CD26 is a leukocyte activation marker that possesses dipeptidyl peptidase IV activity but whose natural substrates and immunological functions have not been clearly defined. Several chemo-kines, including RANTES (regulated on activation, normal T cell expressed and secreted), have now been shown to be substrates for recombinant soluble human CD26. The truncated RANTES(3–68) lacked the ability of native RANTES(1–68) to increase the cytosolic calcium concentration in human monocytes, but still induced this response in macrophages activated with macrophage colony-stimulating factor. Analysis of chemokine receptor messenger RNAs and patterns of desensitization of chemokine responses showed that the differential activity of the truncated molecule results from an altered receptor specificity. RANTES(3–68) showed a reduced activity, relative to that of RANTES(1–68), with cells expressing the recombinant CCR1 chemokine receptor, but retained the ability to stimulate CCR5 receptors and to inhibit the cytopathic effects of HIV-1. Our results indicate that CD26-mediated processing together with cell activation–induced changes in receptor expression provides an integrated mechanism for differential cell recruitment and for the regulation of target cell specificity of RANTES, and possibly other chemokines.

Hydrogen peroxide (H 2 O 2 ) triggers a redox cycle between ferric and ferryl hemoglobin (Hb) leading to the formation of a transient protein radical and a covalent hemeprotein cross-link. Addition of H 2 O 2 to highly purified human hemoglobin (HbA 0 ) induced structural changes that primarily resided within ␤ subunits followed by the internalization of the heme moiety within ␣ subunits. These modifications were observed when an equal molar concentration of H 2 O 2 was added to HbA 0 yet became more abundant with greater concentrations of H 2 O 2 . Mass spectrometric and amino acid analysis revealed for the first time that ␤Cys-93 and ␤Cys-112 were oxidized extensively and irreversibly to cysteic acid when HbA 0 was treated with H 2 O 2 . Oxidation of further amino acids in HbA 0 exclusive to the ␤-globin chain included modification of ␤Trp-15 to oxyindolyl and kynureninyl products as well as ␤Met-55 to methionine sulfoxide. These findings may therefore explain the premature collapse of the ␤ subunits as a result of the H 2 O 2 attack. Analysis of a tryptic digest of the main reversed phase-high pressure liquid chromatography fraction revealed two ␣-peptide fragments (␣128 -␣139) and a heme moiety with the loss of iron, cross-linked between ␣Ser-138 and the porphyrin ring. The novel oxidative pathway of HbA 0 modification detailed here may explain the diverse oxidative, toxic, and potentially immunogenic effects associated with the release of hemoglobin from red blood cells during hemolytic diseases and/or when cell-free Hb is used as a blood substitute. . is generated and trapped in the heme pocket (7,8). Interestingly, the processes of heme degradation can be enhanced unintentionally when Hb is chemically modified and used as an HBOC, thus the nature of chemical modification greatly influences the geometry of heme and its susceptibility to oxidation and degradation (9

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...