Torben Plesner

In situ immunological characterization of Langerhans cells with monoclonal antibodies: Comparison with other dendritic cells in skin and lymph nodes

The antigenic properties of epidermal Langerhans cells (LC) were determined and compared with those of non-lymphoid dendritic dermal cells (DDC), interdigitating reticulum cells (IRC), dendritic reticulum cells (DRC), and histiocytic reticulum cells (HRC) by examination of serial and double immunoenzymatic and -fluorescence stained frozen sections of skin and lymph node biopsies. All of these cell types expressed leucocyte common antigen. LC, DDC, and IRC demonstrated similar antigenic phenotypes (HLA-DR+, Leu3+, OKT6+/−, anti-C3 receptor−, R4/23−, Ig-complex−, M02−), whereas the antigenic properties on DRC (HLA-DR−, Leu3−, OKT6−, anti-C3 receptor+, R4/23+, Ig-complex+, M02−) and HRC (HLA-DR+/−, Leu3−, OKT6−, anti-C3 receptor+ R4/23−, Ig-complex+, MO2+) were markedly different. These data suggest that LC, DDC, and IRC are closely interrelated cell types, and support the concept that DRC and HRC are unique cell types which do not appear to be related to LC, DDC, or IRC. The lack of labelling of LC with monoclonal anti-C3b receptor antibody, and polyclonal antiserum recognizing C3b, C3bi, and C3d receptors strongly indicate that the EAC-rosetting of LC previously described is not due to the presence of C3 receptors on these cells. Alternatively, LC may express C3 receptor molecules different from those previously identified (C3b, C3bi, and C3d).

T cell activation. II. Activation of human T lymphoma cells by cross-linking of their MHC class I antigens.

The present work demonstrates that antibody-induced cross-linking of MHC class I antigens on Jurkat T lymphoma cells leads to a rise in intracellular calcium (Cai2+) and, in the presence of phorbol ester (PMA), to IL-2 production and IL-2 receptor expression. The rise in Cai2+ exhibited a profile very different from that obtained after anti-CD3 antibody-induced activation suggesting that activation signals are transduced differently after binding of anti-CD3 antibody and class I cross-linking, respectively. However, when Cai2+ was examined in individual Jurkat cells by means of a digital image processing system no differences were observed after cross-linking with anti-CD3 and anti-MHC class I antibodies, respectively. Two CD3-negative mutant lymphoma lines were nearly totally refractory to class I cross-linking. Taken together our results may indicate the existence of a functional linkage between the T cell receptor complex and MHC class I molecules.

Subcellular Distribution of Urokinase and Urokinase Receptor in Human Neutrophils Determined by Immunoelectron Microscopy

A high-affinity receptor for urokinase-type plasminogen activator (uPAR) has been identified on the plasmamembrane of a number of different cell types, and has been shown to be important for plasminogen activation, cell adhesion, and possibly signal transduction. uPAR and uPA cosediment with secretory vesicles and specific granules by subcellular fractionation and translocate to the plasma membrane upon activation of neutrophils. Here the subcellular distribution of uPAR and uPA is studied by electron microscopy of neutrophils using immunogold double labeling for uPAR and uPA and a set of markers for well-defined subtypes of granules: matrix metalloproteinase type-9 (MMP-9) for gelatinase granules, lactoferrin (LF) for specific granules, and myeloperoxidase (MPO) and neutrophil elastase (NE) for primary granules. With this technique uPAR colocalizes with uPA in 71% of labeled granules. In granules containing uPAR the degree of coexpression with MMP-9, MPO and NE was 19, 66, and 74%, respectively. In granules labeled for uPA the corresponding overlap with MMP-9, MPO and NE was 24, 64, and 51%, respectively. Low levels of co-localization were found for uPAR and LF (7%) and for uPA and lactoferrin (5%). The results indicate that uPAR and uPA arepresent in gelatinase granules and primary granules, but rarely in specific granules. The demonstration of uPAR and uPA in primary granules is of particular interest, and may indicate that uPAR and uPA participate in the activation of latent hepatocyte growth factor of neutrophils.

Cytoplasmic inclusions in lymphocytes of chronic lymphocytic leukaemia

Peripheral blood from 90 CLL patients was examined by light- and electron-microscopy for the occurrence of crystalline inclusions in lymphocytes. Inclusions were demonstrated in 10 patients (11%). In these patients the inclusions were present in 5–45% of peripheral blood lymphocytes. In the light microscope the inclusions appeared as rectangular, unstained structures in May-Grünewald Giemsa and PAS stains. In the electron microscope the inclusions appeared as intracytoplasmic, completely partially membrane-bound bodies, which were often associated with dilated profiles of rough endoplasmic reticulum. The ultrastructure of the inclusions was granular. In immunofluorescence staining the inclusions were found to contain immunoglobulin of the same type and class as the surface membrane-bound immunoglobulin of the neoplastic lymphocytes, most frequently IgMlambda. The lymphocytes of one case with kappa light chains at the cell surface membrane contained inclusions of the same ultrastructural morphology as those of the other cases with lambda light chains. The presence of inclusions was not associated with any specific clinical or prognostic features. The inclusions persisted during antileukaemic therapy. Their formation may be related to a dysfunction in the synthesis of surface membrane-bound immunoglobulins.

Characterisation of Non-P-Glycoprotein multidrug-resistant Ehrlich ascites tumour cells selected for resistance to mitoxantrone

An Ehrlich ascites tumour cell line (EHR2) was selected in vivo for resistance to mitoxantrone (MITOX). The resistant cell line (EHR2/MITOX) was 6123-, 33-, and 30-fold-resistant to mitoxantrone, daunorubicin, and etoposide, respectively, but retained sensitivity to vincristine. The resistant cells showed moderate sensitisation to mitoxantrone on treatment with verapamil or cyclosporin A. Compared with EHR2, the multidrug resistance-associated protein mRNA was increased 13-fold in EHR2/MITOX. Western blot analysis showed an unchanged, weak expression of P-glycoprotein. Topoisomerase IIalpha was reduced to one-third in EHR2/MITOX relative to EHR2 cells, whereas topoisomerase IIbeta was present in EHR2 but could not be detected in EHR2/MITOX. In the resistant subline, net accumulation of MITOX (120 min) and daunorubicin (60 min) was reduced by 43% and 27%, respectively, as compared with EHR2. The efflux of daunorubicin from preloaded EHR2/MITOX cells was significantly increased. EHR2/MITOX microsomes had a significant basal unstimulated ATPase activity. The apparent K(i) value for vanadate inhibition of the ATPase activity in EHR2/MITOX microsomes was not significantly different from the K(i) value for P-glycoprotein-positive cells. However, whereas verapamil (50 microM) inhibited the ATPase activity of EHR2/MITOX microsomes, it stimulated the ATPase activity of microsomes derived from P-glycoprotein-positive cells. In conclusion, the resistance in EHR2/MITOX was multifactorial and appeared to be associated with: 1) a quantitative reduction in topoisomerase IIalpha and beta protein; 2) reduced drug accumulation, probably as a result of increased expression of a novel transport protein with ATPase activity; and 3) increased expression of MRP mRNA.

Transmembrane Signalling via HLA‐DR Molecules on T Cells from a Sezary T‐Cell Leukaemia Line

The human Sezary T‐cell leukaemia line. HUT.78, represents a population of activated T cells, i.e. they are HLA‐DR+ and IL‐2R+. We have analysed the capacity of HUT.78cells (1) to stimulate HLA‐DR‐specific T‐cell lines or clones and (2) to be induced to synthesize IL‐2 by anti‐HLA‐DR monoclonal antibodies. The results of our experiments show that HLA‐DR molecules on HUT.78 cells can stimulate at least one HLA‐DR‐specific T‐cell clone and can act as transmembrane signal transmitters.

Failure to Synthesize the Human T‐Cell CD3‐ζ Chain and Its Consequence for the T‐Cell Receptor‐CD3 Complex Expression

The T‐cell antigen receptor is composed of two variable chains (α and β, termed TcR) which confer ligand specificity, and four constant chains (γ, δ,ɛ, and ζ, collectively termed CD3) whose functions are not Cully understood. To explore the role of the individual CD3 components, the human T‐cell tumour line Jurkat was chemically mutagenized followed by negative selection with F101.01 (a monoclonal antibody against the TcR‐CD3 complex), and cloning. Growing clones were analysed for TcR‐CD3 expression by immunofluorescence. One clone, J79, was found to express greatly diminished levels of TcR‐CD3. This clone produced all the TcR‐CD3 components except the CD3‐ζ, as demonstrated by metabolic labelling and immunoprecipitation followed by one‐ and two‐dimensional sodium dodecyl sulphate‐polyacrylamide gel electrophoresis. These data indicate that the CD3‐ζ determines the normal intracellular fate of the TcR‐CD3 complex, and that the CD3‐ζ is necessary for the intracellular transport and expression at the cell surface of the TcR‐CD3 complex.

T-Cell Activation. III. Attempts to Activate MHC Class I-Negative and Class I-Transfected EL4 T-Lymphoma Cells by Immobilized Anti-CD3 Antibody

The aim of this study was to examine whether the unresponsiveness of MHC class I‐negative subclones of the EL‐4 thymoma lo CD3 cross‐linking can be restored by transfection of class I genes into the H‐2‐negative cells. Cell activation experiments with selected MHC class I‐negative subclones and H‐2b‐and H‐2Ld‐positive transfectants showed that these cells are equally capable of secreting interleukin 2 (IL‐2) after exposure to the phorbol ester phorbol 12‐mvristalc 13‐acetale (PMA) and ionomycin. In contrast, only the parental H‐2‐positive EL4 cells are capable of responding to treatment with immobilized anti‐CD3 antibody with IL‐2 secretion and IL‐2 receptor expression. Measurements of intracellular free Ca2+ (Ca2+i) following anti‐CD3 antibody‐induced cross‐linking of parental EL4 cells and H‐2‐negativeand H‐2b gene‐transfected subclones showed that the parental cells and two of the class I transfectants, one H‐2‐positiveand one H‐2‐negative. responded with a slow rise in Ca2+,. whereas one H‐2‐positive transfected cell clone was completely refractory to CD3 cross‐linking. Modulation experiments using parental EL 4 cells, H‐2‐negative subclones and H‐2‐positive transfectants demonstrated that the CD3 and class I molecules of these different cells are modulated to the same extent after exposure lo specific antibodies. The present findings thus indicate that the unresponsiveness of H‐2‐negative EL4 subclone cells not CD3 cross‐linking is not functionally associated with a lack of class I surface expression.

Quantitative buffy coat analysis in haematological patients compared to standard laboratory methods.

We have tested a Quantitative Buffy Coat (QBC) instrument (Becton Dickinson, USA), and compared results obtained with this instrument to results obtained with standard methods in a haematology clinic. The basic principle of the method is a classical haematocrit centrifuge. The analysis provides a haematocrit value, platelet count, a total white blood count, and separates the white blood cells in granulocytes and mononuclear cells (lymphocytes plus monocytes). The instrument is easy to use but requires a trained observer. All results are available in 15 min. We have found the accuracy of the method good for all parameters. The precision of the instrument is good but for estimation of granulocytes and lymphocytes plus monocytes we did not find the high sensitivity claimed by the manufacturer (manufacturers lower limit 0.02 x 10(9)/l; standard deviation for low levels of granulocytes: 0.300 x 10(9)/l and lymphocytes plus monocytes: 0.235 x 10(9)/l). A large fraction of samples (leukocytes 27%, platelets 40%) from a haematological clinic falls beyond the limits for reliable results set by the manufacturer, which reduces the utility of the instrument in such patients. Furthermore, in 21% and 10% of samples within the recommended range for leukocytes and platelets, respectively, QBC results could not be read owing to ill-defined boundaries. For granulocytes and lymphocytes plus monocytes 25% and 34% of the samples, respectively, could not be read owing to ill-defined boundaries. The instrument is not constructed to protect against blood contamination of the centrifuge and, therefore, the safety of the instrument is not satisfactory.