Erythropoietin (EPO) is both hematopoietic and tissue protective, putatively through interaction with different receptors. We generated receptor subtype-selective ligands allowing the separation of EPO's bioactivities at the cellular level and in animals. Carbamylated EPO (CEPO) or certain EPO mutants did not bind to the classical EPO receptor (EPOR) and did not show any hematopoietic activity in human cell signaling assays or upon chronic dosing in different animal species. Nevertheless, CEPO and various nonhematopoietic mutants were cytoprotective in vitro and conferred neuroprotection against stroke, spinal cord compression, diabetic neuropathy, and experimental autoimmune encephalomyelitis at a potency and efficacy comparable to EPO.

Erythropoietin (EPO) is a tissue-protective cytokine preventing vascular spasm, apoptosis, and inflammatory responses. Although best known for its role in hematopoietic lineages, EPO also affects other tissues, including those of the nervous system. Enthusiasm for recombinant human erythropoietin (rhEPO) as a potential neuroprotective therapeutic must be tempered, however, by the knowledge it also enlarges circulating red cell mass and increases platelet aggregability. Here we examined whether erythropoietic and tissue-protective activities of rhEPO might be dissociated by a variation of the molecule. We demonstrate that asialoerythropoietin (asialoEPO), generated by total enzymatic desialylation of rhEPO, possesses a very short plasma half-life and is fully neuroprotective. In marked contrast with rhEPO, this molecule at doses and frequencies at which rhEPO exhibited erythropoiesis, did not increase the hematocrit of mice or rats. AsialoEPO appeared promptly within the cerebrospinal fluid after i.v. administration; intravenously administered radioiodine-labeled asialoEPO bound to neurons within the hippocampus and cortex in a pattern corresponding to the distribution of the EPO receptor. Most importantly, asialoEPO exhibits a broad spectrum of neuroprotective activities, as demonstrated in models of cerebral ischemia, spinal cord compression, and sciatic nerve crush. These data suggest that nonerythropoietic variants of rhEPO can cross the blood–brain barrier and provide neuroprotection.

Ischemic brain injury resulting from stroke arises from primary neuronal losses and by inflammatory responses. Previous studies suggest that erythropoietin (EPO) attenuates both processes. Although EPO is clearly antiapoptotic for neurons after experimental stroke, it is unknown whether EPO also directly modulates EPO receptor (EPO-R)–expressing glia, microglia, and other inflammatory cells. In these experiments, we show that recombinant human EPO (rhEPO; 5,000 U/kg body weight, i.p.) markedly reduces astrocyte activation and the recruitment of leukocytes and microglia into an infarction produced by middle cerebral artery occlusion in rats. In addition, ischemia-induced production of the proinflammatory cytokines tumor necrosis factor, interleukin 6, and monocyte chemoattractant protein 1 concentration is reduced by >50% after rhEPO administration. Similar results were also observed in mixed neuronal-glial cocultures exposed to the neuronal-selective toxin trimethyl tin. In contrast, rhEPO did not inhibit cytokine production by astrocyte cultures exposed to neuronal homogenates or modulate the response of human peripheral blood mononuclear cells, rat glial cells, or the brain to lipopolysaccharide. These findings suggest that rhEPO attenuates ischemia-induced inflammation by reducing neuronal death rather than by direct effects upon EPO-R–expressing inflammatory cells.

The chemokine CXC ligand 8 (CXCL8)͞IL-8 and related agonists recruit and activate polymorphonuclear cells by binding the CXC chemokine receptor 1 (CXCR1) and CXCR2. Here we characterize the unique mode of action of a small-molecule inhibitor (Repertaxin) of CXCR1 and CXCR2. Structural and biochemical data are consistent with a noncompetitive allosteric mode of interaction between CXCR1 and Repertaxin, which, by locking CXCR1 in an inactive conformation, prevents signaling. Repertaxin is an effective inhibitor of polymorphonuclear cell recruitment in vivo and protects organs against reperfusion injury. Targeting the Repertaxin interaction site of CXCR1 represents a general strategy to modulate the activity of chemoattractant receptors. L eukocyte trafficking into tissue sites of inflammation is directed by chemokines. Chemokines are grouped into four families based on a cysteine motif in the amino terminus of the protein (1, 2). Human CXC ligand 8 (CXCL8)͞IL-8 and related molecules are polymorphonuclear cells (PMN) chemoattractants. Two high-affinity human CXCL8 receptors are known, CXC chemokine receptor 1 (CXCR1) and CXC chemokine receptor 2 (CXCR2). Only one corresponding receptor has been identified in the mouse, and this is recognized by ligands that act as neutrophil attractant, although a mouse orthologue of CXCL8 has not been identified. By recruiting and activating PMN, CXCL8 and related rodent molecules have been implicated in a wide range of disease states characterized by PMN infiltration in organs, including reperfusion injury (RI) (3).G protein-coupled receptors (GPCR) are a prime target for the development of new strategies to control diverse pathologies (4-6). Antichemokine strategies include antibodies, N-terminal modified chemokines, and small-molecule antagonists (7-9). Here we describe a class of GPCR inhibitors that specifically block the inflammatory CXCL8 chemokine receptors CXCR1 and CXCR2 by means of an allosteric noncompetitive mode of interaction and protection against RI. Materials and MethodsReagents. Repertaxin (R)(Ϫ)-2-(4-isobutylphenyl)propionyl methansulfonamide) salified with L-lysine was dissolved in saline. Chemokines were from PeproTech (London). Chemicals, cell culture reagents, and protease inhibitors were from Sigma.Migration. Cell migration of human PMN and monocytes and rodent peritoneal PMN were evaluated in a 48-well microchemotaxis chamber with or without Repertaxin. Agonists (1 nM CXCL8, 10 nM N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), 10 nM CXCL1, 2.5 nM CCL2, 1 nM C5a, 5 nM rat and mouse CXCL1, and 2.5 nM rat and mouse CXCL2) were seeded in the lower compartment. The chemotaxis chamber was incubated for 45 min (human PMN), 1 h (rodent PMN), or 2 h (monocytes). L1.2 migration was evaluated by using 5-m pore-size Transwell filters (Costar) (10). Mutation Analysis of CXCR1 and Signaling. The human CXCR1 ORF was PCR amplified from a CXCR1͞pCEP4 plasmid (kindly provided by P. M. Murphy, National Institutes of Health, Bethesda). Receptor mutants and chimeric re...