Christoph Holzinger

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.

Nifedipine reduces the incidence of myocardial infarction and transient ischemia in patients undergoing coronary bypass grafting.

A randomized study was performed on 104 patients undergoing elective coronary artery bypass grafting to examine whether the infusion of nifedipine (n = 53) reduces the incidence of perioperative myocardial ischemia and necrosis in the early postoperative period. Continuous hemodynamic and three-channel Holter monitoring was performed for 24 hours and serial assessment of serum enzymes and 12-lead electrocardiography were performed for 36 hours postoperatively. Nifedipine (minimum dose, 10 micrograms/kg/hr for 24 hours) was applied from the onset of extracorporal circulation. The control group (n = 51) received nitroglycerin (minimum dose, 1 micrograms/kg/min for 24 hours). Using the combined analyses of electrocardiography and Holter recordings, myocardial ischemia was defined as being either a transient ischemic event (TIE), transient coronary spasm (TCS), or myocardial infarction (MI). The two groups did not differ with respect to preoperative New York Heart Association classification, age, history of myocardial infarction, extracorporal circulation and aortic cross-clamp time, number of distal anastomoses, or systemic and pulmonary hemodynamics. The incidence of perioperative myocardial ischemia was substantially lower in the nifedipine than in the nitroglycerin group [TIE: three of 53 patients (6%) versus nine of 50 patients (18%), p less than 0.001; MI: two of 53 patients (4%) versus six of 50 patients (12%), p less than 0.001; and TCS: none of 53 patients (0%) versus two of 50 patients (4%), p = NS].(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of interleukin-1, -2, -4, -6, interferon-gamma and granulocyte/macrophage colony stimulating factor on human vascular endothelial cells

Human vascular endothelial cells (HUVEC) exhibit various immunological functions, i.e. expression of HLA class-II antigens after incubation with IFN-gamma or antigen presenting function. It has also been reported that HUVEC are able to produce IL-1, IL-6, GM-CSF and immunologically active cleavage products of arachidonic acid. In our study we investigated whether various cytokines, namely IL-1, IL-2, IL-6, GM-CSF and IFN-gamma, do alter the proliferative capacity of HUVEC, the production of van Willebrandt factor (vWF) and the expression of MHC class-II antigens. HUVEC were prepared by the collagenase digestion of human umbilical veins. Monolayers of cells were incubated with cytokines in different concentrations for 24 and 48 h. IFN-gamma inhibits the HUVEC [3H]thymidine uptake in a dose-dependent manner. Suppression of proliferation (40.1%) could be observed after 24 h incubation with 100 U IFN-gamma/ml. IL-1 was a more effective inhibitor of HUVEC proliferation (54% at 10 U/ml and 24 h incubation and 48.4% after 48 h) than IFN-gamma. IL-6 and GM-CSF showed an increasing effect on proliferation with 226% and 151% of the control group, respectively. IFN-gamma after an incubation period of 12 h and IL-1 after 24 h reduced the vWF content by about 30%. Bright MHC class-II expression was induced only by IFN-gamma. In conclusion, some of the immunoregulative cytokines might play an important role in the control of HUVEC proliferation.

Phenotypic Patterns of Mononuclear Cells in Dilated Cardiomyopathy

BACKGROUND Immunological factors in the pathogenesis of idiopathic dilated cardiomyopathy (IDC) were suggested previously on the basis of the demonstration of mononuclear cell infiltrates and autoantibodies against the myocardium. The present study investigated whether tissue leukocyte subpopulations isolated from hearts with IDC (n = 6) differ in phenotype from those of tissues without IDC (n = 7). METHODS AND RESULTS Leukocytes were quantified as reactive cells per square millimeter in perivascular, interstitial, and parenchymal tissue sections. Freshly isolated heart-tissue T cells and peripheral-blood T cells from the same patients were analyzed by triple staining and flow cytometry to identify T-cell subpopulations as well as their states of differentiation (expression of CD45RA and Leu-8 versus CD45RO) and activation (IL-2R, IL-7R, very late antigen-1, HLA-DR). All types of infiltrating cells (T cells, B cells, macrophages, granulocytes) are increased in hearts with IDC compared with normal hearts, but only CD8+ T cells and macrophages are increased relative to the other leukocyte subpopulations. CD45RO+/CD45RA-/Leu-8- cells constitute the majority of heart-tissue T cells in both normal hearts and hearts with IDC. Strikingly, hearts with IDC are infiltrated by eightfold greater numbers of perivascularly located IL-2R(+)- (26% of all T cells) and CD45RO(+)-activated memory T cells; moreover, in contrast to normal heart, approximately 40% of both CD4+ and CD8+ heart-tissue T cells express activation markers. CONCLUSIONS Both normal hearts and hearts with IDC are populated by leukocytes. The quantitative increase in IDC, associated with a dramatically altered activation status of heart-tissue T cells, suggests a direct role of infiltrating leukocytes in the pathogenesis of IDC.

Treatment of non-healing skin ulcers with autologous activated mononuclear cells

The aim of this study was to investigate whether cultured autologous mononuclear cells (MNC) effectively initiate, accelerate and improve granulation and epithelialisation of skin ulcers. Thirty-three patients with chronic arterial occlusive disease (CAOD; n = 21) or venous post-thrombotic syndrome (PTS; n = 12) were treated with autologous MNC and compared with a control group of 30 patients who received tissue culture medium alone. Previous treatments had been unsuccessful for a mean of 9.23 (3-19) months. MNC were harvested from the peripheral blood of each patient by standard techniques, cultured for three days and applied to the ulcer twice a week. After 4.6 +/- 1.9 weeks, 29/33 ulcers were closed in the MNC group. Patients in the control group took 8.1 +/- 1.2 weeks for 17/30 ulcers. Thus ulcer healing was significantly speedier with MNC seeding; 48% of all ulcers were closed after 30 days of MNC treatment and 92% after 60 days. Patients with PTS responded significantly faster than patients with CAOD. In 90% of patients with painful ulcers MNC treatment resulted in pain relief, whereas in the control group only 50% of patients became pain-free.

Infusion of nifedipine after coronary artery bypass grafting decreases the incidence of early postoperative myocardial ischemia

We performed a randomized study on patients undergoing elective coronary bypass grafting to examine whether postoperative infusion of nifedipine (n = 25) could reduce the incidence of isolated transient myocardial ischemia, myocardial infarction, or both. The control group (n = 25) received nitroglycerin. Hemodynamic and Holter monitoring and serial assessment of enzymatic and electrocardiographic changes were performed for all patients. Both groups showed comparable preoperative and operative data. The incidence of myocardial infarction was significantly lower in the nifedipine group (n = 1) as compared with the control group (n = 4), whereas the number of patients with isolated transient myocardial ischemia was similar in both groups (nifedipine, 3; control, 4). At the time of peak activity, levels of creatine kinase (350 +/- 129 versus 511 +/- 287 IU/mL), creatine kinase-MB (8.4 +/- 5.4 versus 17.1 +/- 11.0 IU/mL), and glutamate-oxaloacetate-transaminase (30.4 +/- 4.4 versus 41.0 +/- 7.9 IU/mL) were markedly lower in the nifedipine group (p less than 0.05). We conclude that infusion of nifedipine after elective coronary artery bypass grafting effectively decreases the incidence of myocardial infarction and the extent of myocardial necrosis during the early postoperative period.

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.

Are T cells from healthy heart really only passengers? Characterization of cardiac tissue T cells

Numerous studies have dealt with occurrence of dendritic cells in various nonlymphoid organs such as kidney, liver or heart, whereas lymphocyte patterns in these organs have not been analyzed in detail. In the present study, leukocytes were quantified as cells/mm2 in the perivascular, interstitial and parenchymal tissue sections of normal heart. We measured an overall mean leukocyte count in normal heart tissue of 17.0 +/- 2.7 CD45+ leukocytes/mm2, 9.1 +/- 1.8 thereof being CD4+ T-helper cells (Th). By comparison, CD8+ T-cytotoxic/suppressor cells (Ts) and CD14+ macrophages each accounted for only approximately 2.5 cells/mm2, and CD20+ B cells for only 1.3 cells/mm2. These T cells were further characterized as either CD45RA+ naive T cells or as CD45RO+ memory T cells. Segmentation of the tissue as defined in Section 2 yielded an ascending number of CD45RO+ memory T cells from perivascular (0.4 +/- 0.2 cells/mm2) through parenchymal (12.8 +/- 3.0 cells/mm2) to interstitial (21.0 +/- 5.3/mm2). By contrast, the number of CD45RA+ and Leu-8+ cells decreased from perivascular to parenchymal. Peripheral T cells showed a reverse pattern of CD45RA/CD45RO antigen expression. Only approximately 3% of T cells expressed activation markers IL-2R and IL7R. Our data demonstrate that the majority of T cells in normal heart tissue are resting memory tissue T cells and are not contaminating T cells from the peripheral blood. The increase in CD45RO+ cells from perivascular to parenchymal with a corresponding decrease in CD45RO+ and Leu-8+ heart-tissue T cells argues in favor of T-cell traffic in normal heart tissue.

THE PROGRESSION OF MILD ACUTE CARDIAC REJECTION EVALUATED BY RISK FACTOR ANALYSIS : THE IMPACT OF MAINTENANCE STEROIDS AND SERUM CREATININE

The natural course of mild acute cardiac allograft rejection (MAR) under cyclosporine-based therapy is generally considered benign, and usually antirejection therapy is not instituted. The present study was undertaken to determine the frequency of and the risk factors for progression of MAR into a clinically significant (moderate or severe) rejection on subsequent endomyocardial biopsy (EMB). Among 167 cardiac recipients, transplanted from 3/1984 to 4/1990, MAR under cyclosporine-based therapy was diagnosed on 220 EMBs. Depending upon the outcome on the subsequent EMB, MAR was categorized as progressive or nonprogressive. This served as the dependent variable for a stepwise logistic regression analysis evaluating 11 covariates as potential risk factors: perioperative antibody prophylaxis (ATG vs. OKT3), maintenance therapy, underlying disease, HLA-mismatches for A- and B + DR-loci, serum creatinine (mg/dl) and cyclosporine HPLC blood level (ng/ml) at diagnosis of MAR and at subsequent biopsy, recipient age, donor age. 40 (18.2%) of 220 MARs became progressive as opposed to 37 (7.3%) of a control cohort of 507 negative EMBs (P less than 0.0001). Stepwise logistic regression yielded the type of maintenance therapy (P = 0.0019) and serum creatinine level at diagnosis of MAR (P = 0.0615) as independent predictors of progression of MAR. After adjustment for influence of maintenance therapy and serum creatinine none of the cyclosporine variables provided any additional information. MARs without maintenance steroids and low serum creatinine levels had the highest risk (37.2% observed incidence) to develop moderate or severe rejection on subsequent EMB. This analysis supports evidence that diagnosis of MAR on EMB is associated with a considerable high progression rate into clinically significant rejection when compared to negative EMBs. Progression particularly occurs in MAR under steroid-free maintenance therapy and suggests early augmentation of immunosuppression. In terms of progression of MAR serum creatinine values, obviously indicating cyclosporine nephrotoxicity, appear to reflect the extent of cyclosporine-mediated immunosuppressive activity more properly than parameters of its bioavailability by measuring cyclosporine HPLC blood levels.

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.