Interleukin-10 has different effects on proliferation of Listeria monocytogenes in livers and spleens of mice.

The aim of this study was to investigate the effect of interleukin-10 (IL-10) on the course of Listeria monocytogenes infection in naive and immune mice. Treatment with IL-10 during the course of a primary infection significantly decreased the number of bacteria in the spleen and did not affect the number in the liver. During a secondary infection in immune mice treated with IL-10, the number of bacteria was significantly lower in the spleen but significantly higher in the liver in comparison to mock-treated immune mice. IL-10 treatment during a primary Listeria infection decreased the concentration of gamma interferon (IFN-gamma) in plasma and the toxoplasmastatic activity of macrophages, whereas it increased the percentage of mildly CD3-positive T cells in the spleen. During a secondary infection, the concentration of IFN-gamma in plasma was decreased on day 1 but remained unaffected during later days of infection. From these results, we conclude that IL-10 has different effects on the proliferation of L. monocytogenes in the spleen and liver during primary and secondary Listeria infections.

Expression of zeta molecules is decreased in NK cells from HIV-infected patients.

Cytolysis by natural killer (NK) cells is impaired in HIV infection. We investigated whether the expression of zeta (zeta) molecules, essential elements of signalling initiated upon ligation of, e.g., CD16, is reduced and if so, whether this reduction could be involved in defective cytolysis. FACS analysis revealed significantly lower levels of zeta in NK cells from AIDS patients compared to cells from patients without AIDS and healthy controls. CD16-dependent cytolysis by NK cells correlated with expression of zeta molecules and CD16, the latter possibly related to zeta expression. No correlation was observed between CD16-independent cytolysis and zeta expression. Reduced expression of zeta molecules by NK cells from HIV-infected patients thus correlates with disease progression and may, in part, explain the defective cytolysis by these cells.

Different effect of granulocyte colony-stimulating factor or bacterial infection on bone-marrow cells of cyclophosphamide-treated or irradiated mice.

In the present study, the effect of treatment with granulocyte colony-stimulating factor (G-CSF) on cellular composition of the bone marrow and the number of circulating leucocytes of granulocytopenic mice, whether or not infected with Staphylococcus aureus, was assessed. With two monoclonal antibodies, six morphologically distinct cell populations in the bone marrow could be characterised and quantitated by two-dimensional flow cytometry. Granulocytopenia was induced by cyclophosphamide or sublethal irradiation. Cyclophosphamide predominantly affected the later stages of dividing cells in the bone marrow resulting in a decrease in number of granulocytic cells, monocytic cells, lymphoid cells and myeloid blasts. G-CSF administration to cyclophosphamide-treated mice increased the number of early blasts, myeloid blasts and granulocytic cells in the bone marrow, which indicates that this growth factor stimulates the proliferation of these cells in the bone marrow. During infection in cyclophosphamide-treated mice the number of myeloid blasts increased. However, when an infection was induced in cyclophosphamide and G-CSF-treated mice, the proliferation of bone-marrow cells was not changed compared to that in noninfected similarly treated mice. Sublethal irradiation affected all bone-marrow cell populations, including the early blasts. G-CSF-treatment of irradiated mice increased only the number of myeloid blasts slightly, whereas an infection in irradiated mice, whether or not treated with G-CSF, did not affect the number of bone-marrow cells. Together, these studies demonstrated that irradiation affects the early blasts and myeloid blasts in the bone marrow more severely than treatment with cyclophosphamide. Irradiation probably depletes the bone marrow from G-CSF-responsive cells, while cyclophosphamide spared G-CSF responsive cells, thus enabling the enhanced G-CSF-mediated recovery after cyclophosphamide treatment. Only in these mice, bone marrow recovery is followed by a strong mobilisation of mature granulocytes and their band forms from the bone marrow into the circulation during a bacterial infection.

Decreased expression of zeta molecules by T lymphocytes is correlated with disease progression in human immunodeficiency virus-infected persons.

In human immunodeficiency virus type 1 (HIV-1) infection, functional activities of T lymphocytes are impaired. Analogous to tumor-infiltrating T lymphocytes from cancer patients, in whom poor proliferative responses are associated with fewer zeta molecules, this study compared expression of CD3zeta molecules by T lymphocytes from HIV-infected persons and healthy controls. Flow cytometry and immunoblotting revealed significantly diminished zeta expression by CD3, CD4, and CD8 T lymphocytes from AIDS patients but not from persons without AIDS. zeta-mRNA levels were also decreased in cells from AIDS patients. CD3zeta expression correlated significantly with CD4 cell counts and HIV-1 RNA levels; impaired expression of CD3zeta molecules appeared to be reversible upon virus load reduction following highly active antiretroviral treatment (HAART). Thus, reduced expression of CD3zeta molecules by T lymphocytes from HIV-infected persons correlates with disease status. Investigations into CD3zeta expression by subpopulations of peripheral T lymphocytes and by T lymphocytes in lymphoid tissues will contribute to the understanding of immune reconstitution of HIV-infected patients following HAART.

Arachidonic acid, but not its metabolites, is essential for FcgammaR-stimulated intracellular killing of Staphylococcus aureus by human monocytes.

Since arachidonic acid (AA) production by phospholipase A2 (PLA2) is essential for the Fcgamma receptor (FcgammaR)-mediated respiratory burst and phagocytosis of opsonized erythrocytes by monocytes and macrophages, we focused in this study on the role of AA and its metabolites in the FcgammaR-stimulated intracellular killing of Staphylococcus aureus by human monocytes. The results revealed that the PLA2 inhibitors, but not inhibitors of cyclo-oxygenase and lipoxygenase, markedly suppressed the FcgammaR-mediated killing process. The production of O-2 by monocytes upon FcgammaR cross-linking was inhibited by 4-bromophenacyl bromide in a dose-dependent fashion, indicating that inhibition of PLA2 activity impairs the oxygen-dependent bactericidal mechanisms of monocytes, which could be partially restored by addition of exogenous AA and docosahexaenoic acid, but not myristic acid. These polyunsaturated fatty acids, but not myristic acid, stimulated the intracellular killing of S. aureus by monocytes, although not as effectively as FcgammaR cross-linking. Furthermore, FcgammaR cross-linking stimulated the release of AA from monocytes. Studies with selective inhibitors revealed that the FcgammaR-mediated activation of PLA2 is dependent on Ca2+ and tyrosine kinase activity. Together these results indicate a key role for PLA2/AA, but not its major metabolites, in mediating the FcgammaR-stimulated intracellular killing of S. aureus by monocytes.

Monocytes incubated with surfactant: a model for human alveolar macrophages?

Monocytes migrate to the lungs and enter the alveoli where they come into contact with surfactant and differentiate into alveolar macrophages. This study focused on the question of the extent to which monocytes and monocyte-derived macrophages (MDM) incubated with surfactant resemble alveolar macrophages. Surfactant-incubated monocytes shared with alveolar macrophages the intracellular presence of surfactant, efficient phagocytosis of opsonized Staphylococcus aureus, and poor intracellular killing of ingested bacteria. The suppressive effect of surfactant on bactericidal activities of monocytes could not be attributed to either the surfactant lipid fraction or surfactant protein A. Monocytes incubated with surfactant differed from alveolar macrophages with respect to expression of various Fc and complement receptors involved in intracellular killing of bacteria. Surfactant-incubated monocytes produced significantly more H2O2 upon stimulation with phorbol ester than alveolar macrophages, but significantly less than control monocytes. Together, monocytes and MDM incubated with surfactant, although similar to alveolar macrophages in some aspects, are not an adequate model for alveolar macrophages. Most likely, factors other than surfactant in the microenvironment of the alveoli, such as oxygen tension, play a role in the differentiation of monocytes to alveolar macrophages as well.

Defective TCR-mediated signaling in synovial T cells in rheumatoid arthritis.

In rheumatoid arthritis (RA), the functional status of T cells is incompletely understood. Synovial T cells display phenotypic evidence of former activation, but there is poor production of T cell-derived cytokines in the synovium. In addition, synovial T cell proliferation upon mitogenic and antigenic stimulation was decreased compared with that in peripheral blood T cells. Moreover, previous reports revealed that early Ca2+ rises induced by TCR/CD3 stimulation were decreased in RA T cells compared with those in healthy controls. To investigate the molecular mechanisms of RA synovial T cell hyporesponsiveness, we analyzed the TCR/CD3-mediated protein tyrosine phosphorylation in RA peripheral blood and synovial fluid (SF) T cells. SF T cells exhibited a decreased overall tyrosine phosphorylation pattern upon stimulation. Most notably, the induction of phosphorylation of p38 was virtually absent. Moreover, we found that tyrosine phosphorylation of the TCR zeta-chain, one of the most proximal events in TCR signaling, is clearly diminished in RA SF T cells. The decrease in tyrosine phosphorylation was accompanied by a decrease in detectable levels of zeta-protein within synovial T cells. These results suggest that a defective TCR signaling underlies the hyporesponsiveness of synovial T cells in RA.

Phagocytosis and intracellular killing of serum-opsonized Staphylococcus aureus by mouse fibroblasts expressing human Fcgamma receptor type IIa (CD32).

Phagocytes bear more than one class of receptors for the Fc domain of IgG (FcgammaR). In addition the same ligand can interact with different classes of FcgammaR. This complexity makes it difficult to study the contribution of the various classes of FcgammaR to antimicrobial functions. To circumvent this difficulty, in the present study mouse 3T6 fibroblasts transfected with cDNA encoding for human FcgammaR type IIa (FcgammaRIIa-expressing cells) were used to determine the role of this receptor in phagocytosis and intracellular killing of serum-opsonized Staphylococcus aureus. Experiments using microbiological and fluorescent techniques to discriminate between cell-adherent and intracellular bacteria revealed that serum-opsonized bacteria are phagocytized by FcgammaRIIa-expressing cells, but not by parental fibroblasts. Non-opsonized bacteria were poorly internalized by FcgammaRIIa-expressing as well as parental fibroblasts. Furthermore, incubation of FcgammaRIIa-expressing cells with opsonized bacteria at 4oC and incubation of FcgammaRIIa-expressing cells with cytochalasin E prior to addition of opsonized bacteria inhibited the phagocytosis of these bacteria almost completely. Phagocytosis of opsonized bacteria by FcgammaRIIa-expressing cells was partly inhibited by selective inhibition of protein tyrosine kinases (PTK). FcgammaRIIa cross-linking initiated transient tyrosine phosphorylation of various proteins in FcgammaRIIa-expressing cells. These data indicate that activation of PTK is involved in the FcgammaRIIa-mediated phagocytosis of opsonized S. aureus by transfected fibroblasts. Human serum from normal individuals and agammaglobulinemic patients triggered the intracellular killing of S. aureus by FcgammaRIIa-expressing fibroblasts. Surprisingly, heat-inactivated human serum, IgG and incubation with anti-FcgammaRII antibodies followed by a bridging secondary antibody did not stimulate the killing process. The possibility that these ligands did not interact with FcgammaRIIa on the cells can be excluded since they induced tyrosine phosphorylation of cellular proteins. The serum factor that stimulates the intracellular killing of bacteria by FcgammaRIIa-expressing cells is not yet identified. Oxygen-independent mechanisms are thought to be responsible for the killing of intracellular bacteria by these cells since the NADPH oxidase inhibitor diphenylene iodonium did not affect the serum-stimulated intracellular killing of S. aureus and no reactive oxygen and nitrogen intermediates were produced by FcgammaRIIa-expressing cells after appropiate stimulation. Taken together, these data show that phagocytosis but not intracellular killing of S. aureus is mediated via FcgammaRIIa on cells expressing this receptor.

Binding of surfactant protein A to C1q receptors mediates phagocytosis of Staphylococcus aureus by monocytes.

During both steady-state conditions and inflammatory reactions in the lower airways, monocytes migrate to the alveoli where they come into contact with surfactant. Surfactant is composed of phospholipids, neutral lipids, and specific proteins, and its main function is to reduce surface tension in the alveoli. The most abundant glycoprotein surfactant protein A (SP-A) affects the structure, function, and metabolism of pulmonary surfactant. The aim of the present study was to determine whether SP-A plays a role in the antibacterial activities of human monocytes and whether this is mediated by a receptor for SP-A on these cells. The results showed that SP-A binds to both Staphylococcus aureus and monocytes and mediates the phagocytosis of the bacteria by these cells. SP-A does not stimulate the intracellular killing of bacteria by monocytes, and SP-A-opsonized S. aureus do not induce the production of reactive oxygen intermediates. SP-A binds to the C1q receptor (C1qR) on monocytes, since its binding was inhibited by C1q and the SP-A-enhanced association of S. aureus with these cells was completely abolished when monocytes were adherent to surfaces coated with C1q or anti-C1qR monoclonal antibody. Furthermore, the binding of SP-A to monocytes results in an increased intracellular concentration of adenosine 3',5'-cyclic monophosphate. Together, these results demonstrate that C1qR mediates the phagocytosis of SP-A-opsonized S. aureus by monocytes.

Pulmonary surfactant inhibits monocyte bactericidal functions by altering activation of protein kinase A and C.

Pulmonary surfactant, the main function of which is to reduce surface tension in the alveoli, is also known to affect the functions of monocytes. Two protein kinases play a role in the regulation of the bactericidal functions of phagocytes, i.e. cAMP-dependent protein kinase A (PKA), which is involved in inhibition, and Ca2+/phospholipid-dependent PKC, which is involved in stimulation of these functions. In the present study we investigated whether altered activation of PKA and/or PKC plays a role in the surfactant-induced inhibition of both the intracellular killing of Staphylococcus aureus and the production of reactive oxygen intermediates (ROI) by monocytes. The significance of increased activation of PKA was demonstrated by the following findings. Firstly, surfactant induced a sustained increase in the intracellular cAMP concentration in monocytes. Secondly, dibutyryl-cAMP (db-cAMP), a membrane-permeable cAMP analogue, mimicked the inhibitory effects of surfactant on both the killing capacity and the production of ROI by monocytes. Thirdly, an inhibitor of PKA partially restored the impaired bactericidal functions of monocytes incubated with surfactant. The involvement of decreased activation of PKC in the impaired bactericidal functions of monocytes incubated with surfactant was evident from two findings. Firstly, surfactant attenuated the phorbol myristate acetate (PMA)-mediated translocation of PKC. Secondly, surfactant inhibited the production of O2- by monocytes upon stimulation with PMA. Therefore, the mechanism involved in the surfactant-induced inhibition of the bactericidal functions of monocytes comprises both activation of an inhibitory pathway, which includes cAMP and PKA, and inactivation of a stimulatory pathway, in which PKC is involved.

Pulmonary surfactant inhibits activation of human monocytes by recombinant interferon-gamma.

During an inflammatory reaction in the alveoli, the functional activities of monocytes, macrophages and granulocytes are regulated by a complex network of inflammatory mediators. The primary cytokine involved in activation of these phagocytes is interferon-gamma (IFN-gamma). The possible influence of local factors, such as pulmonary surfactant, on the activation process has not been studied until now. The aim of the present study was to investigate the effects of surfactant on the activation of monocytes by recombinant (r)IFN-gamma. The results revealed that human surfactant significantly inhibited both the increase in the expression of the high-affinity receptor for IgG, i.e. Fc gamma RI, and the production of H2O2 by rIFN-gamma-activated monocytes. Since our surfactant preparation stimulated the basal production of tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) by monocytes, the effect of Survanta, a surfactant extract, on the rIFN-gamma-induced production of these cytokines by monocytes was studied. The results revealed that Survanta caused 80-90% inhibition of the rIFN-gamma-induced production of TNF-alpha and IL-1 beta by these cells. Together, these results could mean that surfactant is involved in the protection of the alveolar epithelium against injury caused by reactive oxygen intermediates (ROI) and TNF-alpha, and in the down-regulation of the production of inflammatory mediators. In view of these considerations, surfactant therapy may not only improve lung compliance and gas exchange but may also be beneficial in reducing the inflammatory reaction in the lungs.

Lung surfactant suppresses oxygen-dependent bactericidal functions of human blood monocytes by inhibiting the assembly of the NADPH oxidase.

Surfactant is known to lower the surface tension in alveoli and affects the antibacterial functions of alveolar and peritoneal macrophages. We investigated the effects of surfactant on the bactericidal functions and oxidative metabolism of human blood monocytes and granulocytes. Monocytes incubated with surfactant ingest this material and subsequently exhibit an impaired ability to kill ingested bacteria. Granulocytes incubated with surfactant do not ingest this material, and their bactericidal functions are not affected. However, granulocytes that have ingested surfactant-coated Staphylococcus aureus display an impaired ability to kill these bacteria. Moreover, in monocytes and granulocytes that contain surfactant--the latter by ingestion of surfactant-coated S. aureus--the intracellular production of H2O2 is impaired due to inhibition of the assembly of the NADPH oxidase. Together these results demonstrate that surfactant inside monocytes and granulocytes inhibits the capacity of these cells to kill bacteria intracellularly by impairing oxygen-dependent killing mechanisms.

Interferon-gamma-activated human granulocytes kill ingested Mycobacterium fortuitum more efficiently than normal granulocytes.

Although shortly after the onset of a mycobacterial infection granulocytes are present at the site of inflammation, the role of granulocytes in the elimination of mycobacteria is not well understood. In vitro studies with, for example Mycobacterium tuberculosis or M. bovis, are hampered by the slow proliferation and clumping of the bacteria. To avoid these disadvantages, we developed a model using the atypical mycobacterium M. fortuitum. The present study concerned two questions: whether human granulocytes are able to phagocytose and intracellularly kill opsonized M. fortuitum and whether intracellular killing of these bacteria can be enhanced by treatment of the granulocytes with recombinant human interferon-gamma (rIFN-gamma). The results showed that normal granulocytes phagocytosed opsonized M. fortuitum rapidly, but did not kill these bacteria effectively. The intracellular killing of M. fortuitum was significantly enhanced by incubation of the granulocytes with rIFN-gamma for 18 h before the start of the killing assay. Since these rIFN-gamma-pretreated granulocytes did not release more O2- and H2O2 upon stimulation with phorbol 12-myristate 13-acetate or opsonized M. fortuitum than control granulocytes, non-oxidative killing mechanisms are probably involved in the enhanced killing of M. fortuitum.

Plasma disposition and renal clearance of sulphadimidine and its metabolites in laying hens.

Plasma disposition of sulphadimidine (SDM) and its metabolites was studied in laying hens after 100 mg SDM kg-1 doses were administered as a single intravenous dose, a single oral dose and multiple oral doses once daily for five consecutive days. SDM was extensively metabolised by acetylation and hydroxylation. In plasma, the metabolite observed with the highest concentration was N4-acetylsulphadimidine (N4-SDM) followed by hydroxymethylsulphadimidine (CH2OH) and 5-hydroxysulphadimidine. Following intravenous administration a biphasic elimination (as seen for a capacity limited reaction) pattern for SDM and its metabolites was observed. Multiple (5x) SDM dosing revealed plasma SDM concentrations ranging between 7 and 108 micrograms ml-1; within 96 hours of termination of the multiple SDM dosing, the plasma SDM concentration was below 0.01 micrograms ml-1. The renal clearances of N4-SDM and the hydroxy metabolites were approximately 10 times greater than that of SDM. The SDM mass balance (faecal/urinary recovery) showed a loss of 56 per cent after intravenous dosage and of 67 per cent after a single oral dosage; the hydroxy metabolites accounted for the highest percentage in faeces/urine. Thus additional metabolic pathways must exist in laying hens.

Residues of sulphadimidine and its metabolites in eggs following oral sulphadimidine medication of hens.

The depletion of sulphadimidine (SDM) and its N4-acetyl and hydroxy metabolites was studied in eggs laid by hens after administration of either a single or multiple oral dosages of 100 mg SDM/kg. During medication and until 1 day after the last dose, the SDM and its metabolite concentrations in the egg white exceeded those in the egg yolk and reflected the plasma levels. In the period starting 2 days after the (last) dosage, the SDM concentration in the yolk became higher than in the egg white, and the drug depletion curves ran parallel. The mean maximum amount of SDM found in the whole egg was 1500 micrograms after a single and 1280 micrograms after multiple dosage. In eggs, traces of the N4-acetyl and 6-methylhydroxy metabolites could be detected (mainly in the egg white), and their concentrations were approximately 40 times lower than those of the parent drug. A highly significant correlation (P less than 0.005) was found between the development stage of the oocyte at the time of (last) medication and the amount of SDM found in the egg that developed from it. A period of 7 or 8 days after the (last) dosage of 100 mg SDM/kg/day is required to obtain SDM levels below 0.1 micrograms/g egg.