Othmar Förster

Phenotype and Function of Peripheral and Prostatic Lymphocytes in Patients with Benign Prostatic Hyperplasia

In a previous report, we demonstrated intense lymphocytic infiltration of all benign prostatic hypertrophy (BPH) tissues analyzed in conjunction with HLA-DR expression on normally MHC-class-II-negative prostate epithelial cells. The composition of these infiltrates (70 to 80% CD3+ T-cells, but no granulocytes) resembles the situation seen in immune responses against altered self or self rather than against foreign antigens (infection). In the present study, phenotypic and functional immunoassays were used in order to investigate whether T-cells in BPH are indeed activated, and whether this activation is systemic or restricted locally to the prostate. Analysis of T-cell activation marker expression and proliferation requirements provided substantial evidence that these infiltrating lymphocytes, in contrast to their peripheral counterparts, are chronically activated. Since local accumulation of activated lymphocytes can cause tissue destruction, high concentrations of cytokines, and consequently tissue rebuilding, this process might contribute to the pathogenesis of BPH.

Differentiation of Rat Bone Marrow Cells Into Macrophages Under the Influence of Mouse L929 Cell Supernatant

Bone marrow cells (BMC) flushed from femora of Lewis rats were cultured in Dulbeccos modification of Eagles medium supplemented with mouse L929 cell supernatant as a source of colony‐stimulating factor (CSF). Differentiation of macrophage progenitor cells into macrophages (Mø) and expression of various markers were kinetically assessed. The proportion of Mø increases from approximately 4% in freshly isolated BMC to 100% after 7‐8 days of cell culture. These cells, termed bone marrow cell‐derived macrophages (BMDMø), adhere to and spread on plastic surface; exhibit Mø morphology; stain intensely for nonspecific esterase; are able to phagocytose latex particles, IgG‐sensitized erythrocytes, and C3‐coated red cells; and express receptors for IgG and C3. A subpopulation of BMDMø expresses MHC class II antigens as demonstrated by immunofluorescence using MRC OX6 and MRC OX17 monoclonal antibodies which recognize antigens coded in the I‐A or I‐E subregion of the MHC, respectively.

Self-renewal, maturation, and differentiation of the rat myelomonocytic hematopoietic stem cell

Hematopoiesis is viewed as a differentiating system emanating from a pluripotent hematopoietic stem cell capable of both self‐renewal and differentiation. By identifying and characterizing a novel and highly specific in vitro mitogenic response to the N‐acetyl glucosamyl/sialic acid specific, stem cell‐binding lectin wheat germ agglutinin (WGA), we demonstrate the existance of a rare (0.1%), plastic adherent precursor in rat bone marrow capable of proliferation (two to seven divisions) in response to WGA. Stimulated cells possess a lineage (lin)low/− immunophenotype and immature blastoid morphology (WGA blasts). A subsequent proliferative response to stem cell factor (SCF), the ligand for the proto‐oncogene receptor tyrosine kinase c‐kit, is characterized by an initial maturation in immunophenotype and subsequent self‐renewal of cells (SCF blasts) without differentiation for at least 50 generations. Although granulocyte colony‐stimulating factor (G‐CSF), interleukin (IL) ‐6, IL‐7, and IL‐11 synergize with SCF to increase blast colony formation, cytokines such as granulocyte‐macrophage CSF or IL‐3 are without significant effect. At all time points in culture, however, cells rapidly differentiate to mature neutrophils with dexamethasone or to mainly monocytes/macrophages in the presence of 1α,25‐dihydroxyvitamin D3, characterized by cell morphology and cytochemistry. Removal of SCF during blast maturation, self‐renewal, or induction of differentiation phases results in apoptotic cell death. Data indicate a pivotal role for SCF/c‐kit interaction during antigenic maturation, self‐renewal, and apoptotic protection of these lineage‐restricted progenitors during non‐CSF‐mediated induction of differentiation. This approach provides a source of many normal, proliferating myelomonocytic precursor cells, and introduces possible clinical applications of ex vivo expanded myeloid stem cells.—Lucas, T., Krugluger, W., Samorapoompichit, P., Gamperl, R., Beug, H., Förster, O. Self‐renewal, maturation, and differentiation of the rat myelomonocytic hematopoietic stem cell. FASEB J. 13, 263–272 (1999)

Galectin-3 inhibits granulocyte-macrophage colony-stimulating factor (GM-CSF)-driven rat bone marrow cell proliferation and GM-CSF-induced gene transcription.

The expression of galectin-3 (formerly known as IgE-binding protein or Mac-2) in rat bone marrow (BM) was investigated by FACS, immunocytochemical and immunoblot analysis. The functional significance of rat recombinant galectin-3 on mouse recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF)-driven proliferation of macrophage progenitors and gene transcription was further examined. Immunocytochemical analysis of in situ BM sections demonstrated galectin-3 in myelopoietic cells and surrounding stroma, whereas erythropoietic and lymphopoietic environments essentially lacked galectin-3 expression. FACS analysis demonstrated that incubation of freshly isolated BMC with lactose, a competing ligand for galectin-3 binding to glycoconjugates, decreased binding of antigalectin antibodies to cells primarily expressing the myeloid antigen recognized by mAb His-54. Similarly, lectin-mediated binding of exogenous galectin-3 to myeloid lineage cells was also demonstrated. Immunoblot analysis of BM eluates demonstrated galectin-3 both in the extracellular matrix and in a lactose elutable form, bound to the surface of BMC. [3H]Thymidine incorporation studies on BMC cultured in the presence of galectin-3 demonstrated suppression of GM-CSF-induced proliferation by galectin-3. In addition, differential display analysis of immediate early gene expression in BMC cultured in the presence of galectin-3 revealed a 76.2% inhibition of GM-CSF-induced gene transcription by galectin-3 assessed by the number of PCR-fragments generated. Our data suggest a role for galectin-3 in the organization of myelopoietic compartments in rat BM and regulation of the action of growth factors on myelopoietic precursor cells.

Expression of the VEP13 Antigen (CD16) on Native Human Alveolar Macrophages and Cultured Blood Monocytes

Human alveolar macrophages (AM phi) from thirteen patients, who were suffering from various lung diseases were harvested by bronchoalveolar lavage. Peripheral blood monocytes from eight healthy donors were isolated by Ficoll-Hypaque gradient centrifugation and adherence to plastic surface. To detect the VEP13 antigen (CD16) on these cells, a rosette assay employing ox erythrocytes coated by the CrCl3 method with purified VEP13 monoclonal antibody (Eo-VEP13) was used. A mean of 31.3% of freshly isolated AM phi and 3.9% of blood monocytes formed Eo-VEP13 rosettes. Monocytes cultured for 3 or 6 days in the presence of a supernatant from mouse L929 cells, which had been shown previously to improve long-term viability of human monocytes in culture, showed 12.5% and 25.3% Eo-VEP13 rosettes, respectively. No significant increase in VEP13 antigen expression was noted by culturing monocytes without L929 cell supernatant. The factor in L929 supernatant that induces VEP13 antigen expression has not been identified. Tunicamycin at 10 micrograms/ml inhibited significantly VEP13 antigen expression on monocytes. In contrast, IgG rosette formation was not reduced by tunicamycin. Our data show that subpopulations of native human AM phi and peripheral blood monocytes cultured in presence of a supernatant of L929 fibroblasts containing mainly murine CSF may express the CD16 antigen, which is normally found on large granular lymphocytes (LGL). Suppression by tunicamycin indicates that Fc receptor glycosylation takes place during a later differentiation step of mononuclear phagocytes.

Lectin binding of rat bone marrow cells during colony-stimulating factor type 1--induced differentiation: soybean agglutinin as a marker of mature rat macrophages.

Rat bone marrow cells (BMC) cultured in the presence of murine colony‐stimulating factor type 1 (CSF‐1) differentiate within 7 days into a cell population containing 96–100% macrophages (Mφ). In this study, binding of 10 different fluorescein isothiocyanate (FITC)‐conjugated lectins to cultured BMC at various stages of differentiation into Mφ was investigated. Only soybean agglutinin (SBA) showed a binding pattern that was significantly correlated to Mφ differentiation. Nearly all adherent (A) cells bound SBA. Binding of SBA to nonadherent (NA) cells increased from 20% on day 0 to 80% on day 8. NA cells were separated by means of a fluorescence‐activated cell sorter into SBA‐, SBA±, and SBA+ fractions. On day 0 and day 2, Mφ, blast‐like cells, eosinophils, and some neutrophils were found in the SBA+ population. From day 4 onwards, the SBA+ fraction contained almost exclusively Mφ. Neutrophils and some blasts were found in the SBA‐population on days 0 and 2. In the SBA± fraction, mainly blasts and lymphocytes were identified. With increasing time of culture, Mφ or Mφ precursors prevailed also in the SBA‐/± cell population. Cells forming colonies in soft agar in the presence of CSF‐1 were highly enriched in the SBA± fraction. A large number of cells in S‐G2/M phases but few colony‐forming cells were found in the SBA+ population. Our data suggest that SBA is a useful additional tool to define late stages of Mφ differentiation.

Specificity of ganglioside binding to rat macrophages

The binding specificity of rat alveolar macrophages (AM phi) for sheep erythrocytes (E) coated with gangliosides GM1, GM2, GM3, GD1a, GD1b or GT1b was analyzed in a rosette assay by studying the inhibitory effect of gangliosides, various carbohydrates, IgG, C3b-like C3, and fibronectin in this assay. The uptake of gangliosides by E was calculated from radioactivity measurements using 3H-labeled gangliosides. The different gangliosides were taken up by E at 37 degrees C to a similar extent. Uptake of 3H-labeled GM2 correlated linearly to its concn in the incubation medium. Erythrocytes pretreated with the same molar concn of GM2, GD1a, GD1b or GT1b were bound to AM phi to the same degree reaching a maximum of about 90% rosette forming cells. A mean of 17.8% AM phi-bound GM3-coated E. Treatment of E with asialo-GM2 (GA2) or GM1 did not induce significant rosette formation. A dose-dependent inhibition of rosette formation was observed when AM phi were preincubated at 0 degree C with GM2, GM3, GD1a, GD1b or GT1b, but not with GM1 or GA2 Of the tested carbohydrates, sialyl-lactose had a strong inhibitory effect, while lactose was completely ineffective. N-acetyl-neuraminic acid, N-glycolyl-neuraminic acid and N-acetyl-galactosamine were slightly inhibitory. A series of other carbohydrates including highly negatively charged compounds, as well as fibronectin, IgG or C3b-like C3 did not show significant inhibition. Our data indicate the expression of a receptor on rat AM phi recognizing carbohydrates containing sialic acid at or near the non-reducing terminus.

Suramin affects human peripheral blood mononuclear cells in vitro: inhibition of T cell growth and modulation of cytokine secretion.

Suramin, a polyanionic compound, which has been in clinical use for the treatment of African trypanosomiasis for several decades, has recently been introduced in clinical oncology. Its effects on the immune system seemed therefore of interest. In the present study we tried to elucidate in vitro how suramin affected different functions of human peripheral blood mononuclear cells (PBMC). Suramin suppressed the proliferation of PBMC in response to various stimuli, including OKT3, phytohemagglutinin (PHA), phorbolmyristate-acetate (PMA) and ionomycin, purified protein derivate of Mycobacterium tuberculosis (PPD) and antibodies against CD2. It also inhibited the binding of monoclonal antibodies to T cell surface antigens. This effect was not dependent on the isotype of the antibody, but seemed to be highly epitope-specific. Among a panel of antibodies against one antigen, only a few were affected by the compound. Whereas the binding of Leu3a and OKT3 was for instance fully suppressed by suramin, OKT4 and Leu4 binding was only slightly affected. Suramin also decreased the expression of T cell surface molecules such as CD2, CD25 and CD4 in preactivated PBMC and had pronounced effects on cytokine production. Interestingly the compound had adverse regulatory effects on different cytokines. Whereas the secretion of interferon-gamma was completely suppressed by suramin, interleukin-2 (IL-2) and IL-4 production was stimulated. These results demonstrate that suramin affects T cell function in multiple different ways. This will have to be considered, when suramin is used in the treatment of cancer patients.

Mannose-binding lectin: comparison of two assays for the quantification of MBL in the serum of pediatric patients

Individuals with mannose-binding lectin (MBL)-deficiency are at an increased risk from infections with mannose-bearing microorganisms. We have investigated two quantitative research assays for measuring MBL protein in serum for routine diagnosis. The evaluation of 817 serum samples with a nephelometric assay revealed two deficiencies, a number far below the postulated 5-10% of the population. Reevaluation of 102 serum samples with an MBL-ELISA detected low levels in 27 cases (26.4%) and clear deficiencies in 21 samples (20.4%). In our hands, the MBL-ELISA permitted the detection of decreased levels of MBL in serum, as occurs in individuals with homozygous or heterozygous MBL gene mutations; in contrast, the nephelometric assay appeared to be unsuitable for the detection of MBL deficiencies. We support the routine measurement of MBL in serum, especially in children with frequent infections.

Expression and function of sialoadhesin in rat alveolar macrophages

Alveolar macrophages (Amφ) represent an immunologically distinct sub-population within the reticuloendothelial system. Phagocytosis and possibly antigen presentation by Amφ are essential components of specific and innate primary immune defence processes against inhaled material. The mφ-restricted sheep erythrocyte receptor sialoadhesin (Sn) is a member of the immunglobulin superfamily and binds specifically to sialic acid-containing structures such as selectins and was originally identified as the sheep erythrocyte receptor (SER) responsible for sialic acid-dependent binding of native sheep erythrocytes (SE) to resident murine bone marrow macrophages in rosetting assays. Sn expression has been demonstrated on murine and rat mφ in lymphatic organs and is recognised by the monoclonal antibody (mAb) ED3 in the rat. In addition, sialic acid-dependent receptor (SAR) activities that mediate rosette formation of alveolar, peritoneal, splenic and bone marrow-resident rat mφ with SE pretreated with gangliosides and SER-like activities between native SE and trypsinised Amφ, have been described. The binding activities of both SAR and Sn show similar characteristics suggesting that these molecules are closely structurally related or identical. To clarify the relationship between Sn, SAR and SER-like activities, the binding of mAb ED3 to isolated rat Amφ was investigated by flow cytometry and rosetting assays. It is demonstrated that rat Amφ express Sn and evidence is provided that SAR and SER-like activities are mediated by Sn.