differ between the groups. Receiver operator characteristics (ROC) analysis revealed that frequencies of PPD specific IFN-γ/IL-2 dual-positive T cells below 56% were an accurate marker for active TB (specificity 100%, sensitivity 70%) enabling effective discrimination from non-active states. In conclusion, a frequency lower than 56% IFN-γ/IL-2 dual positive PPD-specific circulating CD4 T-cells is strongly indicative of active TB.