Martin Foerster

Human γδ-T Lymphocytes Express and Synthesize Connective Tissue Growth Factor: Effect of IL-15 and TGF-β1 and Comparison with αβ-T Lymphocytes

T lymphocytes bearing the γδ-TCR accumulate during wound healing and inflammation. However, the role of γδ-T lymphocytes in fibrogenic tissue reactions is not well understood. Therefore, we addressed the question of whether human γδ-T cells express and synthesize connective tissue growth factor (CTGF), a factor known to regulate fibrogenesis and wound healing. In addition, the lymphoblastic leukemia T cell line (Loucy) that possesses characteristics typical of γδ-T cells was used as a model to evaluate the regulation of CTGF gene expression. Blood γδ-T cells isolated from healthy donors were grown in the presence of IL-15/TGF-β1 for 48 h and assessed for the expression and synthesis of CTGF. Nonstimulated human blood γδ-T cells and Loucy γδ-T cells expressed low levels of CTGF mRNA. Costimulation of the cells with IL-15 and TGF-β1 resulted in a substantially increased level of CTGF mRNA expression within 4–8 h, and it remained elevated for at least 48 h. In contrast, no CTGF mRNA was detected when nonstimulated and stimulated human CD4+ αβ-T cells were analyzed. In addition, Western blot analysis of human γδ-T cell lysates prepared 4 days following stimulation with IL-15 and TGF-β1 revealed a 38-kDa CTGF protein in cell lysates of human γδ-T cells. Detection was confirmed using Colo 849 fibroblasts, which can constitutively express high levels of CTGF. In conclusion, we herein present novel evidence that in contrast to CD4+ αβ-T cells human γδ-T cells are capable of expressing CTGF mRNA and synthesizing its corresponding protein, which supports the concept that γδ-T cells may contribute to wound healing or tissue fibrotic processes.

Differential regulation of CD95 (Fas/APO-1) expression in human blood eosinophils

CD95 (Fas, APO‐1) is a cell surface receptor expressed on many cells including eosinophils which mediates apoptosis when ligated by agonistic antibodies or its natural ligand FasL. Since inhibition of apoptosis may play an important role in controlling tissue eosinophilia, we investigated the expression of CD95 on purified peripheral blood eosinophils from normal donors. Freshly isolated eosinophils expressed CD95 on the cell surface as well as CD95‐specific mRNA at low levels which did not change during 24‐h culture. Incubation of eosinophils with IL‐3, IL‐5 and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) did not modulate the basal expression of CD95. IFN‐γ as well as TNF‐α, however, induced a significant, dose‐ and time‐dependent increase in CD95 mRNA and cell surface expression as measured by reverse transcription‐PCR and flow cytometry. Co‐stimulation with IFN‐γ and TNF‐α had synergistic effects on the CD95 surface expression on eosinophils. Addition of IL‐3, IL‐5 or GM‐CSF to IFN‐γ‐ and TNF‐α‐stimulated eosinophils caused in a reduction of CD95 expression. Functional activity for CD95 following incubation with IFN‐γ and TNF‐α was demonstrated by increased apoptosis in response to cross‐linking with FasL. From these data, we conclude that IFN‐γ and TNF‐α can up‐regulate cell surface expression of CD95 on eosinophils, which leads to an increased susceptibility of eosinophils to Fas‐mediated apoptosis. Thus, our results suggest that receptors involved in eosinophil apoptosis can be regulated by antagonistic cytokines.

Toward a Spectroscopic Hemogram: Raman Spectroscopic Differentiation of the Two Most Abundant Leukocytes from Peripheral Blood

The first response to infection in the blood is mediated by leukocytes. As a result crucial information can be gained from a hemogram. Conventional methods such as blood smears and automated sorting procedures are not capable of recording detailed biochemical information of the different leukocytes. In this study, Raman spectroscopy has been applied to investigate the differences between the leukocyte subtypes which have been obtained from healthy donors. Raman imaging was able to visualize the same morphological features as standard staining methods without the need of any label. Unsupervised statistical methods such as principal component analysis and hierarchical cluster analysis were able to separate Raman spectra of the two most abundant leukocytes, the neutrophils and lymphocytes (with a special focus on CD4(+) T-lymphocytes). For the same cells a classification model was built to allow an automated Raman-based differentiation of the cell type in the future. The classification model could achieve an accuracy of 94% in the validation step and could predict the identity of unknown cells from a completely different donor with an accuracy of 81% when using single spectra and with an accuracy of 97% when using the majority vote from all individual spectra of the cell. This marks a promising step toward automated Raman spectroscopic blood analysis which holds the potential not only to assign the numbers of the cells but also to yield important biochemical information.

Expression and synthesis of fibroblast growth factor-9 in human γδ T-lymphocytes. Response to isopentenyl pyrophosphate and TGF-β1/IL-15

γδ T‐lymphocytes are believed to play a role in maintaining the normal configuration of epithelial tissue. As little is known about the factors mediating this function, we addressed the question of whether γδ T‐lymphocytes produce fibroblast growth factor (FGF)‐9 as well as two other growth factors associated with epithelial tissue reconstitution. Blood γδ T cells isolated from healthy donors were grown in the presence of isopentenyl pyrophosphate (IPP) or transforming growth factor‐β1 (TGF‐β1)/interleukin‐15 (IL‐15) for 24 h and were assessed for the expression and synthesis of FGF‐9, keratinocyte growth factor (KGF), and epidermal growth factor (EGF). Resting human γδ T cells constitutively expressed KGF and FGF‐9 mRNA but no EGF mRNA. In the presence of IPP, FGF‐9 mRNA expression significantly increased in a dose‐dependent manner, expression of KGF remained unaltered, and EGF mRNA could not be detected. In contrast to IPP, stimulation of the cells with TGF‐β1/IL‐15 did not alter FGF‐9 expression. Moreover, stimulation with anti‐CD3 does not induce FGF‐9 expression but triggers a high signal of interferon‐γ mRNA. Western blot analysis of γδ T cell lysates, prepared 4 days following stimulation with IPP, showed an increase of FGF‐9 protein as compared with control cells. In conclusion, the results demonstrate for the first time that human blood and bronchoalveolar lavage γδ T‐lymphocytes are capable of expressing FGF‐9. The data also provide novel evidence that immunoregulatory cells can synthesize FGF‐9.

Interferon-αcon-1 Treatment of Three Patients with Severe Glucocorticoid-Dependent Asthma

We report herein the therapeutic effect of interferon (IFN)-αcon in three patients with severe persistent asthma and long-term oral glucocorticoid treatment. IFN-αcon (9 µg) administered subcutaneously thrice a week over a period of more than 24 months led to a substantial clinical improvement with regard to the number of daily asthma attacks, nighttime disturbance, emergency visits and hospitalizations. In addition, lung function and exercise capacity improved. At the same time, treatment with IFN allowed discontinuation of the daily glucocorticoid dose in all patients for the first time in more than 8 years. Our findings suggest that IFN-αcon leads to a significant clinical improvement while at the same time allowing reduction and discontinuation of the glucocorticoid treatment in severe persistent glucocorticoid-dependent asthma.

BCG strain S4-Jena: An early BCG strain is capable to reduce the proliferation of bladder cancer cells by induction of apoptosis.

BackgroundIntravesical immunotherapy with Mycobacterium bovis bacillus Calmette-Guérin has been established as the most effective adjuvant treatment for high risk non-muscle-invasive bladder cancer (NMIBC). We investigated the differences between the S4-Jena BCG strain and commercially available BCG strains. We tested the genotypic varieties between S4-Jena and other BCG strains and analysed the effect of the BCG strains TICE and S4-Jena on two bladder cancer cell lines.ResultsIn contrast to commercially available BCG strains the S4-Jena strain shows genotypic differences. Spoligotyping verifies the S4-Jena strain as a BCG strain. Infection with viable S4-Jena or TICE decreased proliferation in the T24 cell line. Additionally, hallmarks of apoptosis were detectable. In contrast, Cal29 cells showed only a slightly decreased proliferation with TICE. Cal29 cells infected with S4-Jena, though, showed a significantly decreased proliferation in contrast to TICE. Concordantly with these results, infection with TICE had no effect on the morphology and hallmarks of apoptosis of Cal29 cells. However, S4-Jena strain led to clearly visible morphological changes and caspases 3/7 activation and PS flip.ConclusionsS4-Jena strain has a direct influence on bladder cancer cell lines as shown by inhibition of cell proliferation and induction of apoptosis. The data implicate that the T24 cells are responder for S4-Jena and TICE BCG. However, the Cal29 cells are only responder for S4-Jena and they are non-responder for TICE BCG. S4-Jena strain may represent an effective therapeutic agent for NMIBC.

Phenotypic and molecular characterization of CD103+ CD4+ T cells in bronchoalveolar lavage from patients with interstitial lung diseases

The integrin CD103 is preferentially expressed on intraepithelial T lymphocytes, and cells expressing this integrin may play a regulatory role in the microenvironment of the epithelial cell layer.

Synovial fibroblasts and synovial macrophages from patients with rheumatoid arthritis and other inflammatory joint diseases show chromosomal aberrations

Chromosomal aberrations were investigated in nuclei extracted from synovial tissue and first‐passage synovial fibroblasts (P‐1 SFB, 98% enrichment) or macrophages (P‐1 Mϕ) from patients with rheumatoid arthritis (n = 10). The findings were compared with those in other rheumatic diseases (osteoarthritis, n = 14; reactive arthritis, n = 1), as well as with those in chronic obstructive pulmonary disease (n = 8). Controls were paired peripheral blood lymphocytes from arthritic patients, synovial tissue or SFB/Mϕ from joint trauma/normals (n = 9), and peripheral blood monocytes from normal donors (n = 10). GTG banding of metaphase chromosomes and interphase fluorescence in situ hybridization with centromere‐specific probes were used. Comparable chromosomal aberrations were observed in synovial tissue and P‐1 SFB of patients with rheumatoid arthritis, osteoarthritis, and reactive arthritis (polysomy 7 and aneusomies of chromosomes 4, 8, 9, 12, and 18). Notably, aneusomies of chromosomes 4, 6, 7, 8, 9, 11, 12, and/or X were also detected in P‐1 synovial Mϕ from rheumatoid arthritis (90% of the cases), osteoarthritis (93%), and reactive arthritis (1/1), as well as bronchial Mϕ from chronic obstructive pulmonary disease (25%). No aberrations were detected in paired peripheral blood lymphocytes (except for one osteoarthritis case with a karyotype 45,X[10]/46,XX[17]), or in peripheral blood monocytes and synovial tissue of normals/joint trauma. Because Mϕ aberrations were common to chronic joint and pulmonary disease, chronic inflammatory stress may induce chromosomal aberrations with potential functional relevance in local mesenchymal cells and infiltrating leukocytes in an organ‐independent fashion.

Bcl‐2‐Mediated Regulation of CD69‐Induced Apoptosis of Human Eosinophils: Identification and Characterization of a Novel Receptor‐Induced Mechanism and Relationship to CD95‐Transduced Signalling

Elimination of the eosinophils from the airways by selective induction of apoptosis represents a therapeutic approach for asthma. Here we report on a possible target molecule, the surface receptor CD69. To simulate an asthmatic response, segmental allergen challenge in mild asthmatics was performed. Eosinophil numbers increased in bronchoalveolar lavage (BAL) at 18 h. In contrast to blood cells, BAL eosinophils expressed the activation marker CD69. Purified blood eosinophils stimulated with granulocyte/macrophage colony‐stimulating factor (GM‐CSF) or interferon‐γ (IFN‐γ) expressed CD69 and showed prolonged viability. Only IFN‐γ enhanced constitutive CD95 expression. Coincubation with anti‐CD69 or anti‐CD95 monoclonal antibody (MoAb) induced apoptosis, as revealed by propidium iodide incorporation, membrane blebbing and nuclear fragmentation. Additionally, both anti‐CD69 and anti‐CD95 MoAb reduced cytokine‐enhanced Bcl‐2 expression. In conclusion, CD69 transduces a Bcl‐2‐dependent death signal when ligated by a specific antibody. As, in contrast to the ubiquitous death‐inducer CD95, the function of CD69 appears to be restricted to activated eosinophils, it represents an ideal target for therapeutic intervention in asthma.

DIFFERENTIAL REGULATION OF AMINOPEPTIDASE N (CD13) BY TRANSENDOTHELIAL MIGRATION AND CYTOKINES ON HUMAN EOSINOPHILS

Aminopeptidase N (CD13) is a cell surface metalloprotease involved in growth regulation, tumor invasion, and down-regulation of regulatory peptides. CD13 expression on eosinophils in bronchoalveolar lavage (BAL) of asthmatics 10 minutes and 18 hours after segmental allergen provocation was significantly increased (+225% to +294%) compared to blood eosinophils. In vitro CD13 expression could be induced on blood eosinophils by transendothelial migration of the cells across interlenkin (IL) 1β-activated human umbilical cord vein endothelial cells (HUVECs) as well as by the exposure to the cytokines IL-3, IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF). The cytokines GM-CSF and IL-5 were significantly less effective in inducing CD13 compared to IL-3. The IL-3-induced expression of CD13 was decreased in the presence of the protein-synthesis inhibitor cycloheximide (−8.8%). Moreover, blocking of CD13 by the protease inhibitors actinonin and bestatin significantly enhanced migration (+40.0% to +80.0%) of eosinophils across HUVEC monolayers. In summary, the data suggest that CD13 is regulated both by the process of transmigration and by the cytokine IL-3. Further, CD13 itself seems to be involved in the process of eosinophil transmigration.