Joachim Roesler

A fast and easy method to determine the production of reactive oxygen intermediates by human and murine phagocytes using dihydrorhodamine 123

Analysis of the functional activity of phagocytes is of great importance in the differential diagnosis of patients with recurrent bacterial infections. Here we describe a method to determine the production of reactive oxygen intermediates (ROI) by microcytofluorometry using dihydrorhodamine 123, a derivative of rhodamine 123. Using this method the ROI production of erythrocyte-depleted whole blood samples can be measured without further time-consuming purification steps. Possible harmful manipulation of the isolated cells can also be avoided and highly reproducible and significant results are obtained in the minimum of time. This assay provides a very sensitive alternative to the clinically used NBT test in the diagnosis of patients with chronic granulomatous disease (CGD). Moreover, the analysis of oxygen-dependent effector functions of murine effector cells and cell lines may be important in investigating resistance to certain microbes (e.g., Candida albicans, Staphylococcus aureus or different protozoa such as Toxoplasma gondii or Leishmania species).

Application of purified polysaccharides from cell cultures of the plant Echinacea purpurea to test subjects mediates activation of the phagocyte system

Polysaccharides purified from large-scale cell cultures of the plant Echinacea purpurea were tested for their ability to activate human phagocytes in vitro and in vivo. These substances enhanced the spontaneous motility of PMN under soft agar and increased the ability of these cells to kill staphylococci. Monocytes were activated to secrete TNF-alpha, IL-6 and IL-1 whereas class II expression was unaffected. Intravenous application of the polysaccharides to test subjects immediately induced a fall in the number of PMN in the peripheral blood, indicating activation of adherence to endothelial cells. This fall was followed by a leukocytosis due to an increase in the number of PMN and a lesser increase of monocytes. The appearance of stab cells and some juvenile forms and even myelocytes indicated the migration of cells from the bone marrow into the peripheral blood. The acute phase C-reactive protein (CRP) was induced, probably due to activation of monocytes and macrophages to produce IL-6. In addition a moderate acceleration of the erythrocyte sedimentation rate was observed. Altogether, as in mice, the polysaccharides could induce acute phase reactions and activation of phagocytes in humans. The possibility of clinical use is discussed.

Polysaccharides isolated from plant cell cultures of Echinacea purpurea enhance the resistance of immunosuppressed mice against systemic infections with Candida albicans and Listeria monocytogenes.

Polysaccharides (EP) isolated from large scale plant cell cultures of Echinacea purpurea, have been shown to activate human and murine phagocytes. In this study we investigated the influence of EP on the nonspecific immunity in immunodeficient mice. EP was effective in activating peritoneal macrophages isolated from animals after administration of cyclophosphamide (CP) or cyclosporin A (CsA). EP-treated macrophages exhibited increased production of tumor necrosis factor-alpha (TNF) and enhanced cytotoxicity against tumor target WEHI 164 as well as against the intracellular parasite Leishmania enrietti. After a CP-mediated reduction of leukocytes in the peripheral blood, the polysaccharides induced an earlier influx of neutrophil granulocytes as compared to PBS-treated controls. EP treatment of mice, immunosuppressed with CP or CsA, restored their resistance against lethal infections with the predominantly macrophage-dependent pathogen Listeria monocytogenes and predominantly granulocyte-dependent Candida albicans. Further, the effects of EP in allogeneic bone marrow chimeric mice are discussed. These findings may have therapeutical implications in prophylactic treatment of opportunistic infections.

Diagnosis of chronic granulomatous disease and of its mode of inheritance by dihydrorhodamine 123 and flow microcytofluorometry

Dihydrorhodamine 123 (DHR) attached to membranes of granulocytes (PMN) and monocytes is caused to fluoresce by reactive oxygen intermediates (ROI) indicating the ability of phagocytes to produce these microbicide metabolites in a flow microcytofluorimeter. Whole blood samples from five boys with known chronic granulomatous disease (CGD) and from their mothers (and from one father and one grandmother), were examined following erythrocyte lysis in order to test this new method. An incubation period of 10 min with phorbol-myristate-acetate, followed by another 15 min incubation period with DHR before flow microcytofluorimetric analysis of 5 or 10×103 phagocytes, was sufficient to obtain the following results. PMN and monocytes from four patients with CGD could clearly not produce any ROI whereas cells from one patient displayed decreased activity in ROI production as compared to cells from a healthy donor. The X-linked mode of inheritance was detected in six carriers by the presence of two different cell populations (one normal ROI-producing and one negative or less active population). All the phagocytes from one mother produced ROI in normal amounts suggesting an autosomal mode of inheritance. All in all, the method presented provides a fast and most simple tool to diagnose CGD, to determine a decrease or total lack of ROI production and to establish the mode of inheritance of the disease.

Altered surface marker expression and function of G-CSF-induced neutrophils from test subjects and patients under chemotherapy

We have previously reported an altered surface marker expression and chemotaxis of G‐CSF‐induced neutrophils from patients with severe congenital neutropenia. However, effects of G‐CSF and influence of the underlying disease on neutrophils could not be discerned. In this study we have evaluated the effects of G‐CSF on neutrophil phenotype and function in patients under chemotherapy and in healthy test subjects. We found a significantly enhanced expression of FcγRI, CD14 and CD54 and a decrease in the level of FcγRIII during G‐CSF treatment. In addition, motility of G‐CSF‐induced neutrophils was significantly decreased. The effects were seen in patients under cytotoxic chemotherapy and in healthy test subjects. Surface marker alterations and neutrophil motility were affected by G‐CSF administration in a dose‐dependent manner. Kinetic studies on neutrophils from healthy test subjects demonstrated that all effects could be seen after a single administration of 300 μg G‐CSF and began to appear within 4 h. Release of partially immature neutrophils from the bone marrow and indirect activation of these cells by G‐CSF are discussed as possible reasons for the findings presented. They demonstrate that G‐CSF has profound effects on neutrophil phenotype and function in vivo which might have clinical implications.

Heterogeneity in the mobilization of cytoplasmic calcium by human polymorphonuclear leukocytes in response to fMLP, C5a and IL-8/NAP--1

The visible excitation and emission wavelengths of the recently developed fluorescent Ca2+ indicator fluo‐3 permit analysis of the intracellular Ca2+ concentration, [Ca2+]i, in flow cytometry with a 488‐nm argon laser. The role of [Ca2+]i in human polymorphonuclear leukocyte heterogeneity was investigated in response to formyl‐methionyl‐leucyl‐phenylalanine (fMLP), C5a, and interleukin 8/neutrophil attractant/activation protein 1 (IL‐8/NAP‐1) by flow cytometry. The [Ca2+]i changes in different subpopulations within a heterogeneous cell suspension were resolved upon stimulation with fMLP. Using an anti‐CD16 phycoerythrin‐conjugated antibody and fluo‐3 simultaneously, neutrophils affected and nonaffected in Ca2+ mobilization were distinguished in two patients suffering from glycogen storage disease type lb. In normal neutrophils, a different time course of Ca2+ mobilization of neutrophil subpopulations immediately after stimulation with fMLP was detected. In addition, after stimulation with a low concentration of IL‐8/NAP‐1 (10‐10 M) two subsets of neutrophils appeared; one of them showed an increase in [Ca2+]i, while the other did not. These results indicate heterogeneity in the neutrophil signal transduction process involved in Ca2+ mobilization. Therefore, flow cytometric analyses can resolve changes in single‐cell [Ca2+]i distribution patterns, which is important for the understanding of [Ca2+]i in neutrophil heterogeneous activation processes.

In vitro functions of neutrophils induced by treatment with rhG‐CSF in severe congenital neutropenia

Neutrophils (and monocytes) from 5 patients suffering from severe congenital neutropenia (SCN) were investigated in vitro after induction of this cell type by treatment with recombinant human granulocyte colony‐stimulating factor (rh G‐CSF) in vivo. Some abnormal morphological features were seen, such as anomalies of nuclei of phagocytes. No limitations were found 1) in the ability of neutrophils and monocytes to produce reactive oxygen intermediates (ROI) following stimulation with phorbolmyristate‐acetate (PMA), 2) in the ability of neutrophils to phagocytose particles of zymosan A and to produce ROI simultaneously and 3) in the ability to kill bacteria of the species staphylococcus aureus. However the specific migration of neutrophils in a gradient of FMLP under soft agar was decreased to approximately 50% in all patients tested as compared to healthy controls. In addition, spontaneous motility was decreased in one patient. Nevertheless, the good clinical improvements of patients suffering from SCN after treatment with rh G‐CSF appeared to be due to induction of neutrophils displaying overall good functional activities with respect to natural defense.

Macrophage function in murine allogeneic bone marrow radiation chimeras in the early phase after transplantation

We tested several of the functions of macrophages (Mφ) in the early phase after allogeneic bone marrow transfer to get information about this important aspect of the nonspecific immune system in the T‐cell‐deficient recipient. On days 3‐5 after transfer, the number of Mφ was reduced in the spleen, liver, lungs, and peritoneal cavity (Pe). The phagocytosis of sheep red blood cells (SRBC) by these Mφ was normal or even enhanced, as in the case of Pe‐Mφ. Already on days 8‐12 after transfer, the number of Mφ in spleen and liver exceeded that of controls, whereas the number was still reduced in lungs and Pe. We examined their ability to kill P815 tumor cells, to produce tumor necrosis factor‐α (TNFα), to phagocytose SRBC, to produce reactive oxygen intermediates (ROI) in vitro and to kill Listeria monocytogenes in vivo. Most functions were normal and often even enhanced, depending on the organ origin, but the ability of Pe‐Mφ to produce ROI was reduced. Proliferative response to macrophage colony‐stimulating factor (M‐CSF) and killing of YAC‐1 tumor cells revealed a high frequency of macrophage precursor cells in the spleen and liver and a high natural killer (NK) activity in the liver. Altogether, enhanced nonspecific immune function, especially preactivated Mφ, may enable chimeras to survive attacks by opportunistic pathogens.

Changes in light-scatter profile, membrane depolarization and calcium mobilization of neutrophils induced by G-CSF in vivo.

Summary. To extend our studies about phenotypical and functional alterations of G‐CSF‐induced neutrophils we have evaluated their light‐scatter profile, mobilization of intracellular calcium ([Ca2+]i) and membrane depolarization after stimulation. A significant increase in the forward scatter signals could be demonstrated in such neutrophils from patients with neutropenias of various origin and from healthy test subjects. This increase began 4 h and returned to normal 96 h after G‐CSF injection in the latter group. We found an impairment of [Ca2+]i mobilization in neutrophils from patients with glycogen storage disease type IB after stimulation of these cells with fMLP, It was even more pronounced than in severe congenital neutropenia (SCN). However, [Ca2+]i fluxes were normal when ionomycin was used. Neutrophils from patients with cyclic neutropenia (cyNP) and chemotherapy‐induced neutropenia (chNP) mobilized [Ca2+]i similar to those from healthy donors. Furthermore, we found a decreased percentage of neutrophils depolarizing after stimulation with fMLP and PMA in patients with SCN, whereas membrane depolarization was normal in patients with chNP and cyNP. All the alterations found here are suggested to be caused by a partial immaturity of the neutrophils, although in vivo activation and a direct effect of G‐CSF on myeloid precursors might be involved.