Surface phenotype analysis of human monocyte to macrophage maturation.

Cells of the mononuclear phagocyte system arise from circulating blood monocytes. Upon emigration from the vasculature, monocytes differentiate into macrophages, a process that monocytes similarly undergo in vitro. We have established primary cultures from elutriated or adherence-purified blood monocytes and analyzed the antigenic modulation during monocyte to macrophage transformation, which could be followed by the expression of specific antigens and which required as yet unknown inducer signals present in the serum. It is shown that in the absence of serum monocytes only survive in vitro when cultured adherent to plastic but rapidly die in suspension culture. Starting at 0.5%, serum induced maturation dose-dependently, with the optimal concentration being 2 to 5%. Of those antigens not present on monocyte, the low-affinity Fc receptor (CD16), the alpha-chain of the vitronectin receptor (CD51), gp65-MAX.1, and gp68-MAX.3 were expressed only upon serum-induced macrophage differentiation, whereas the transferrin receptor (CD71), MAX.26, and to some degree also gp65-MAX.11 appeared to be independent of maturation and were also found on primary cultures of adherent monocytes under serum-free conditions. In addition, the rapid induction of HLA class II antigens (within 24 hr) was similar with and without serum, as was the continued high-density expression in long-term culture. The monocyte-specific CD14 antigen was down-regulated in the absence of serum but kept its level of expression on differentiated macrophages. In comparison, alveolar and peritoneal macrophages, respectively, differed in their antigenic phenotype: Alveolar macrophages expressed high HLA class II antigens but low CD14, whereas for peritoneal macrophages the opposite was found. Both interferon-gamma and -alpha suppressed macrophage maturation in vitro but had contrary effects on HLA class II and CD16 expression: Interferon-gamma up-regulated the two types of antigens, which, in contrast, were down-regulated by interferon-alpha.     

Partial purification of an iron-dependent L-serine dehydratase from Clostridium sticklandii

An oxygen-sensitive and highly unstable L -serine dehydratase was partially purified from the Grampositive anaerobe Clostridium sticklandii. The final active preparation contained five proteins of 27, 30, 44.5, 46, and 58 kDa as judged by SDS-PAGE. The N-terminal sequence of the 30 kDa subunit showed some similarity to the α-subunits of the iron-containing L -serine dehydratases from Clostridium propionicum and Peptostreptococcus asaccharolyticus. Oxygen-inactivated L -serine dehydratase from C. sticklandii was reactivated by incubation with Fe²⁺ under reducing conditions. Furthermore, the enzyme was inactivated by iron-chelating substances like phenanthroline and EDTA. Pyridoxal-5-phosphate (PLP) did not stimulate the activity, and known inhibitors of PLP-containing enzymes such as NaBH₄ had no effect on the activity of L -serine dehydratase from C. sticklandii.     

Large-scale in vitro expansion of polyclonal human CD4(+)CD25high regulatory T cells

CD4(+)CD25+ regulatory T (Treg) cells are pivotal for the maintenance of self-tolerance, and their adoptive transfer gives protection from autoimmune diseases and pathogenic alloresponses after solid organ or bone marrow transplantation in murine model systems. In vitro, human CD4(+)CD25+ Treg cells display phenotypic and functional characteristics similar to those of murine CD4(+)CD25+ Treg cells: namely, hyporesponsiveness to T-cell receptor (TCR) stimulation and suppression of CD25- T cells. Thus far, the detailed characterization and potential clinical application of human CD4(+)CD25+ Treg cells have been hampered by their paucity in peripheral blood and the lack of appropriate expansion protocols. Here we describe the up to 40 000-fold expansion of highly purified human CD4(+)CD25high T cells in vitro through the use of artificial antigen-presenting cells for repeated stimulation via CD3 and CD28 in the presence of high-dose interleukin 2 (IL-2). Expanded CD4(+)CD25high T cells were polyclonal, maintained their phenotype, exceeded the suppressive activity of freshly isolated CD4(+)CD25high T cells, and maintained expression of the lymph node homing receptors L-selectin (CD62L) and CCR7. The ability to rapidly expand human CD4(+)CD25high Treg cells on a large scale will not only facilitate their further exploration but also accelerate their potential clinical application in T cell-mediated diseases and transplantation medicine.     

Isolated cerebral manifestation of Epstein–Barr virus‐associated post‐transplant lymphoproliferative disorder after allogeneic hematopoietic stem cell transplantation: a case of clinical and diagnostic challenges

N.A. Kittan, F. Beier, K. Kurz, H.H. Niller, L. Egger, W. Jilg, R. Andreesen, E. Holler, G.C. Hildebrandt. Isolated cerebral manifestation of Epstein–Barr virus‐associated post‐transplant lymphoproliferative disorder after allogeneic hematopoietic stem cell transplantation: a case of clinical and diagnostic challenges.
Transpl Infect Dis 2011: 13: 524–530. All rights reserved Abstract: We present the case of a 49‐year‐old male patient with Epstein–Barr virus (EBV)‐associated post‐transplant lymphoproliferative disorder (PTLD) limited to the brain that occurred 6 months after allogeneic hematopoietic stem cell transplantation (HSCT). Clinical symptoms included mental confusion, ataxia, and diplopia. Magnetic resonance imaging (MRI) revealed cerebellar and periventricular lesions consistent with an inflammatory process. Cerebrospinal fluid (CSF) analysis, but not peripheral blood, was positive for EBV‐DNA, but no malignant cells were found. Brain biopsy was not feasible because of low platelet counts. As we considered a diagnosis of either EBV‐associated encephalitis or PTLD, the patient was treated with rituximab combined with antiviral therapy. However, the cerebral lesions progressed and follow‐up CSF testing revealed immunoglobulin H clonality as evidence of a malignant process. Subsequent treatment attempts included 2 donor lymphocyte infusions (DLI). Despite treatment, the patient died from autopsy‐proven PTLD within 8 weeks of the onset of symptoms. This case demonstrates the clinical and diagnostic challenges of primary cerebral PTLD in a patient following allogeneic HSCT.     

Interleukin-10 promotes NK cell killing of autologous macrophages by stimulating expression of NKG2D ligands

Under inflammatory conditions, the pleiotropic cytokine interleukin-10 (IL-10) is released in many tissues. It mediates anti-inflammatory effects in particular by inhibiting the release of T helper type 1 (Th1) cytokines. In contrast, we show here that NK cell cytotoxicity against autologous macrophages is elevated if both cell types are cultured with IL-10. The expression of most activatory NK receptors is increased after culture in the presence of IL-10. On the other hand, macrophages cultured in the presence of IL-10 show elevated expression of the NKG2D ligands major histocompatibility complex (MHC) class 1-like molecules (MIC) - A and - B, as well as UL-16 binding proteins (ULBP) - ULBP-1, ULBP-2 and ULBP-3. By masking the interaction of NK cells with macrophages through interruption of the NKG2D receptor with its ligands, we could reverse the IL-10-induced lysis of macrophages. Our data therefore reveal that IL-10 may exert a novel immunomodulatory role by stimulating NKG2D ligand expression on macrophages, thereby rendering them susceptible to NK cell elimination. This suggests that NK cells would delete macrophages and potentially other immature antigen-presenting cells (APC) or their precursors under inflammatory conditions as a feedback mechanism to shut off uncontrolled immune responses.     

Whole-body UVB irradiation during allogeneic hematopoietic cell transplantation is safe and decreases acute graft-versus-host disease

Depletion of host Langerhans cells (LCs) prevents cutaneous graft-versus-host disease (GvHD) in mice. We analyzed whether UVB irradiation is tolerated during the course of human allogeneic hematopoietic cell transplantation and whether depletion of LCs by broadband UVB could improve GvHD outcome. A total of 17 patients received six whole-body UVB irradiations with 75% of the individually determined minimal erythemal dose after conditioning with a reduced intensity protocol. LCs, dermal dendritic cells (DCs), and macrophages were analyzed before and after UVB irradiation by immunohistochemical analysis. Circulating blood cells and serum factors were analyzed in parallel. In striking contrast to previous data, our irradiation protocol was well tolerated in all patients. UVB treatment decreased the number of LCs and also affected dermal DCs. UVB-treated patients also had significantly higher 25-hydroxyvitamin D3 serum levels and higher numbers of circulating CD4+ FoxP3+ regulatory T cells. Strikingly, nine out of nine patients with complete LC depletion (<1 LC per field) developed only grade I GvHD or no GvHD up to day 100. Our results strongly suggest that prophylactic UVB irradiation post transplant is safe and should be further explored as a clinical strategy to prevent acute (skin) GvHD.     

Monoclonal antibodies against differentiation antigens on human macrophages

Human macrophages matured in vitro from blood monocytes without additional exogenous activation were used to produce monoclonal antibodies (mAbs). Ten of them were selected for high avidity and the best discrimination between mature macrophages and a panel of other cells including freshly prepared monocytes, B cells, T cells, granulocytes and thrombocytes. Five mAbs (MAX. 1, MAX. 2, MAX. 3, MAX. 11, MAX. 24) were found to detect macrophage differentiation antigens. One mAb (MAX. 26) detects an antigen which is shared by mature macrophages and T cells.     

Generation of cytokines in human visceral leishmaniasis: dissociation of endogenous TNF-alpha and IL-1 beta production

The role of TNF-alpha in visceral leishmaniasis is ambivalent, the eventual outcome of this infection, cure or generalization, being determined by the relative amounts of cytokines produced in vivo. Since release, by monocytes/macrophages, of TNF-alpha and interleukins 1 beta (IL-1 beta) and IL-6 is important in both the induction and effector phases of the immune responses, these mediators were determined in sera and cell culture supernatants of seventeen L. donovani infected patients in Brazil. The results are compared to those of a local control group. Circulating immunoreactive TNF-alpha in patients (median, 140 pg ml-1) was increased ten-fold over controls (median 16 pg ml-1, p less than or equal to 0.0001). In contrast, serum IL-1 beta was less than 20 pg ml-1 in all patients, although detectable in sera of 3/16 Brazilian controls (chi 2 = 3.5, p less than 0.1). Mitogen induced in vitro release of IL-1 beta and IL-6 by patients' circulating mononuclear cells was significantly reduced, and the capacity of patients' peripheral monocytes for H2O2 generation in response to opsonized zymosan was significantly diminished. In the patients, serum TNF-alpha levels were inversely related to IL-1 beta release in vitro (rho = -0.57, p less than or equal to 0.01).     

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