Differential expression of Ia antigens by rheumatoid synovial lining cells.

The differential expression of Ia antigens was studied in freshly isolated rheumatoid nonlymphoid synovial lining cells (SLC) and rheumatoid synovial fibroblast cell lines cultured in the presence of Interferon-gamma, using a large panel of anti-Ia reagents with monomorphic or polymorphic specificities. All the HLA-DR or -DQ specificities detectable on the corresponding peripheral blood B cells were also expressed in freshly isolated SLC. However, in all instances, the number of DR-positive SLC exceeded the percentage of cells expressing DQ antigens. In addition, the epitope expression of Ia antigens varied within the DR or DQ populations of Ia molecules as revealed by polymorphic reagents. Double-label experiments or using the ingestion of Latex particles as a marker demonstrated that the synovial macrophages (type I SLC) primarily bear the DR+DQ+ phenotype, while there is an additional population of nonphagocytic SLC (previously termed type II SLC) that has a DR+ and monocyte marker negative phenotype but did not have detectable levels of DQ antigens as analyzed by both fluorescence microscopy and cell sorter analysis. This latter population frequently had a morphology showing dendritic processes and rapidly lost the expression of Ia antigens upon culture. Cells with a similar, primarily DR+ phenotype were readily obtained in synovial fibroblast cultures after treatment with Interferon-gamma. These data suggest that there are two populations of Ia+ synovial lining cells: the synovial macrophages (type I cells) with the DR+DQ+ phenotype, and cells probably related to fibroblasts with a DR+ phenotype without detectable DQ antigens (type II cells). The fact that the latter phenotype could be induced by Interferon-gamma treatment of cultured synovial fibroblasts suggests that this mediator may have a similar role in vivo in the activation of certain synovial cell populations.

A multidrug-resistance protein (MRP)-like transmembrane pump is highly expressed by resting murine T helper (Th) 2, but not Th1 cells, and is induced to equal expression levels in Th1 and Th2 cells after antigenic stimulation in vivo.

A transmembrane pump for organic anions was identified in resting murine T helper (Th) 2, but not Th1 lymphocyte cell clones, as revealed by extrusion of a fluorescent dye. Dye extrusion inhibition studies suggested that the pump may be the multidrug-resistance protein (MRP). The different expression of the pump in resting Th1 and Th2 cell clones correlated with their respective levels of MRP mRNA. The pump was inducible in Th1 cells by antigenic stimulation in vitro leading to equal expression in activated Th1 and Th2 cell clones. This suggested that dye extrusion might allow the detection of Th2 (resting or activated) or of activated Th1 cells ex vivo based on a functional parameter. To test this, mice were infected with Leishmania major parasites to activate L. major-specific T cells of either Th1 (C57BL/6 mice) or Th2 (BALB/c mice) phenotype: 2-3% of CD4+ lymph node T cells of both strains of mice extruded the dye, defining a cell subset that did not coincide with subsets defined by other activation markers. Fluorescence-activated cell-sorting revealed that the lymphokine response (Th1 or Th2, respectively) to L. major antigens was restricted to this dye-extruding subset.

Antibodies TC-12 ("unique") and TH-111 (CD96) characterize T-cell acute lymphoblastic leukemia and a subgroup of acute myeloid leukemia.

To extend the panel of monoclonal antibodies useful for immunophenotyping of acute leukemias, two new reagents, TC-12 and TH-111, were developed. TC-12 was found "unique," and TH-111 was assigned to the recently defined CD96 cluster. Both reagents show little reactivity with blood and bone marrow nucleated cells but define a major (TH-111: 78.3%) or an important (TC-12: 45.6%) subset of T-cell acute lymphoblastic leukemia (ALL). In addition, in acute myeloid leukemia (AML), the expression of TC-12 was found in 64 (20.2%) of 317 and TH-111 in 97 (29.1%) of 333 of these patients. TC-12 positivity in AML was virtually restricted to the Fab subtypes M0, M1, M2, and M6. In the group of immature AML characterized by the coexpression of CD7 as well as CD117 and CD34 positivity, leukemic blasts frequently disclosed the TC-12 and TH-111 antigen. Although the TC-12 antigen could not be determined, TH-111 immunoprecipitated the TACTILE (CD96) antigen and, when expressed, was found to be associated with the transferrin receptor. These reagents may help not only to define and dissect T-cell ALL, but also to characterize a subgroup of immature AML at the divergence of T-cell and myeloid lineage.

Studies on the mechanism of polyclonal B cell stimulation by TH2 cells.

Recently, it has been shown that cloned L1/1 T helper cells of type 2 (TH2-cells), when stimulated with antigen, are able to induce polyclonal B cell proliferation. Here we present evidence that this process is dependent on direct cell-cell interaction between T and B cells, which in the effector phase, i.e., during stimulation of the B cells by activated T cells, can be mediated by a mechanism other than cognate interaction. This conclusion is derived from experiments in which highly purified, small B cells of high density were polyclonally stimulated by L1/1 T cells triggered by an anti-T3 monoclonal antibody in the absence of antigen. The triggering process was independent of the presence of the Fc part of the antibody and occurred in cultures devoid of macrophages. Thus, the well-established cognate recognition does not appear to be the only mechanism of B cell induction by T helper cells.

The role of the transferrin receptor for the activation of human lymphocytes.

The proliferative response of peripheral blood mononuclear cells (PBMC) in synthetic serum-free media depends on the presence of sufficient amounts of transferrin (Tf). In the present communication we show that the reduction of Tf concentration in culture media results in a decreased proliferation, whereas lymphokine production and the expression of activation markers (IL-2 receptor; transferrin receptor, (TfR); HLA class II) remain unchanged. To examine whether this effect is due to iron depletion we added iron chelates (ferric citrate, FeCi; ferric nitrilotriacetic acid, FeNTA) which can be internalized by cells without the requirement for Tf. The iron chelates could fully restore the proliferative response even in complete absence of Tf, suggesting that the observed inhibitory effect was indeed caused by iron depletion. Addition of a monoclonal TfR antibody, J 64, also caused a marked inhibition of proliferation of PBMC in regular serum-containing medium as well as in Tf-free synthetic medium; this effect could not be overcome by any of the tested iron chelates. Therefore, growth inhibition caused by J 64 cannot simply be attributed to iron starvation. These data suggest that J 64 may interfere with processes others than iron uptake and that the TfR might confer a necessary promoting signal for lymphocyte proliferation.

Changes in cell surface antigen expression on human articular chondrocytes induced by gamma-interferon. Induction of Ia antigens.

Ia antigens (class II HLA molecules) have been detected on cells eluted from affected human cartilage in certain disease states, but not on normal cartilage cells. Because the presence of Ia antigens on chondrocytes may play an important role in rheumatic diseases, we investigated the induction of these molecules by gamma-interferon (gamma-IFN), a potent Ia-inducing lymphokine. Human articular chondrocytes were incubated with recombinant gamma-IFN, and the expression of Ia antigens was studied by cell sorter analysis, using a panel of reagents that detect monomorphic and polymorphic specificities of the DR and DQ Ia antigen families. While the induction of DR antigens, including polymorphic DR specificities, was readily obtained with gamma-IFN (50-95% positive cells), DQ antigens were negative or were displayed only on a lower percentage of chondrocytes (5-60%). In addition, incubation with gamma-IFN led to an increased expression of HLA class I antigens. The expression of various other surface markers either remained unchanged (as in 4F2 and BA-2) or showed tendencies toward decreased percentages (as in 83c2) or increased percentages (as in M phi R-17). No apparent change in cell morphology or growth pattern was observed.

Kinetics of cell-mediated immunity developing during the course of Leishmania major infection in 'healer' and 'non-healer' mice: progressive impairment of response to and generation of interleukin-2.

Leishmania major (L. major)-infected mice of 'non-healer' (BALB/c) and 'healer' (C57BL/6) mouse-strain origin were studied with regard to the kinetics of cell-mediated immunity developing during the course of the disease. Cells obtained from lymph nodes draining L. major-infected footpads were comparatively analysed for their representation in the respective L3T4+, Lyt-2+ and sIg+ lymphocyte subsets; they were studied for their capacity to release interleukin-2 and to proliferate in response to L. major antigen and concanavalin A, including the determination of the frequencies of T cells proliferating antigen-specifically with or without an exogenous source of IL-2. The data obtained indicate L. major infection-induced long-lasting alterations in the cellular composition of the lymph node in both 'healer' and 'non-healer' mice. Moreover, they suggest that the inability of 'non-healer' mice to recover from L. major infection is associated with a progressive impairment of their lymph node T cells to release interleukin-2 in the culture supernatant and to respond to this lymphokine in vitro.

Functional and phenotypical characterization of activated T cells from intra-articular sites in inflammatory joint diseases. Possible modulation of the CD3 antigen.

The presence of activated T lymphocytes bearing interleukin 2 (IL-2) receptors and HLA class II (Ia) antigens accompanied by impaired T cell functions such as a decreased mitogenic responsiveness are characteristic findings, especially in intra-articular sites in chronic inflammatory joint diseases. The objective of the present study was to further characterize these in vivo activated T cells by the investigation of IL-2 production and a possible T cell receptor modulation. IL-2 receptors were found to be expressed primarily in the CD4+ subset. The Ia+ subset expressing both DR and DQ antigens showed a weaker mitogen-induced response as compared to the Ia- fraction. A decreased mitogen-induced IL-2 production and a lower response to anti-CD3 monoclonal antibodies was observed with synovial T lymphocytes as compared to peripheral blood T cells. The density of the CD3 molecule, known to be closely associated with the T cell receptor, was significantly lower in intra-articular sites, while other T cell-specific surface molecules were expressed to a similar extent in both compartments. The decreased synovial T cell mitogenesis was not restored by the addition of lymphokines (IL-1 and IL-2) or blood monocytes, nor by removing CD8+ T cells. These data present further evidence for a significant T cell activation in intra-articular sites in chronic inflammatory joint diseases. The decreased expression of the CD3 glycoprotein suggests a modulation by so far unidentified antigen(s), which could also be responsible for the weak T cell response elicited by polyclonal mitogens.

CD80 expression on monocytes in HIV-infected patients.

A prominent feature of HIV infection is a progressive anergy of T cells and an increase of activation-dependent T-cell death. CD80 (B7.1), the ligand of CD28, is an important co-stimulatory molecule on antigen-presenting cells that delivers an essential second signal for T-cell activation. To test whether immunologic dysfunction in HIV disease involves CD80, we studied CD80 expression on circulating monocytes in heparinized whole blood of 33 HIV-infected patients and 13 controls. Most monocytes in patients and controls expressed significant amounts of CD80. There was no statistical difference of mean fluorescence intensity (MFI) of CD80 expression when all HIV-infected patients were compared with healthy controls. However, asymptomatic patients in clinical Centers for Disease Control and Prevention (CDC) stage A showed a significantly stronger CD80 expression than did healthy controls. Additionally, patients receiving antiretroviral therapy exhibited significantly higher CD80 expression than did patients not receiving therapy and healthy controls. We did not find a correlation with the presence of HIV p24 antigenemia and counts of CD4+ and CD8+ T cells. Although we studied CD80 expression only on circulating monocytes and not in HIV-infected monocytes or in activated macrophages, our data do not support a role for a general impairment of CD80 expression in induction of anergy in HIV disease.

UV-B irradiated cell lines execute programmed cell death in various forms.

We investigated changes typical for apoptosis in various cell lines after UV-B irradiation. Using established methods for detection of apoptosis we demonstrate changes of cellular morphology, phosphatidylserine (PS) exposure, ollgonucleosomal DNA fragmentation and generation of hypochrome nuclei. To isolated high-molecular-weight (hmwt) DNA fragments we engaged a new method avoiding pulse field gel electrophoresis. Most UV-B irradiated cell lines showed oligonucleosomal DNA fragmentation, hypochrome nuclei, morphological changes, annexin-V binding and positive TUNEL reaction. However, no oligonucleosomal DNA fragmentation could be detected in Raji and HaCaT cells. Whereas HaCaT cells displayed all other changes typical for apoptosis, Raji cells were TUNEL negative, formed low amounts of hmwt DNA and showed an 'atypically' low hypochrome shift. Nevertheless, UV-B irradiated Raji cells excluded propidium iodide (PI), bound annexin-V and stopped proliferation. This suggests that Raji cells underwent growth arrest with exposure of PS being the only feature of apoptosis. However, in the presence of phagocytes expressing the phosphatidylserine receptor these cells would share the removal pathway with apoptotic cells. Since UV-B induced programmed cell death differs in dependence of cells under investigation, the failure to detect oligonucleosomal DNA fragmentation or chromatin condensation is not suitable to exclude programmed (apoptotic?) cell death.

Costimulation via TCR and IL-1 receptor reveals a novel IL-1alpha-mediated autocrine pathway of Th2 cell proliferation.

Previous studies have shown that triggering of Th2 cells via the TCR is sufficient for production of IL-4 but not for proliferation of these cells. Proliferation of Th2 cells occurs only in the additional presence of a costimulatory signal delivered by IL-1. For the majority of Th2 cell clones, this type of proliferation was found to be independent of IL-4. Here, we further investigated the mechanism of IL-4-independent proliferation. We demonstrate that, after costimulation via TCR and IL-1R, but not via either receptor alone, Th2 cells are triggered to produce cell-associated IL-1alpha, as detected at the level of function, protein, and mRNA expression. In the presence of the TCR signal, autocrine IL-1alpha is then able to costimulate IL-4-independent proliferation of Th2 cells and to further enhance its own production. Thus, our results point to a novel, IL-4-independent, self-amplifying autocrine pathway of Th2 cell proliferation that requires a signal via the TCR and a costimulatory signal via IL-1R. This pathway may explain frustrating results in experimental models that attempted to treat established Th2-mediated diseases in vivo with IL-4-neutralizing agents alone.

Stimulation of cyclin-dependent kinase activity and G1- to S-phase transition in human lymphocytes by the human T-cell leukemia/lymphotropic virus type 1 Tax protein.

The human T-cell leukemia/lymphotropic virus type 1 (HTLV-1) induces a malignant lymphocytic disease. The HTLV-1 transactivator protein, Tax, is believed to be crucial for the development of the disease since it is transforming in vitro and induces tumors in transgenic animals. Although the transcriptional modulation of viral and cellular gene expression by Tax has been analyzed thoroughly, it has remained unclear how the Tax functions act on the cell cycle of primary T cells. To investigate the mechanism of Tax-mediated T-cell stimulation, we transduced primary human cord blood T cells with a conditional, tetracycline repressor-based tax expression system. Permanent Tax expression results in an abnormal proliferation of T cells which closely resemble HTLV-1-infected lymphocytes. Suppression of Tax synthesis stopped lymphocyte growth and caused cell cycle arrest in the G1 phase. Upon reinduction of tax expression, the arrested cells entered the S phase. This showed that Tax has mitogenic activity, which is required for stimulating the G1- to S-phase transition of immortalized lymphocytes. In mammalian cells, the G1-phase progression is controlled by the serial activation of several cyclin-dependent kinases (Cdks), starting with Cdk4 and Cdk6. In the presence of Tax, both Cdk4 and Cdk6 were activated. The suppression of Tax synthesis, however, resulted in a significant reduction of the Cdk4 and Cdk6 activities but did not influence the expression of Cdk4, Cdk6, or cognate D-type cyclin proteins. These data suggest that Tax induces Cdk4 and Cdk6 activity in primary human T lymphocytes; this Cdk activation is likely to account for the mitogenic Tax effect and for the abnormal T-cell proliferation of HTLV-1-infected lymphocytes.

Monoclonal antibodies EBU-141 (CDw75) and EBU-65 allow reliable distinction between mature and pre-B-cell tumors in suspension and on tissue sections.

Two new monoclonal antibodies, EBU-65 and EBU-141, were raised by immunization with plasma cell line U-266. Both antibodies strongly react with B lymphocytes in immunofluorescent staining as well as on paraffin-embedded sections. More than 200 leukemias and lymphomas were tested, and for both antibodies reactivity was found only with "mature" B-cell tumors but not with precursor B-cell leukemias. None of the non-B-lineage hematolymphatic tumors tested was stained by EBU-141 or EBU-65. A subpopulation of T lymphocytes particularly present in nonmalignant pleural effusions was detected by EBU-65 additionally. Although EBU-141 was clustered as CDw75 and EBU-65 as "unique," a close relationship of the staining pattern was found and both antibodies react with a sialyltransferase. In particular, CDw75 antibody EBU-141 was demonstrated to be very useful for immunophenotyping of B-cell neoplasias, while EBU-65 reacted with most multiple myelomas and a subgroup of "activated"-appearing T cells.

[Prostaglandin E2, interleukin 1 and gamma interferon production of mononuclear cells of patients with inflammatory and degenerative joint diseases]. / Prostaglandin-E2-, Interleukin-1- und Interferon-Gamma-Produktion mononukleärer Zellen von Patienten mit entzündlichen und degenerativen Gelenkerkrankungen.

Inflammatory joint diseases exhibit distinct pathohistological and immunological characteristics. The studies performed demonstrated that in comparison to normal controls peripheral blood mononuclear cells from patients with rheumatoid arthritis (RA) presented an increased percentage of monocytic cells. Peripheral blood mononuclear cells from patients with RA produced significantly increased amounts of prostaglandin E2 and significantly decreased amounts of interferon-gamma following mitogen stimulation with LPS or PWM respectively. The spontaneous production of interleukin 1 was found to be elevated. A significantly increased LPS induced production of prostaglandin E2 could also be observed in monocyte depleted rheumatoid peripheral cells and in peripheral cells of patients with osteoarthritis and HLAB27 associated joint diseases. Mononuclear cells from rheumatoid synovial tissue produced increased amounts of prostaglandin E2 and decreased amounts of interferon-gamma; the spontaneous prostaglandin E2 production was similar to the values obtained by mitogen stimulation which may originate from the distinct cellular composition of synovial tissue.

Phorbol ester enhances both interleukin 2 receptor expression and immunoglobulin secretion in human Epstein-Barr virus-immortalized B cells.

The enhanced expression of interleukin 2 (IL2) receptors by 12-O-tetradecanoylphorbol 13-acetate (TPA) on sublines of an Epstein-Barr virus-immortalized human B17 B cell line (M. Steinitz et al., Immunobiology 1979. 156: 41.) was studied by immunofluorescence using the anti-Tac monoclonal antibody and by binding studies with purified radiolabeled IL2. These studies show that TPA at a final concentration of 5 ng/ml greatly increased Tac antigen expression on a number of sublines of B17. IL2-binding studies revealed that TPA induced an increase in not only the number of IL2 receptors per cell but also the affinity of the receptors for IL2. The number and affinity of IL2 receptors on the C76 subline treated with TPA appear to be similar to those of activated normal human peripheral T cells. Furthermore, TPA-induced differentiation of these B cell lines was measured by induction of immunoglobulin secretion using an enzyme-linked immunosorbent assay. The capacity of TPA to induce differentiation in human B cells and the biological significance of IL2 receptor expression by activated B cells are discussed.

Determination of the electrophoretic mobility of chromosomes by free flow electrophoresis. I. Morphology and stability.

Isolated metaphase chromosomes of several fibroblastoid cell lines (Chinese hamster, Chinese hamster x human hybrid) were subjected to free flow electrophoresis (FFE) to study their electrophoretic mobility (EM). The morphology and stability of the chromosomes were unaffected by FFE as examined by cytogenetic methods and flow cytometry. The chromosomes of the complement all showed similar EM under most of the conditions applied. At neutral pH the EM of the chromosomes had the same sign as free DNA and about 2/3 of its magnitude. The variation of EM with buffer parameters such as ionic strength, valence of counterions, buffer capacity and dielectric constant of the solvent were investigated. Thermal denaturation increased the EM of the chromosomes by 20%. Partial denaturation might offer a possibility to separate or enrich large amounts of chromosomes by FFE.

Specific sample preparation in colorectal cancer.

Large tissue samples from ten patients operated for colorectal cancer were prepared in the operating room in iced phosphate buffered saline, containing ethylene diaminetetraacetic acid and protease inhibitors. After cutting the specimens into small fragments, the tissues were gently pressed through a steel mesh. Membranes were permeabilized in chilled ethanol 70% to allow cytosolic fluoresceine isothiocyanate labeling, performed with anti-cytokeratin (CAM 5.2) antibodies. Samples were quantitatively sorted with a fluorescence activated cell sorter (FACS) and denatured before processing separation by two-dimensional electrophoresis on polyacrylamide gels. This procedure made it possible to sample about 4 x 10(7) viable normal and tumoral cells before fixation, and up to 4 x 10(6) cells after FACS. The gels run before and after fixation showed no major differences. The rate of cytokeratin-positive cells in the samples was the following (mean, CI 5-95%): mucosa 29.5% (8.9-66.7%), tumor 44.3% (6.6-94.8%). The epithelial cell content in colorectal cancer and normal mucosa shows important intersample variations. This is important for any comparison of fresh samples, whether at DNA, RNA, or at the protein level. We propose a method allowing the preparation of pure epithelial cell samples from normal and tumoral colonic fresh mucosa.

[Phenotypic analysis in colorectal carcinoma: an international interdisciplinary project]. / Phänotypische Analyse beim kolorektalen Karzinom: ein internationales interdisziplinäres Projekt.

An European research network grouping surgeons, pathologists, biochemists and molecular biologists is presented. The aim of this network is to define new diagnostic, prognostic and therapeutic markers at protein and RNA levels in colorectal cancer. The methodology is based on specific sample preparation techniques, allowing the isolation of pure epithelial cells, and on differential-display techniques, such as two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and reverse arbitrarily-primed polymerase chain reaction (RAP-PCR).