A B S T R A C T By comparing natural immunity to Aspergillus fumigatus (AF) in vivo with the action of human or mouse phagocytes against AF in vitro, we delineated two sequential lines of defense against AF. The first line of defense was formed by macrophages and directed against spores. Macrophages prevented germination and killed spores in vitro and rapidly eradicated conidia in vivo, even in neutropenic and athymic mice. The second was the neutrophilic granulocyte (PMN), which protected against the hyphal form of AF. Human and mouse PMN killed mycelia in vitro. Normal, but not neutropenic mice, stopped hyphal growth, and eradicated mycelia. Either line of defense acting alone protected mice from high challenge doses. Natural immunity collapsed only when both the reticuloendothelial system and PMN were impaired. These findings are in keeping with the clinical observation that high doses of cortisone and neutropenia are the main risk factors for invasive aspergillosis. Cortisone inhibited the conidiacidal activity of mouse macrophages in vivo and of human or mouse mononuclear phagocytes in vitro. Cortisone damaged this first line of defense directly and not through the influence of T lymphocytes or other systems modifying macrophage function as shown in athymic mice and in vitro. In addition, daily high doses of cortisone in mice reduced the mobilization of PMN so that the second line of defense was also impaired. Thus, cortisone can break down natural resistance on its own. Myelosuppression rendered mice susceptible only when the first line of defense was overpowered by high chal-

Nitric oxide synthase (NOS) has received immense interest as an antimicrobial and antitumoral effector system of mononuclear phagocytes from rodents. Because there is increasing doubt that an analogous system exists in human macrophages, NOS was reexamined in these cells. Under tightly controlled conditions, with murine macrophages as positive controls, human macrophages failed to secrete nitric oxide «0.1 #Lmol/106 cells/24 h), even after activation with endotoxin, intcrferon-v, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor-a, bacteria, or proliferating lymphocytes. The discrepancy between murine and human macrophages depended on neither the anatomic source (blood, peritoneum), the agent used for activation, nor the duration of activation. NOS activity was paralleled by metabolization of L-arginine to L-citrulline. Exogenous tetrahydrobiopterin, an essential cofactor of NOS not synthesized by human macrophages, did not support NOS activity in human macrophages. Also, no NOS activity was found in cellular subfractions of human macrophages. It appears that in humans, the inducible high-output NOS is not conserved as an antimicrobial system of macrophages.Nitric oxide (NO) has recently been brought into focus as an antimicrobial and antitumoral effector system ofmononuclear phagocytes with activity against fungi [1,2], bacteria [3,4], parasites [5][6][7][8], and tumor cells [9][10][11][12][13][14][15]. While there is general agreement that phagocytes from mice and rats synthesize abundant NO from L-arginine [I, 7, 16-19], demonstration of high-output NO synthase (NOS) activity in human mononuclear phagocytes remains controversial. On one hand, it has been proposed that the antimicrobial activity of human blood-derived macrophages, seen after prolonged activation against Mycobacterium aviuni-Mycobacteriurn intracellulare [3] or Trypanosoma cruzii [20], depends on NO. On the other hand, human peritoneal, alveolar, and bloodderived macrophages have not been found to secrete substantial amounts of NO [21][22][23][24], even after treatment with endotoxin (lipopolysaccharide, LPS) and interferon-v (IFN-'Y). Also nitrite, a descendant of NO that is unstable under physiologic conditions, was not detectable in supernatants from human macrophages stimulated with LPS and IFN-')" [23][24][25]. Furthermore, alveolar and peritoneal macrophages have not been found to metabolize appreciable amounts of L-arginine, the substrate of NOS [21].Because of the significance attributed to the effector function of NO produced by mouse and rat macrophages [26][27][28]

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 receptordependent 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 ␣␣-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 ␤-Cys93 can reduce or enhance this pathway of uptake. In human macrophages, Hp-complex formation critically enhances Hb uptake at low (1 g/mL), but not at high (greater than 100 g/mL), ligand concentrations, lending support for a concentrationdependent 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. IntroductionHeme, which is mainly derived from hemoglobin (Hb), is a strong oxidant and has potent pro-inflammatory properties. These properties become apparent if the intricate intra-erythrocytic compartmentalization of heme is compromised after the destruction of erythrocytes. [1][2][3][4] Large quantities of free hemoglobin can be found in the circulation of patients who have undergone transfusion with cell-free hemoglobin products as a blood substitute. 5 Macrophages are the primary scavengers of Hb after systemic hemolysis and during wound healing. These cells also play a key role in the clearance of exogenously administered blood substitutes. 6 CD163 is a member of the cysteine-rich scavenger receptor family and is exclusively expressed by cells of monocyte/ macrophage lineage. 7 Resident tissue macrophages contain the highest levels of CD163, most notably Kupffer cells in the liver and macrophages within the bone marrow and spleen red pulp. [8][9][10] To date, the Hb-haptoglobin (Hp) complex is the only known ligand of CD163, 11,12 and neither Hp alone nor free Hb has been found to display high-affinity binding to the receptor. Because the Hb-Hp complex binds to CD163 with high affinity and the receptor system has a high endocytotic capacity, CD163 is thought to mediate the clearance of Hb-Hp complexes from the blood. 13 Several lines of evidence indicate that CD163 plays a key role in the anti-inflammatory and wound-healing process. First, there is a high level of CD163 expression by macrophages during the down-regulatory phase of inflammatory reactions. 8,14 Second, CD163 expression is strongly induced by glucocorticoids 15,16 and...

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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.

The co-localization of activated macrophages and damaged neurons observed in brain injury and degenerative brain diseases may hint to macrophage-induced neuronal cytotoxicity. Recently, macrophages have been found to secrete neurotoxic molecules such as radical oxygen intermediates and glutamate, the latter interacting with N-methyl-D-aspartate (NMDA) receptors. As shown in the present study, brain macrophages termed microglial cells co-cultured with differentiated cerebellar neurons excert potent neurotoxic effects. Neurotoxicity is unlikely to be due to cytokines since tumor necrosis factor (TNF)-alpha, interleukin (IL)-1 beta, IL-6 and interferon (IFN)-alpha/IFN-beta/IFN-gamma had no such effects. In contrast, when treating neurons with H2O2 or oxygen radical-generating systems cytotoxicity was induced. Furthermore, microglia were found to produce O2- and H2O2 when triggered with phorbol 12-myristate 13-acetate. However, in co-cultures of neurons and microglia, oxygen-radical scavengers catalase and superoxide dismutase, failed to protect neurons from microglia-induced killing. Moreover, when using undifferentiated neurons which are susceptible to H2O2 but not to NMDA receptor-dependent killing, microglia did not destroy the neurons. Thus, the amount of reactive oxygen intermediates produced by microglia in co-culture do not reach the critical concentrations required for neurotoxicity. As dibenzocyclohepteneimide, an antagonist to NMDA receptors neutralized neurotoxicity in microglia-neuronal co-cultures, excitatory amino acids released by microglia are suggested to compose the major determinant of neurotoxicity.

By exposing human blood-derived macrophages and alveolar macrophages in vitro to dexamethasone, we showed in these studies that glucocorticoids markedly suppress the antimicrobial activity of macrophages but not macrophage activation by lymphokines. As little as 2.5 X 10-8 mol/liter of dexamethasone prevented macrophages from inhibiting germination of Aspergillus spores or from eliminating ingested bacteria such as Listeria, Nocardia, or Salmonella. Damage to macrophage function was inhibited by progesterone and appeared to be receptor-mediated. In accordance with in vivo observations, dexamethasone required 24-36 h to suppress antimicrobial activity. While glucocorticoids interfered with base-line activity of macrophages, dexamethasone concentrations comparable to drug levels in patients had no effect on macrophage activation. Proliferating lymphocytes and 'y-interferon thus increased the antimicrobial activity of phagocytes exposed to glucocorticoids over that of control cells. Macrophage activation and correction of the dexamethasone effect by 'y-interferon, however, was dependent on the pathogen. The lymphokine enhanced the antimicrobial activity of dexamethasone-treated macrophages against Listeia and Salmonella but not against Aspergillus or Nocardia. Dexamethasone-induced damage to the antimicrobial activity of human macrophages in vitro parallels observations that glucocorticoids render laboratory animals susceptible to listeriosis and aspergillosis by damaging resident macrophages. Suppression of macrophage antimicrobial activity should thus be considered when treating patients with glucocorticoids; its prevention byinterferon might be beneficial for some but not all pathogens.

The purpose of this study was to evaluate fluorine-18 fluorodeoxyglucose positron emission tomography (FDG-PET) for the detection of soft tissue and bone infections. Forty-five PET examinations in 39 patients (26 male, 13 female, age range 27-86 years) with suspected infectious foci were examined with whole- or partial-body PET scans using FDG. Twenty-seven scans were done in patients with soft tissue and 18 in patients with bone infections. Corrected and uncorrected transaxial PET images were acquired. Seven hundred and twelve body regions in these 45 PET scans were evaluated. Pathological findings were graded using a confidence scale from A to E (A, definitive infection; E, no infection). Disease status was defined in all patients by culture, biopsy or surgery and clinical follow-up. In 45 PET scans there were 40 true-positive, four false-positive and one false-negative findings. Twelve foci suspected to be infectious in nature on the basis of other imaging examinations were identified as negative by PET, thus representing true-negative findings. Sensitivities for the patients with soft tissue (STI) and bone infections (BI) and for the pooled data were 96%, 100% and 98%, respectively. As the calculation of specificity is not straightforward, it was calculated on a per lesion as well as on a per body region basis to permit estimation of an upper and a lower limit. On a per lesion basis, specificities were 70% (STI), 83% (BI) and 75% for the pooled data and on a per body region basis (dividing the body into 22 regions) they were 99% (STI), 99% (BI) and 99% for the pooled data. One false-negative result was found in a patient with cholangitis. It is concluded that PET appears to be a highly sensitive method to detect infectious foci. Specificity is more difficult to estimate, but is probably in the range from 70% to above 90%.