In vitro and in vivo effects of pentoxifylline on macrophages and lymphocytes derived from autoimmune MRL-lpr/lpr mice.

MRL-lpr/lpr mice develop an autoimmune disease similar to human systemic lupus erythematosus (SLE). The main characteristics of this disease are increasing autoantibody formation, elevated plasma levels of immune complexes, a massive lymphoproliferation, a rising proteinuria, and arthritic symptoms. Finally, the mice die at an age of about 6 months due to a fatal immune complex glomerulonephritis. Macrophages are involved in the development of SLE due to their functions as antigen-presenting as well as cytokine-producing cells. T and B cells are involved in the disease by secreting cytokines and producing antibodies. Pentoxifylline (PTX), a xanthine derivative, is known to exert different effects on functions of leukocytes and erythrocytes and has been used in clinical studies, e.g., in septic shock syndrome. In our studies we first investigated the in vitro effect of PTX on macrophages and lymphocytes derived from MRL-lpr mice. Our investigations concerning production of superoxide anion and TNF-alpha by LPS and/or IFN-gamma activated bone marrow and peritoneal macrophages, MHC class II expression on these cells, and the proliferative capacity and Il-2 production of mitogen activated lymphocytes, revealed that PTX reduces the activation and the inflammatory response of these cells. Based on these results, we further investigated the effect of in vivo treatment with PTX. MRL-lpr mice treated with PTX showed diminished proteinuria, reduced titer of dsDNA-autoantibodies in the plasma and an increased survival rate. Our data clearly demonstrate that PTX is able to diminish the severity of the disease and to prolong the life of MRL-lpr/lpr mice.

Examination of the mononuclear phagocyte system in lupus-prone male BXSB mice.

During the course of lupus-like autoimmune disease male BXSB mice develop an increasing monocytosis in the peripheral blood. As we demonstrated previously, this monocytosis is parallelled by an expansion of a strain-specific high number of macrophage precursor cells (CFU-M) in the bone marrow of male mice. To test the hypothesis that the expanded mononuclear phagocyte system (MPS) may promote autoimmune disease in these mice the organ-associated macrophage system was examined. Our latest data show that an unusual expansion of CFU-M also appears in spleen and liver of male mice 2 weeks after birth. In addition to a morphological alteration of the organs during the course of the disease there is a change in number and distribution of organ-resident macrophages. Considering these results the possible contribution of the expanded MPS in promoting autoimmune disease is discussed.

Expansion of the liver-associated macrophage system in systemic lupus erythematosus-prone NZB/W mice.

Systemic lupus erythematosus is characterized by profound changes of the immune system. We report on alterations of the macrophage system in the murine NZB/W model of this disease. A greatly increased number of mature macrophages was isolated from the liver of NZB/W mice as compared to BALB/c mice and several other inbred strains used as healthy controls. In addition, the macrophage precursor compartment in the liver of NZB/W mice was expanded severalfold as measured by proliferation of light-fraction nonadherent nonparenchymal cells (NPCs) in response to colony-stimulating factors. Functional properties of the macrophages isolated from various anatomic sites of the lupus-prone mice were tested. Production of monokines by macrophages from liver, spleen, and peritoneal cavity, calculated on a per cell basis, was in the same range as in several healthy control strains tested. Yet the overall production of these immunoregulatory molecules by the increased liver macrophage system, the body's largest compartment of macrophages, is likely to result in increased levels of circulating monokines in the plasma of lupus-prone NZB/W mice. Indeed, significantly elevated levels of interleukin-6, interleukin-1, and colony-stimulating activity could be demonstrated in the plasma of these mice both spontaneously and after stimulation with lipopolysaccharide. A possible contribution of the expansion of the macrophage system to the development of the disease is discussed.

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 wave-lengths 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 1b. 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.

Effects of macrophage-CSF on pulmonary-macrophage repopulation after bone marrow transplantation.

Pulmonary infections are important causes of morbidity and mortality in immunosuppressed patients after transplantation. After experimental irradiation and syngeneic bone marrow transplantation in mice, macrophages show reduced repopulation in the lung compared with that in other tissues. Macrophages are major microbicidal immune effector cells in host pulmonary defense. Therefore, we examined the role of locally applied cytokines for macrophage repopulation in the lung. An accelerated repopulation of macrophages in the lung was observed after intranasal application of macrophage-colony stimulating factor (M-CSF), but this effect was not enhanced by a combination of M-CSF with interleukin (IL)-3. Local proliferation contributed to this effect. Macrophages in the lung tissue of M-CSF-treated mice displayed greater secretion of IL-6, whereas M-CSF treatment did not enhance the gene expression of other macrophage-specific chemokines. The role of M-CSF treatment was determined in pulmonary murine cytomegalovirus infection using an irradiation/reconstitution model. The M-CSF treatment had no effect on virus load in the lung tissue. However, phosphate-buffered saline-treated mice seemed to develop stronger inflammation after viral infection than M-CSF-treated mice. We conclude that local M-CSF treatment modulates cellular inflammation in the lung during immunosuppression.

Macrophage precursor cells produce perforin and perform Yac-1 lytic activity in response to stimulation with interleukin-2.

Macrophage precursor cells, derived from mouse bone marrow culture with granulocyte-macrophage colony-stimulating factor or colony-stimulating factor 1 (CSF-1) as growth factor and interleukin-2 (IL-2) as stimulating factor, were activated by IL-2 to exert strong cytolytic activity against Yac-1 cells. In response to IL-2 stimulation these bone marrow macrophage precursor cells produced perforin as lytic molecules. The purity of the precursor cells for the study was proved as homogeneous positivity for Mac-1, NK-1.1 and negativity for Lyt 1 and 2. The cells express CSF-1 receptors on their surface, are able to proliferate and differentiate into typical macrophages when stimulated with CSF-1, and are therefore members of the macrophage lineage. Perforin transcripts were identified by Northern blot analysis of IL-2-treated macrophage precursor cells, and the presence of perforin protein in the cytoplasmic granules was demonstrated by immunohistochemical staining using a monoclonal antiperforin antibody. In addition, the biological activity of the perforin contained in the macrophage precursor's granules could be documented as calcium-dependent lytic activity using Yac-1 and sheep red blood cells as targets. The results presented in this paper imply the existence of a bipotent precursor cell, which can mature into a typical macrophage if CSF-1 or phorbol 12-myristate 13-acetate is supplied as differentiation stimulating factor but develops into an NK/LAK cell when early activation with IL-2 is provided.

Analysis of leukocyte activation during acute rejection of pulmonary allografts in noninfected and cytomegalovirus-infected rats.

After human lung transplantation acute rejection and cytomegalovirus (CMV) infections may occur, probably contributing to the development of chronic rejection. We established a model of subacute allograft rejection in rats to analyze leukocyte activation and effects of a CMV infection. Histoincompatible lung transplants (BN/LEW) without immunosuppression (group A) and lungs of initially immunosuppressed animals (group B) were analyzed. The production of inflammatory mediators (interleukin-6, tumor necrosis factor alpha, nitric oxides) and the expression of MHC class II antigens by alveolar and lung tissue macrophages were significantly enhanced during the alloresponse. In recipients without immunosuppression (group A) allograft necrosis was detected by day 6, whereas group B allografts were fully rejected by day 25. In allografts of immunosuppressed, CMV-infected animals (group C) the CMV infection was clearly aggravated and the number of activated lung tissue macrophages was increased when compared with noninfected allografts or isografts. The subacute model provides the advantage of allowing us to study mechanisms of acute rejection without the effects of reperfusion injury. Furthermore these findings underline the role of inflammatory mediators produced by macrophages during rejection.

Abnormal regulation in the signal transduction in neutrophils from patients with severe congenital neutropenia: relation of impaired mobilization of cytosolic free calcium to altered chemotaxis, superoxide anion generation and F-actin content.

Severe congenital neutropenia (SCN) can be corrected in vivo by treatment with pharmacological dosages of recombinant human granulocyte colony-stimulating factor (rhG-CSF). In order to analyze the decreased chemotaxis of neutrophils from SCN patients receiving rhG-CSF, neutrophil functions essential for chemotaxis were investigated. The mobilization of cytosolic calcium ([Ca2+]i) and the functional state of cytoskeletal proteins in neutrophils from SCN patients were compared with either neutrophils from healthy donors (or, in selected experiments, from patients with cyclic neutropenia) and neutrophils from patients with chemotherapy-induced neutropenia also receiving rhG-CSF. Using flow cytometric analysis, two neutrophil subpopulations were detected in SCN patients in response to N-formylmethionine leucyl-phenylalanine (FMLP) (10(-9) M to 10(-7) M), one of which was unable to respond to this stimulus with an increase in [Ca2+]i. Whereas a homogeneous increase in [Ca2+]i in normal neutrophils occurred at 10(-9) M FMLP, neutrophils from SCN patients required 10(-6) M FMLP to respond homogeneously with an increase in [Ca2+]i. In contrast, G-CSF induced neutrophils from patients with cyclic neutropenia and from patients with chemotherapy-induced neutropenia showed a normal increase in [Ca2+]i after stimulation. The [Ca2+]i-dependent superoxide anion (O2-) generation in response to FMLP was also significantly diminished in neutrophils from SCN patients compared to normal neutrophils. However, O2- generation elicited by phorbolester (PMA), which directly activates protein kinase C (PKC), was not affected in SCN neutrophils. The total immunoreactive actin content and basal F-actin content in neutrophils from SCN patients were elevated as compared to normal neutrophils and neutrophils from patients with chemotherapy-induced neutropenia. The increase in F-actin content following FMLP activation was much lower in neutrophils from SCN patients as compared with normal neutrophils. These data suggest a defect in the signal transduction pathway in neutrophils from SCN patients between FMLP ligand-receptor interaction and Ca2+ mobilization, whereas upstream of PKC, triggered events seem to be unaffected. Therefore, [Ca2+]i-dependent neutrophil function in response to FMLP, such as actin disassembly, chemotaxis and O2- generation are diminished in SCN neutrophils. The pathomechanism responsible for the defective [Ca2+]i increase might be an initial step in understanding the underlying pathophysiology of SCN.

Effects of bone marrow fibroblasts on the proliferation and differentiation of myeloid leukemic cell lines.

The effects of normal bone marrow fibroblasts (BM FB) on proliferation and differentiation of 10 myeloid leukemic cell lines were investigated in a serum-free co-culture system. The proliferation of three of the cell lines was supported by BM FB. Three of the myeloid cell lines were inhibited 40-70%. The co-culture supernatants were tested for the secretion of hematopoietic cytokines by bioassays. Except for IL-6, which was already produced constitutively by BM FB, only little amounts of interleukin-1 (IL-1), granulocyte colony-stimulating factor (G-CSF), or granulocyte-macrophage colony-stimulating factor (GM-CSF) could be detected in several co-culture supernatants. It could be shown that, according to cytologic and functional criteria, the myeloid leukemic cell lines ML-2 and PLB-985 differentiate along the monocyte-macrophage pathway after co-culture with BM FB. They revealed a histiocytic phenotype and could be induced to produce reactive oxygen intermediates (ROI) after stimulation with zymosan or phorbol-myristate-acetate (PMA). Additional proof for differentiation was obtained from flow cytometric analysis of surface differentiation antigens and adhesion molecules. The neutralization of IL-6 activity in the co-cultures by antibodies resulted in prevention of differentiation of PLB-985 cells, while differentiation of ML-2 cells in the co-cultures was not affected by addition of anti-IL-6 antibodies. Furthermore, in co-culture experiments with fibroblasts from skin and foreskin, we found a differentiation of PLB-985 cells comparable to that in co-cultures with BM FB, but poor differentiation of ML-2 cells. These data suggest that different mechanisms are involved in the differentiation of ML-2 and PLB-985 cells.

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).

Susceptibility of 3H-thymidine-prelabelled Leishmania donovani amastigotes and promastigotes to the cytotoxic activity of peritoneal, splenic and liver macrophages.

C57BL/6 macrophage populations from spleen and liver, the main organs for the manifestation of visceral leishmaniasis, were investigated for their ability to perform spontaneous phagocytosis-associated killing of 3H-thymidine (3H-TdR)-prelabelled L. donovani amastigotes and promastigotes. The results showed that organ macrophages from spleen and liver killed L. donovani amastigotes and promastigotes spontaneously with high efficiency. This consistent finding was first detectable at 2-3 h, and the reaction was completed at 12 h. This type of killing was strongly enhanced when spleen and liver macrophages were activated. This phagocytosis-associated killing mechanism may contribute, to a large extent, in maintaining the infection under control in vivo, by drastically reducing the amount of parasites that is required to establish intracellular parasitism. To be able to assay phagocytosis-associated destruction of both promastigotes and amastigotes, a reproducible system for the production in vitro of Leishmania donovani amastigotes by the macrophage cell-line J774 was developed. The DNA of the Leishmania amastigotes was labelled with 3H-TdR with high efficiency. The spontaneous label release of prelabelled L. donovani amastigotes was comparable to that of prelabelled promastigotes over an assay period of 24 h.

M-CSF transgenic mice: role of M-CSF in infection and autoimmunity.

In this study, transgenic CD2F1 mouse lines (C-1.1-C-1.11) bearing a transgene encoding the murine growth factor M-CSF under the control of the liver specific alpha-1-antitrypsin gene promoter were generated. Transgenic C-1.4 mice showed elevated expression of transgene-encoded M-CSF in the liver and displayed a 2-3-fold increase of M-CSF plasma levels and of macrophage numbers in the liver as compared with non-transgenic littermates. M-CSF transgenic mice showed increased resistance against sublethal i.v. infections with Listeria monocytogenes as compared with infected non-transgenic mice. To investigate the influence of M-CSF in murine systemic lupus erythematosus (SLE), the M-CSF transgenic mouse line C-1.4 was bred into the genetic background of SLE-prone MRL+/+ mice. The resulting C-1.4/MRL transgenic mice bearing increased endogenous M-CSF levels showed consistently lower levels of anti-ss-DNA autoantibodies as compared with non-transgenic MRL+/+ mice. The life span of the C- 1.4/MRL transgenic mice and the severity of the disease in these mice remained unchanged as compared with their non-transgenic littermates. It is concluded that in addition to M-CSF further factors must be involved in the acceleration of the autoimmune disease in SLE prone MRL/lpr mice.

Influence of ibuprofen on the infection with Listeria monocytogenes.

Listeria monocytogenes is a bacterial infection, which is facultatively localized in monocytes and macrophages. The influence of ibuprofen (CAS 15687-27-1), a nonsteroidal anti-inflammatory drug (NSAID), on this bacterial infection in balb/c mice was investigated. One day prior to sublethal infection, balb/c mice were treated intravenously with various therapeutic concentrations of ibuprofen alone or ibuprofen in combination with a suboptimal dosage of murine recombinant interferon gamma, a lymphokine produced by T-helper cells. Three days post-infection, parasite burdens of the mainly infected organs, spleen and liver, were determined by the colony-forming unit assay. It was shown that the prophylactic treatment with ibuprofen in a concentration of 4 mg/kg body weight resulted in a more than 10-fold reduction of viable Listeria monocytogenes in the spleen, whereas in liver 12 mg/kg Ibuprofen was necessary for a comparable kill of viable bacteria. A higher concentration of ibuprofen did not resulted in a higher antibacterial efficacy. In order to clarify the mechanism of ibuprofen action, molecular-biological experiments were performed to measure the messenger RNA (mRNA) induced by ibuprofen. It is presented here that therapeutic concentrations of ibuprofen induced significant higher amounts of mRNA for interleukin-1 in human monocytes compared to untreated cells. These findings support the hypothesis that ibuprofen influences the complex immune system to overcome a bacterial infection.

Kinetics of transfused neutrophils in peripheral blood and BAL fluid of a patient with variant X-linked chronic granulomatous disease.

Patients with chronic granulomatous disease (CGD), an uncommon inherited disorder of phagocytes resulting in the defective production of reactive oxygen intermediates, are prone to bacterial and fungal infections. In the case presented, therapeutic efforts including white cell transfusions, and amphotericin B and IFN gamma administration were undertaken to treat pneumonia caused by Aspergillus fumigatus. During a phase of artificial respiration, transfused white cells in peripheral blood and bronchoalveolar lavage fluid were monitored in order to examine their kinetics and functional activity. Using flowcytometrical methods, host-derived and transfused neutrophils could be distinguished by cytochrome b558 expression using the monoclonal antibody 7D5 for immunofluorescent staining as well as by production of reactive oxygen intermediates. Transfused PMN could be detected in both compartments and their kinetics could be followed up to 24 hours after transfusion. Using flowcytometry, even small numbers of transfused PMN could be measured during episodes of extreme leukocytosis. Since functionally intact transfused PMN were found in the bronchioalveolar lavage fluid, white cell transfusions in combination with antibiotic and immunomodulating therapy should be considered a part of the therapeutic regimens for life-threatening infections in CGD patients.

Expansion and high proliferative potential of the macrophage system throughout life time of lupus-prone NZB/W and MRL lpr/lpr mice. Lack of down-regulation of extramedullar macrophage proliferation in the postnatal period.

Systemic lupus erythematosus is an autoimmune disease, characterized by high titers of autoantibodies against many cell-membrane and intracellular antigens. Polyclonal B cell activation and alterations in the T cell compartment have been described. The present report deals with the organ-associated macrophage (M phi) system of two lupus-prone mouse strains (NZB/W and MRL lpr/lpr) and demonstrates that in both mouse strains the M phi compartment of liver and spleen is clearly expanded. In the liver the number of F 4/80+ M phi is strongly elevated. In addition, presence of early M phi precursors and of extramedullary organ-associated monocyte proliferation in response to colony-stimulating factor (CSF) is documented in liver and spleen of these mice. Further, in normal animals during the first two weeks of life extramedullar monocytopoiesis is present in liver and spleen, which is then down-regulated in the third week of life. In the two lupus-prone mouse strains down-regulation does not occur but extramedullar monocyte proliferation is sustained at high level throughout life time. As possible correlates for the expansion of the M phi system elevated CSF-1 mRNA levels are demonstrated in kidney, spleen and liver of NZB/W mice and elevated CSF serum levels are documented in MRL lpr/lpr mice. The possible contribution of the expanded M phi system to B and T cell dysregulation is discussed.

Evaluation of flow cytometric methods for diagnosis of chronic granulomatous disease variants under routine laboratory conditions.

Neutrophils from 50 pediatric patients with normal phagocyte functions, from 150 healthy adults, from 10 chronic granulomatous disease (CGD)-patients (4 CGD+), and from 18 X-linked carriers for CGD have been tested for their production of H2O2 using staining with dihydrorhodamine 123 and subsequent flow cytometry. Additionally, neutrophils from three patients with myeloperoxidase deficiency were assessed. Cells were activated to produce H2O2 by the phorbol ester phorbol-myristate-acetate (PMA) and by phagocytosis of Escherichia coli bacteria. To evaluate the sensitivity of the method, H2O2-production by neutrophils which was inhibited by different concentrations of diphenyljodonium (DPI) was measured. The results were compared to those from other methods (NBT-testing, cytochrome c-reduction, and especially chemiluminescence). Normal values and ranges of scatter profile were evaluated in terms of peak channel fluorescence: 97% > 700, x = 840 +/- 59 (S.D.), 97% < 890, for pediatric patients. Normal quantitative values also resulted from small blood samples of infants (< 1 year, n = 6, x = 830 +/- 52). For CGD+ (n = 4) the results were clearly far below the normal range. In indicating decreased production of reactive oxygen intermediates the method was at least as sensitive as lucigenin enhanced chemiluminescence. Cytochrome b558-expression of neutrophils from patients and healthy controls was established by flow cytometry following staining with the monoclonal antibody 7D5. The normal range was 97% > 485, 97% < 680, peak channel fluorescence. We conclude that flow cytometric routine diagnostics of CGD can easily enhance the reliability of recognition and the yield of information about this disease compared to conventional methods.

Production of oxygen radicals by fibroblasts and neutrophils from a patient with x-linked chronic granulomatous disease.

Recently, a superoxide-generating NADPH-oxidase system in human fibroblasts has been described. Therefore, we reassessed the possible use of this cell type for prenatal diagnosis of CGD patients comparing normal and CGD peripheral blood neutrophils (PMN) and skin fibroblasts in their reactive oxygen intermediate (ROI)-producing capacity. While PMN of the CGD patient showed a clearly reduced respiratory burst activity, which correlated well with the measured content of cytochrome b558, fibroblasts of the same individual showed no impaired production of superoxide anion or H2O2 upon stimulation by cytokines (TNF and IL-1) or other agents (Ca2+ ionophores and PAF, unpublished results). Furthermore, fibroblasts of the CGD patient or of normal donors could be inhibited in ROI production by diphenylene iodonium (DPI) and 2-iodobiphenyl. In contrast to PMN, no inhibition of the fibroblast NADPH-oxidase system was observed using staurosporin, an inhibitor of proteinkinase C. These data demonstrate, in contrast to previous studies, that fibroblasts are able to produce ROI. Nevertheless, since fibroblasts obtained from a CGD patient exhibited no difference in ROI production compared with fibroblasts obtained from healthy donors, they are not suitable for prenatal diagnosis of CGD.

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 x 10(3) 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.