Inverse regulation of the ADAM-family members, decysin and MADDAM/ADAM19 during monocyte differentiation

Two members of the ADAM (a disintegrin and metalloprotease)-family, MADDAM and decysin, were described as dendritic cell (DC) maturation markers. We are interested in monocyte differentiation and investigated in particular the expression pattern of both genes during the differentiation of human monocytes into DC and macrophages (MAC). Both genes are weakly expressed in freshly isolated monocytes. In immature DC decysin mRNA was absent, even after induction of the terminal differentiation of DC by CD40L or tumour necrosis factor-alpha (TNF-alpha). Only in DC maturated by lipopolysaccharide (LPS) strong signals of decysin mRNA were detected. However, MADDAM mRNA was expressed in immature DC and the expression was markedly increased after induction of the terminal DC differentiation by various stimuli. In contrast, MAC showed a high constitutive decysin mRNA expression, but expressed no MADDAM mRNA. On protein level similar results of MADDAM expression were obtained. Stimulation of MAC by LPS did not induce MADDAM mRNA expression, while decysin mRNA expression was strongly increased. Further investigations revealed that the well-known inducer of MAC differentiation, 1alpha,25-dihydroxyvitamin D3 up-regulated decysin mRNA expression during the differentiation of primary monocytes and myelomonocytic THP-1 cells into MAC. In vivo decysin mRNA expression was only detected in human colon, but not in other tissues we examined. Accordingly, isolated intestinal MAC expressed decysin mRNA. In conclusion, decysin and MADDAM mRNA expression were regulated in an opposite way during monocyte differentiation: MADDAM mRNA and protein was mainly detected in DC, whereas decysin mRNA expression was mainly found in MAC. Therefore only MADDAM, but not decysin is a suitable marker for human monocyte-derived DC.     

Systematic classification of HIV biological subtypes on lymphocytes and monocytes/macrophages

The growth properties and cytopathic effects of several HIV-1 and HIV-2 strains were compared between cultures on human lymphocytes and monocytes/macrophages, respectively. For some isolates (among these three paired isolates from blood and cerebrospinal fluid) replication and cytopathogenicity were comparable between lymphocytes and monocytes/macrophages (dual tropic viruses), while others showed a very specific tropism for only one cell type. Yet another subtype grew neither well on lymphocytes nor on macrophages. Taking into account the growth properties in monocytes/macrophages we propose a classification system for HIV subtypes on these cells (alpha-delta), in analogy to the nomenclature for HIV-subtyping on lymphocytes (a-d). Using this system, some prototypic viruses (LAV/HTLV-IIIB, HIV-2ROD, SIVBK28, HIV-2ALT) as well as several other HIV-1 and HIV-2 isolates were subtyped.     

Activation of human monocyte-derived macrophages cultured on Teflon: response to interferon-gamma during terminal maturation in vitro

Macrophages (M phi) are potential antitumor effector cells derived from circulating blood monocytes (mo). Most studies on human mo/M phi biology and function have been performed using immature mo precursor cells. However, the conclusions drawn may be questionable, as mo have to undergo terminal differentiation before they reach relevant tissue sites of inflammation and immune reaction. We have analyzed the ability of mo-derived, teflon-cultured M phi to respond to activating stimuli with an increased tumor cytotoxic effector cell function using recombinant interferon-gamma (IFN-gamma), IFN-alpha 2, granulocyte/macrophage colony stimulating factor (GM-CSF), interleukin(IL) 2, IL 1 alpha, and bacterial lipopolysaccharides (LPS) as mediator molecules. It could be shown that the response of M phi to the most potent activator molecule, IFN-gamma, depends on the terminal differentiation from the mo stage to the mature M phi. Whereas adherent mo could be activated only moderately, M phi increased their cytotoxicity by a factor of up to 400. IFN-gamma activation positively correlated with the effector cell number, the time of incubation and the dosage used. Activation did not depend on the presence of LPS, and was lost within 24 to 48 h. LPS itself activated cells only in the microgram range. IFN-alpha 2 activated M phi only at a two log higher concentration than IFN-gamma; GM-CSF was only slightly effective, whereas M phi incubation with IL 1 alpha or IL 2 did not result in M phi activation. Thus, the ability of human M phi to become activated appears to be a function of cellular maturation and is acquired during the terminal step of M phi differentiation. Teflon-cultured M phi could facilitate studies of the activation of human M phi and may be more suitable cells for adoptive immunotherapy in cancer patients than blood monocytes.     

Developmental regulation of the cytokine repertoire in human macrophages: IL-1, IL-6, TNF-alpha, and M-CSF

We were interested in the dependence of constitutive and stimulated cytokine secretion on the stage of macrophage (MAC) differentiation in vitro. Elutriation-purified blood MO were cultured up to 28 days and their secretory repertoire was analyzed under adherence conditions at various culture stages. For each of the cytokines tested, interleukin (IL)-1 beta, IL-6, tumor necrosis factor (TNF)-alpha, and macrophage colony-stimulating factor (M-CSF), a different pattern of regulation was observed. During the initial phase of maturation (up to day 7 in culture) within which the characteristics of normal MO to MAC transformation are achieved, M-CSF was the only cytokine to be secreted constitutively. From the LPS-dependent cytokines, IL-1 beta and IL-6 were downregulated whereas TNF-alpha levels increased severalfold. For the release of IL-1 beta, IL-6, and TNF-alpha a synergistic effect of interferon-gamma (IFN-g) and lipopolysaccharide (LPS) was observed. M-CSF release increased until day 7 in culture with LPS being stimulatory for this particular cytokine only during the first days of differentiation. Upon further cultivation of MAC up to 28 days, LPS-induced IL-1 beta levels remained very low, but IL-6 levels increased again reaching that of blood MO, and TNF-alpha continued to rise reaching levels up to 30-fold higher than in blood MO. M-CSF secretion stayed high with LPS even suppressing constitutive secretion. Long-term cultured MAC started to release IL-6 and TNF-alpha also in the absence of a stimulus and, furthermore, became responsive to IFN-g alone. Our data show that the release of each cytokine investigated is differently regulated during maturation. These results document the functional plasticity of human MAC and emphasize the impact that MO to MAC differentiation may have in vivo.     

A Hodgkin cell-specific antigen is expressed on a subset of auto- and alloactivated T (helper) lymphoblasts

A Hodgkin cell-specific antigen detected by the monoclonal antibody Ki- 1 was found on T helper lymphocytes after activation by autologous and allogeneic stimulator cells. About 50% of lymphoblasts generated by auto- and alloactivation reacted with the antibody. In contrast, only less than 6% of lymphoblasts stimulated with Con-A, phytohemagglutinin (PHA), or protein A, and none of lymphoblasts activated by oxidative mitogenesis, expressed this antigen. Among several permanent cell lines tested, the K562, MOLT-4, HL-60, and EBV transformed B lymphoblastoid cells reacted with the Ki-1 antibody. The results may indicate possible relationships between the autoreactive subset of T lymphocytes and the pathogenesis of Hodgkin's disease.     

Regulation of 25-hydroxyvitamin D3-1 alpha-hydroxylase and production of 1 alpha,25-dihydroxyvitamin D3 by human dendritic cells

25-Hydroxyvitamin D3-1 alpha-hydroxylase (25(OH)D3-1 alpha-hydroxylase), the key enzyme of 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) production, is expressed in monocyte-derived macrophages (MACs). Here we show for the first time constitutive expression of 25(OH)D3-1 alpha-hydroxylase in monocyte-derived dendritic cells (DCs), which was increased after stimulation with lipopolysaccharide (LPS). Accordingly, DCs showed low constitutive production of 1,25(OH)2D3, but activation by LPS increased 1,25(OH)2D3 synthesis. In addition, 25(OH)D3-1 alpha-hydroxylase expression was found in blood DCs but not in CD34+-derived DCs. Next we analyzed the functional consequences of these results. Addition of 1,25(OH)2D3 at concentrations comparable with those produced by DCs inhibited the allostimulatory potential of DCs during the early phase of DC differentiation. However, terminal differentiation decreased the responsiveness of DCs to 1,25(OH)2D3. In conclusion, DCs are able to produce 1,25(OH)2D3 especially following stimulation with LPS. Terminal maturation renders DCs unresponsive to the effects of 1,25(OH)2D3, but those cells are able to suppress the differentiation of their own precursor cells in a paracrine way through the production of 1,25(OH)2D3.     

Cytotoxic activity in the serum of a patient with metastasizing nephroblastoma given intravenous infusions of alkyl-lysophospholipids in a phase I study

Summary In a phase I study a cytotoxic activity in the serum of a tumor patient given infusions of synthetic alkyl-lysophospholipid has been demonstrated. Serum samples collected after ALP infusions inhibited ³H-thymidine incorporation by human leukemic cells to an extent that correlated to the dose of ALP administered. Serum taken after the highest dose of ALP given (50 mg/kg body weight) led to complete cell destruction after 72 h in vitro. Whereas cells from the HL60 line were very sensitive to the serum cytotoxicity, K562 cells were much less affected. Cytotoxic activity was found to be cleared from the circulation in a biphasic manner; more than 50% disappeared within 6–8 h but 20–30% was still present after 4 days.Supported by DFG An 111/2     

Expression of transferrin receptors and intracellular ferritin during terminal differentiation of human monocytes

Summary Human blood monocytes when cultured on hydrophobic Teflon membranes differentiate into mature macrophages. The expression of transferrin receptors was monitored by monoclonal antibody (OKT9) binding as detected by immunperoxidase staining. Whereas monocytes were negative, an increasing percentage of macrophages, starting from day 2 in culture, labelled with the anti-transferrin receptor antibody as these cells undergo differentiation. After completion of maturation more than 90% of macrophages expressed transferrin receptors. While 90–95% of macrophages from broncho-alveolar lavage fluids labelled with the OKT9 antibody, only a minor portion of macrophages obtained from peritoneal and pleural cavities did so. In parallel, intracellular ferritin in cells of the monocyte-macrophage lineage increased from 10ng/10⁶ cells to 350–1,500ng/10⁶ cells during maturation in vitro. Alveolar macrophages proved to have the highest ferritin content which ranged from 355–8,400ng/10⁶.The results may indicate that iron uptake and storage is a function of cells at late stages of macrophage maturation and that the occurrence of surface receptors for transferrin can be regarded as differentiation dependent marker.Supported by Deutsche Forschungsgemeinschaft     

Ungew?hnliche Hautver?nderungen bei 72-j?hriger Patientin mit multiplem Myelom

We report on a 72-year-old female patient with multiple myeloma who presented with alopecia and eye-catching alterations of the skin and the nails. A biopsy of the skin could confirm the diagnosis of immunoglobulin light chain (AL) amyloidosis, which was also suspected of having affected other organs. After six cycles of a cytoreductive therapy with bortezomib and dexamethasone a very good partial response of the multiple myeloma was seen and an improvement in the skin and nail alterations could be achieved.