Proteinase-activated receptors induce nonoxidative, antimicrobial peptides and increased antimicrobial activity in human mononuclear phagocytes.

As thrombin and SFLLRNPNDKYEPF (SFLLRN-14), a synthetic ligand, mainly of the proteinase-activated receptor-1 (PAR-1), induce in monocytes the synthesis and secretion of chemokines, the PAR pathway can be viewed as a mononuclear phagocyte-activating principle. Classically, antimicrobial activity of mononuclear phagocytes is the measure for activation. Here, we investigated whether thrombin or SFLLRN-14 increases the antimicrobial activity of human monocytes and compared these effects to those of IFN-gamma. Furthermore, we measured the effects of these agents on the secretion of reactive oxygen intermediates and the antimicrobial activity of acid peptide extracts from monocytes. Human monocytes were exposed to maximally active concentrations of thrombin, SFLLRN-14, and IFN-gamma. Human monocytes treated with thrombin or SFLLRN-14 and then challenged with Salmonella enterica serovar typhimurium, including its attenuated mutant phoP, or Listeria monocytogenes killed, within 3 h, significantly more bacteria than control cells, an effect comparable with or surpassing the effect of IFN-gamma. This finding establishes the proteinase-PAR pathway as a potent, alternate activation pathway of mononuclear phagocytes. Thrombin and SFLLRN-14 had no significant effects on the amount of H(2)O(2) secreted by monocytes. This was in contrast to IFN-gamma, which as expected, increased the secretion of H(2)O(2) by approximately fourfold. Thrombin and SFLLRN-14, but not IFN-gamma, however, significantly increased the antimicrobial activity of acid peptide extracts of monocytes in a radial diffusion assay. Taken together, these findings suggest that IFN-gamma and thrombin differentially regulate oxidative and nonoxidative killing systems of human monocytes.

CD163 is the macrophage scavenger receptor for native and chemically modified hemoglobins in the absence of haptoglobin.

CD163 mediates the internalization of hemoglobin-haptoglobin (Hb-Hp) complexes by macrophages. Because Hp binding capacity is exhausted during severe hemolysis, an Hp-independent Hb-clearance pathway is presumed to exist. We demonstrate that Hb interacts efficiently with CD163 in the absence of Hp. Not only is Hb internalized into an endosomal compartment by CD163 as a result of active receptor-dependent endocytosis; it also inhibits the uptake of Hb-Hp complexes, suggesting a common receptor-binding site. Free Hb further induces heme oxygenase mRNA expression in CD163+ HEK293 cells, but not in CD163- cells. Additional evidence for Hp-independent Hb-CD163 interaction is provided by the demonstration that CD163 mediates the uptake of alpha alpha-DBBF crosslinked Hb, a chemically modified Hb that forms minimal Hp complexes. Moreover, certain modifications to Hb, such as polymerization or the attachment of specific functional groups (3 lysyl residues) to the beta-Cys93 can reduce or enhance this pathway of uptake. In human macrophages, Hp-complex formation critically enhances Hb uptake at low (1 microg/mL), but not at high (greater than 100 microg/mL), ligand concentrations, lending support for a concentration-dependent biphasic model of macrophage Hb-clearance. These results identify CD163 as a scavenger receptor for native Hb and small-molecular-weight Hb-based blood substitutes after Hp depletion.

Regulated expression of platelet factor 4 in human monocytes--role of PARs as a quantitatively important monocyte activation pathway.

Human mononuclear phagocytes have recently been shown to express constitutively and even more so, upon stimulation with bacteria, fungi, lipopolysaccharide (LPS), zymosan, or thrombin platelet basic protein (PBP). This CXC chemokine as well as platelet factor 4 (PF4), which is located genomically at a short distance from the PBP, were previously considered to be specific markers for the megakaryocyte cell lineage. Both chemokines have signaling and antimicrobial activity. In the present studies, transcriptional and expressional regulation of PF4 and related chemokines was studied in human monocytes. As shown by quantitative mRNA analysis, Western blots, radioimmunoprecipitation of cell extracts, and immunofluorescence and quantitatively with enzyme-linked immunosorbent assay, human monocytes express PF4 in the same order of magnitude as the known, regulated CXC chemokine interleukin (IL)-8. Expression of PF4 is up-regulated at the mRNA and protein level by thrombin and mediated by proteinase-activated receptors (PARs), resulting in a 32- to 128-fold higher mRNA level and leading to an up-to-sixfold increase of the peptide concentration in monocyte culture supernatants. Thrombin and the synthetic ligand of PAR-1 and PAR-2, SFLLRN, also induced comparable increases in the levels of mRNA for PBP, IL-8, regulated on activation, normal T expressed and secreted (RANTES), monocyte chemoattractant protein-1, and macrophage-inflammatory protein-1alpha and increased synthesis of these chemokines as shown by immunofluorescence or a quantitative immunobead-based method. The induction of increased mRNA levels for all chemokines by SFLLRN was unsurpassed by LPS, zymosan, interferon-gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and IL-1. Activation of monocytes through PARs represents an alternate activation mechanism, independent from IFN-gamma, TNF-alpha, or other signaling pathways.

Soluble hemoglobin-haptoglobin scavenger receptor CD163 as a lineage-specific marker in the reactive hemophagocytic syndrome.

Reactive hemophagocytic syndrome (RHS) is a disease of overwhelming macrophage activity triggered by infection, malignancy or autoimmune disorders. Currently used laboratory markers for the quantitative assessment of monocyte/macrophage activation lack lineage-restricted expression patterns and thus specificity. Serum levels of the macrophage specific scavenger receptor CD163 were determined by enzyme-linked immunosorbent assay (ELISA) and were found to be highly increased in patients with RHS (median 39.0 mg/L). Significantly lower levels were determined in patients with sepsis (median 9.1 mg/L), acute mononucleosis (median 8.2 mg/L), Leishmania infection (median 6.7 mg/L) and healthy controls (median 1.8 mg/L). Follow-up of patients with a relapsing course of the disease revealed close correlations of sCD163 with clinical disease activity, serum ferritin and other markers of macrophage activity. Large sinusoidal accumulations of CD163 expressing macrophages actively engaged in phagocytosis of blood cells were detected in spleen sections of RHS patients. Our data suggests sCD163 to be a macrophage-specific marker in patients with disorders of inappropriate macrophage activation.

Gene expression profiling of inflamed human endothelial cells and influence of activated protein C.

BACKGROUND: During systemic inflammation, activation of vascular endothelium by proinflammatory cytokines leads to hypotension, microvascular thrombosis, and organ damage. Recent data suggest a link between coagulation and inflammation through the activated protein C (APC) pathway. We studied gene expression profiles in human coronary artery endothelial cells (HCAECs) exposed to proinflammatory stimuli and the influence of APC on expression of candidate genes regulated by these stimuli. METHODS AND RESULTS: HCAECs were stimulated with interleukin-1beta, interferon-gamma, and tumor necrosis factor-alpha. In gene expression profiling, 400 of 8400 genes were regulated >2-fold. Verification of selected candidate genes was achieved by measuring expression of mRNA species by real-time polymerase chain reaction, cytokine secretion by ELISA, and metabolites of tetrahydrobiopterin (BH4) biosynthesis by high-performance liquid chromatography. BH4 synthesis, interleukin-6, interleukin-8, monocyte chemotactic protein-1 (MCP-1), and intercellular adhesion molecule-1 (ICAM-1) were downregulated by APC at the transcriptional and protein level. Endothelial nitric oxide synthase, endothelial adhesion molecule, and vascular cell adhesion molecule-1 were not affected by APC. Activities of transcription factors c-Fos, FosB, and c-Rel were inhibited by APC in inflamed HCAECs. CONCLUSIONS: Our study revealed a novel antiinflammatory mechanism of APC-dependent gene regulation in HCAECs since c-Fos-dependent induction of MCP-1 and ICAM-1 was suppressed. APC downregulates expression and activity of genes related to inflammation, most pronounced under intermediate or mild inflammatory conditions.

Induction and antimicrobial activity of platelet basic protein derivatives in human monocytes.

The antimicrobial activity of a number of chemokines has recently come into focus of research about innate immunity. We have previously shown that platelet basic protein (PBP), which gives rise to several antimicrobial peptides of platelets, is also expressed in human monocytes. In the present studies, we show that exposure of human monocytes to bacteria or microbial components (lipopolysaccharide and zymosan) induces a several-fold greater expression of derivates of PBP. Also, activation of proteinase-activated receptors (PARs) by thrombin or the synthetic peptide ligand SFLLRN of PAR-1 significantly increased PBP expression, presumably on the transcriptional level, as evidenced by higher mRNA levels. Derivates of PBP appeared to reach phago-lysosomes, as higher concentration was found in latex phagosomes isolated by a flotation method. By the gel-overlay technique, two bactericidal derivatives of PBP could be visualized, which were immunoreactive with anti-PBP antibody in Western blots. By matrix-assisted laser desorption/ionization time of flight and surface-enhanced laser desorption and ionization techniques, it was confirmed that the bands corresponded to PBP derivates. After immunofixation with a monoclonal antibody to PBP, the major peptide in zymosan-stimulated monocytes was identified to correspond by molecular weight to connective tissue-activating peptide III, which has been reported to be a major antimicrobial PBP derivate also in platelets. Our observations indicate that PBP and its derivates are constituents of the antimicrobial arsenal of human monocytes. Their increased expression after exposure to microorganisms allows a rapid host response to pathogens.

Functional tetrahydrobiopterin synthesis in human platelets.

BACKGROUND: Previous studies have provided evidence for the importance of platelet-derived nitric oxide (NO) for the regulation of hemostasis. Tetrahydrobiopterin (BH4) is an essential cofactor and regulator of NO synthase activity in the vasculature; however, it is as yet unknown whether platelets dispose over a functional BH4 synthesis. METHODS AND RESULTS: We quantified mRNA expression of genes involved in BH4 synthesis, measured enzymatic activities, and determined intraplatelet levels of pteridines in platelets from healthy volunteers and from patients treated for prolonged periods of time with glucocorticoids. Freshly isolated platelets from healthy volunteers show functional BH4 synthesis, as evidenced by the presence of mRNA species and enzymatic activity of GTP cyclohydrolase I (GTPCH), 6-pyruvoyl tetrahydropterin synthase, and sepiapterin reductase. Biopterin was the major intraplatelet pteridine, whereas no neopterin was found. mRNA expression and enzymatic activity of GTPCH were undetectably low in platelets that had been stored for 5 days, and no pteridines were found in these platelets. Freshly isolated platelets from patients treated with glucocorticoids had decreased mRNA expression and activity of GTPCH compared with platelets from healthy volunteers. CONCLUSIONS: Human platelets dispose over a functional de novo BH4 synthesis. Furthermore, our results indicate the potential of external factors, eg, prolonged storage or glucocorticoid therapy, to significantly affect BH4 synthesis within platelets. Together, these findings offer new insights into the biology and pathobiology of platelet function in humans.

Constitutive and regulated expression of platelet basic protein in human monocytes.

Platelet basic protein (PBP) and several of its derivatives are known for their broad range of functions as signaling molecules and cationic antimicrobial peptides and were considered hitherto megakaryocyte- and platelet-specific. In search of glucocorticoid-regulated antimicrobial systems of monocytes, we found a 15-fold down-regulation of PBP mRNA by differential display. Regulation was confirmed in vivo even at low prednisone doses. Quantitative mRNA analyses confirmed down-regulation also for platelets. Western blotting and immunostains showed down-regulation at the protein level. Pro-PBP derivatives were in the size range of 7.5-14 kD and in immunostains, gave granular cytoplasmatic patterns. Interleukin (IL)-4 and IL-10 induced a similar down-regulation. Phagocytosis resulted in an increase of smaller derivatives in the range of 7.5 kD. Stimulation with interferon-gamma and lipopolysaccharide did decrease expression of PBP and affected derivatization. Expression of PBP and its derivatives is not restricted to the megakaryocytic cell lineage. PBP and some of its derivatives might contribute to the antimicrobial armamentarium of mononuclear phagocytes or have monokine functions. Our studies define PBPs as one among the many immunosuppressive targets of glucocorticoids.

Listeria species escape from the phagosomes of interleukin-4-deactivated human macrophages independent of listeriolysin.

Listeria monocytogenes is the causative agent of infections like sepsis and meningitis, especially in immunocompromised hosts. Human macrophages are able to phagocytose and digest L. monocytogenes but IL-4 prevents human macrophages from killing the bacteria, the mechanisms of which are unknown. In the present study, we examined various listeria species and strains including wild-type and deletion mutants in human macrophages pretreated with IL-4. To analyse the IL-4-mediated deactivation process, we combined quantitative infection assays with various morphologic methods. IL-4 facilitates survival and escape of the pathogenic L. monocytogenes wild-type strain 10403S from the macrophage phagosomes. In untreated macrophages, the isogenic listeriolysin deletion mutant strain DP-L2161 was killed and did not escape from the phagolysosomes. However, after macrophage deactivation with IL-4 DP-L2161 survived and escaped from the phagosomes. This was also the case, but to a lesser extent, even for the naturally avirulent L. innocua. As detected by confocal laser-scanning fluorescence microscopy and electron microscopy, IL-4 permitted the escape of all listeria species tested, including DP-L2161 and L. innocua from the phagosomal compartment of the macrophages. We conclude that escape from the phagosome and survival of the listeria species tested in IL-4-deactivated human macrophages is independent of the virulence factor listeriolysin.

Critical role of interleukin-1beta for transcriptional regulation of endothelial 6-pyruvoyltetrahydropterin synthase.

OBJECTIVE: Synthesis of tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide synthases, is strongly induced on immunostimulation in vascular endothelial cells (VECs). Expression of GTP cyclohydrolase I (GTPCH), the first enzyme in BH4 biosynthesis, is regulated by cytokines and considered rate-limiting. Herein we investigated the molecular mechanism and relevance of cytokine-dependent regulation of 6-pyruvoyltetrahydropterin synthase (PTPS), the second enzyme in BH4 synthesis, in human coronary artery endothelial cells (HCAECs). METHODS AND RESULTS: Real-time polymerase chain reaction revealed a 4-fold induction of PTPS and a 300-fold induction of GTPCH expression by interleukin (IL)-1beta/tumor necrosis factor-alpha/interferon-gamma, mainly through de novo transcription. On immunostimulation, PTPS became rate-limiting. Importantly, IL-1beta induced PTPS rather than GTPCH. As a result, IL-1beta contributed significantly to the amount of BH4 produced (+40%) but concomitantly reduced the accumulation of the GTPCH intermediate, 7,8-dihydroneopterin triphosphate (-50%). CONCLUSIONS: Our data show that PTPS induction is necessary for optimized BH4 synthesis in cytokine-stimulated HCAECs and point to IL-1beta as a leading cytokine in this process.

Continuous infusion of escalated doses of amphotericin B deoxycholate: an open-label observational study.

Amphotericin B deoxycholate (AmB-d) remains a mainstay of antifungal therapy for immunocompromised patients, despite being associated with significant therapy-related toxicity. Because continuous infusion of AmB-d is better tolerated than traditional administration over 2-6 hours, we evaluated escalation of the AmB-d dose in 33 patients (31 of whom were neutropenic), for whom the initial dosage of AmB-d (1 mg/kg/day) was gradually increased to 2.0 mg/kg/day when renal function remained stable and the drug was tolerated. Dose escalation was possible without delay in 28 patients. Median duration of AmB-d therapy was 16 days (range, 7-72 days). Infusion-related reactions accompanied <18% of AmB-d infusions. Twenty-seven patients had a decrease in creatinine clearance while receiving AmB-d therapy. A >2-fold decrease in creatine clearance was observed in 5 patients, and the decrease was dose-limiting in only 1 patient; no dialysis was required. In conclusion, continuous infusion of AmB-d escalated to 2.0 mg/kg/day seems not to cause additional impairment of vital organ functions and to be well tolerated by most patients.

Nephrotoxicity of cyclosporine A and amphotericin B-deoxycholate as continuous infusion in allogenic stem cell transplantation.

BACKGROUND: Nephrotoxicity is an important side effect of amphothericin B deoxycholate (ampho B) and cyclosporine A (CsA). The combined administration of these drugs is frequent in patients with haematological diseases undergoing allogeneic stem cell transplantation. AIM: To assess the additional renal toxicity of ampho B given as a continuous infusion in addition to CsA. METHODS: In a retrospective study renal function was investigated in patients receiving CsA alone or in combination with ampho B (24-hour infusion) after allogeneic stem cell transplantation between January 1998 and April 2001. RESULTS: Of a total of 84 patients, 22 were treated with ampho B. There was a statistically significant decline in renal function in comparison to the 62 patients receiving CsA alone. However, renal insufficiency in all patients remained in a clinically acceptable range and was reversible. The residual renal dysfunction at the end of the hospitalisation was mainly due to continuing therapy with CsA. CONCLUSION: Amphotericin B deoxycholate in addition to CsA leads to a statistically significant but clinically tolerable worsening of renal function. Using a 24-hour infusion and strict salt repletion, amphotericin B can be administered safely as deoxycholate in bone marrow transplant patients in conjunction with CsA for proven or suspected fungal infections.

Induction of the CD163-dependent haemoglobin uptake by macrophages as a novel anti-inflammatory action of glucocorticoids.

Highly efficient systems remove toxic and pro-inflammatory haemoglobin (Hb) from the circulation and local sites of tissue damage. Macrophages are major Hb-clearing cells; CD163 was recently recognized as the specific haemoglobin-haptoglobin scavenger receptor (HSR). We show that dexamethasone strongly induced the specific uptake of haemoglobin-haptoglobin complexes, CD163 mRNA transcription (13-fold) and cell surface expression (10-fold) by human macrophages. In contrast, the TH2-cytokine interleukin 4 (IL-4) completely suppressed functional CD163 expression. The range of functional receptor modulation reached a factor of 100 after 4 h of macrophage-ligand interaction. Based on these results, we propose the augmentation of Hb clearance as a novel anti-inflammatory action of glucocorticoids.

Host-parasite relation in invasive aspergillosis.

Invasive aspergillosis has become one of the most important infectious complications of intensive modern medicine often limiting the success of oncological treatments and transplantation. The rationale for this is found in the effects of immunosuppressive therapies and to a lesser degree underlying disease processes as well as in properties of the fungus. The double pronged nature of host defenses directed against spores and hyphae of Aspergilli is affected by glucocorticoids and myeloablative therapy. Resident alveolar macrophages are the major players eliminating inhaled conidia. Neutrophils, efficiently killing hyphae. These defense mechanisms are so solid that even after the inhalation of billions of spores infection is reliably prevented in man. Mechanisms by which glucocorticoids prevent macrophages from first inhibiting germination of ingested conidia and then killing them have not been elucidated. Mobilization of neutrophils is also hampered by glucocorticoids that have the potential to suppress the expression of an array of neutrophil chemokines by macrophages. Glucocorticoids are thus able to abrogate defenses against Aspergilli on their own, affecting both lines of defense. In neutrophil granulocytes oxidative killing systems directed against hyphae are of major importance as pointed out by infections in children with chronic granulomatous disease, but other killing systems such as defensins and possibly thrombocidins are also of importance. To date it remains speculative that platelet derived thrombocidins play an important role, but such speculations are tempting in view of the angio-invasive nature of the fungus and the coexisting thrombocytopenia in many patients with aspergillosis.

Assessment of digital clubbing in medical inpatients by digital photography and computerized analysis.

BACKGROUND: Digital clubbing has been associated with a large number of disorders. To overcome the limitation of subjective clinical assessment, several objective measurements have been developed among which the hyponychial angle was considered most accurate for quantification of finger clubbing. METHODS AND RESULTS: Here we investigated hyponychial angles in 123 healthy subjects and 515 medical inpatients from a tertiary hospital. Healthy subjects had a mean angle of 178.87 +/- 4.70 degrees (range: 164.78-192.10 degrees ), a finding that is well in accordance with previous results obtained using other techniques, underlining the accuracy of the chosen method of assessment. The mean angle of patients was 181.65 +/- 7.18 degrees (range: 162.22-209.19; p <0.0001 compared to healthy controls). When the upper limit of normality, i.e. 192.10 degrees, was used to define digital clubbing, the prevalence of digital clubbing in our patients was 8.9%; the percentage of clubbed fingers varied substantially among the various disease states (up to 80% in patients with cystic fibrosis). CONCLUSION: The use of digital photography with computerised analysis was found to be an easy, fast and inexpensive method for the quantification of hyponychial angles with excellent intra and inter observer reliability whilst causing no discomfort to patients. This tool may therefore be useful in further longitudinal and cross-sectional studies of finger morphology and may become an accepted standard in the diagnosis of digital clubbing.