T. P. L. Zomerdijk

Divergent Role for TNF-α in IFN-γ-Induced Killing of Toxoplasma gondii and Salmonella typhimurium Contributes to Selective Susceptibility of Patients with Partial IFN-γ Receptor 1 Deficiency

Patients with defects in IFN-γ- or IL-12-mediated immunity are susceptible to infections with Salmonella and non-tuberculous mycobacteria, but rarely suffer from infections with other intracellular pathogens such as Toxoplasma gondii. Here we describe macrophage and T cell function in eight individuals with partial IFN-γ receptor 1 (IFN-γR1) deficiency due to a mutation that results in elevated cell surface expression of a truncated IFN-γR1 receptor that lacks the intracellular domain. We show that various effector mechanisms dependent on IFN-γR signaling are affected to different extents. Whereas TNF-α production was normally up-regulated in response to IFN-γ, IL-12 production and CD64 up-regulation were strongly reduced, and IFN-γ-mediated killing of the intracellular pathogens Salmonella typhimurium and T. gondii was completely abrogated in patient’s macrophages. Since these patients suffer selectively from infections with non-tuberculous mycobacteria and Salmonella, but not T. gondii, despite sero-immunity in six of eight patients, which indicates previous contact with this pathogen, we next studied the role of TNF-α as a possible immune compensatory mechanism. IFN-γ-induced killing of T. gondii appeared to be partially mediated by TNF-α, and addition of TNF-α could compensate for the abrogated killing of T. gondii in the patient’s macrophages. In contrast, IFN-γ-mediated killing of S. typhimurium appeared to be independent of TNF-α. We propose that the divergent role of TNF-α in IFN-γ-induced killing of T. gondii and S. typhimurium may at least partially explain the highly selective susceptibility of patients.

Mean cell volume of human blood leucocytes and resident and activated murine macrophages

The mean cell volume (MCV) of human blood leucocytes and resident and activated murine macrophages was measured with a Coulter counter connected to a 256 channelyzer. The values found for human blood monocytes, granulocytes, and lymphocytes were 421 +/- 24 femtolitre (fl), 334 +/- 32 fl, and 204 +/- 19 fl, respectively. Resident murine peritoneal macrophages were significantly smaller than rIFN-gamma-activated and BCG/PPD-activated peritoneal macrophages and resident alveolar macrophages.

Beneficial effects of fumarate therapy in psoriasis vulgaris patients coincide with downregulation of type 1 cytokines

Background Fumarates have been shown to be effective in psoriasis vulgaris.

Intracellular signalling by binding sites for the antipsoriatic agent monomethylfumarate on human granulocytes.

Monomethylfumarate (MMF), the most active metabolite of the new antipsoriasis drug FumadermR, stimulates an anti‐inflammatory mediator profile in human leucocytes and inhibits the proliferation of keratinocytes. These effects of MMF on cells in vitro may in part explain the beneficial action of FumadermR in patients. In addition, we have reported that MMF stimulates an increase in the intracellular free Ca2+ concentration ([Ca2+]i) and the cyclic adenosine monophosphate (cAMP) concentration in granulocytes and keratinocytes. Because Ca2+ and cAMP control many physiological cellular responses, including cell proliferation and inflammatory mediator production, the present study focused on the intracellular signal transduction pathway which links interaction between MMF and granulocytes with increases in [Ca2+]i and the cAMP concentration. The increase in [Ca2+]i in granulocytes after MMF depended both on extracellular Ca2+ and Ca2+ from intracellular stores. Ca2+ is essential for the increase in the cAMP concentration after stimulation with MMF. The results found for pharmacological inhibitors of various protein kinases suggest that a staurosporine‐sensitive protein kinase different from protein kinase C (PKC) and protein kinase A is involved in the MMF‐induced increase in [Ca2+]i in granulocytes. As MMF activated protein tyrosine kinase (PTK), and inhibition of this protein kinase partially reduced the increase in [Ca2+]i in granulocytes, PTK activitiy most likely is involved. In addition, activation of protein kinase histone 4 (PKH4) probably plays a part in the MMF ‐stimulated increase in [Ca2+]i in granulocytes as well. As MMF stimulated an increase in the GTP ‐ase activity of membranes and pertussis toxin (PTX) inhibited the increase in the [Ca2+]i and PKH4 activity of granulocytes stimulated by this compound, it is concluded that MMF activates PTX‐sensitive G proteins. Competition binding studies with radiolabelled dimethylfumarate (DMF) and unlabelled DMF and MMF revealed the presence of specific binding sites for methylated fumarates on granulocytes. In summary, MMF binds to specific sites on the plasma membrane of cells. This interaction activates pertussis toxin‐sensitive G proteins which then stimulate an increase in PTK and PKH4 activity. These protein kinases may regulate the rise in [Ca2+]i and the intracellular cAMP concentration. Elevated [Ca2+]i and intracellular cAMP concentration stimulate protein kinases that regulate transcription factors. Activation of these factors results in induction of downstream gene expression and thus controls cell functions, e.g. cell proliferation and production of inflammatory mediators, as has been found for cells incubated with MMF.

A Competition Binding Assay for Determination of the Inositol (1,4,5)-Trisphosphate Content of Human Leucocytes

We developed a competition binding assay for estimation of the intracellular inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3) and optimalized it for the measurement of the Ins(1,4,5)P3 content of human blood leucocytes. The present method is considerably cheaper and requires five times fewer cells than the commercial Ins(1,4,5)P3 kit. The mean Ins(1,4,5)P3 content of human blood monocytes, granulocytes, and lymphocytes amounted to 3.3 +/- 1.2 microM, 3.1 +/- 1.4 microM, and 4.6 +/- 1.5 microM, respectively. After stimulation with formyl-methionyl-leucyl-phenylalanine (f-MLP) the Ins(1,4,5)P3 content of human granulocytes and monocytes increased 2-3 times within 10 sec and then gradually decreased, returning to basal values at 60 sec. Lymphocytes did not respond to f-MLP with an increase in their Ins(1,4,5)P3 content.

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

Since arachidonic acid (AA) production by phospholipase A2 (PLA2) is essential for the Fcγ receptor (FcγR)‐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 FcγR‐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 FcγR‐mediated killing process. The production of O−2 by monocytes upon FcγR 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 FcγR cross‐linking. Furthermore, FcγR cross‐linking stimulated the release of AA from monocytes. Studies with selective inhibitors revealed that the FcγR‐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 FcγR‐stimulated intracellular killing of S. aureus by monocytes.

A new competition binding assay for determination of the cAMP content of human leukocytes.

We developed a competition binding assay for measurement of the cAMP content of human cells based on specific binding sites for this messenger in a crude bovine adrenocortical preparation. The mean cAMP content of human granulocytes and lymphocytes was 1.13 +/- 0.32 microM and 1.81 +/- 0.23 microM, respectively. Stimulation of granulocytes with formyl-methionyl-leucyl-phenylalanine (f-MLP) induced a 2-3 fold increase in cAMP between 10 and 45 sec, which returned to resting values after 1 min. Lymphocytes did not react to f-MLP with a transient rise in intracellular cAMP content. This competition binding assay for cAMP is in essence similar to that for Ins(1,4,5)P3 (7), but conditions for the simultaneous assessment of both messengers could not be found. The present assay is rapid, easy to perform, sensitive and considerably cheaper than commercial kits for assessment of the cAMP content of cells.

Signal transduction in resident and activated macrophages

During an infection with (facultative) intracellular bacteria, macrophages become activated, displaying multiple biochemical changes which include the synthesis and secretion of various biochemical effector molecules (1, 2). These changes mediate the acquisition of several functional characteristics, such as an enhanced bactericidal activity (3–6), the ability to inhibit the intracellular proliferation of various protozoa (7–9), and the destruction of tumor cells (10, 11). The features most widely used as criteria for macrophage activation are: the ability to inhibit intracellular proliferation of protozoa (7–9); the enhanced expression of Ia antigen (10, 12); and the increased production of reactive oxygen intermediates (ROI) (13). In an earlier study we showed that the level of reactive nitrogen intermediates (RNI), measured as NO - 2 production, in murine peritoneal macrophages correlates with these classical criteria for macrophage activation (14).