Chemokines are essential mediators of normal leukocyte trafficking as well as of leukocyte recruitment during inflammation. We describe here a novel non-ELR CXC chemokine identified through sequence analysis of cDNAs derived from cytokine-activated primary human astrocytes. This novel chemokine, referred to as I-TAC (interferon-inducible T cell alpha chemoattractant), is regulated by interferon (IFN) and has potent chemoattractant activity for interleukin (IL)-2–activated T cells, but not for freshly isolated unstimulated T cells, neutrophils, or monocytes. I-TAC interacts selectively with CXCR3, which is the receptor for two other IFN-inducible chemokines, the IFN-γ–inducible 10-kD protein (IP-10) and IFN-γ– induced human monokine (HuMig), but with a significantly higher affinity. In addition, higher potency and efficacy of I-TAC over IP-10 and HuMig is demonstrated by transient mobilization of intracellular calcium as well as chemotactic migration in both activated T cells and transfected cell lines expressing CXCR3. Stimulation of astrocytes with IFN-γ and IL-1 together results in an ∼400,000-fold increase in I-TAC mRNA expression, whereas stimulating monocytes with either of the cytokines alone or in combination results in only a 100-fold increase in the level of I-TAC transcript. Moderate expression is also observed in pancreas, lung, thymus, and spleen. The high level of expression in IFN- and IL-1–stimulated astrocytes suggests that I-TAC could be a major chemoattractant for effector T cells involved in the pathophysiology of neuroinflammatory disorders, although I-TAC may also play a role in the migration of activated T cells during IFN-dominated immune responses.

Azithromycin, a novel azalide antibiotic, concentrated in human and mouse polymorphonuclear leukocytes (PMNs), murine peritoneal macrophages, and mouse and rat alveolar macrophages, attaining intracellular concentrations up to 226 times the external concentration in vitro. In murine peritoneal macrophages, azithromycin achieved concentration gradients (internal to external) up to 26 times higher than erythromycin. The cellular uptake of azithromycin was dependent on temperature, viability, and pH and was decreased by 2,4-dinitrophenol. Azithromycin did not decrease phagocyte-mediated bactericidal activity or affect PMN or macrophage oxidative burst activity (H202 release or Nitro Blue Tetrazolium reduction, respectively). Azithromycin remained in cells for several hours, even after extracellular drug was removed. However, its release was significantly enhanced by phagocytosis of Staphylococcus aureus (82 versus 23% by 1.5 h). In vivo, 0.05 ,ig of azithromycin was found in peritoneal fluids of mice 20 h after oral treatment with a dose of 50 mg/kg. Following caseinate-induced PMN infiltration, there was a sixfold increase in peritoneal cavity azithromycin to 0.32 ,ug, most of which was intracellular. Therefore, the uptake, transport, and later release of azithromycin by these cells demonstrate that phagocytes may deliver active drug to sites of infection.

Eosinophils are recruited to sites of inflammation via the action of a number of chemical mediators, including PAF, leukotrienes, eotaxins, ECF-A and histamine. Although many of the cell-surface receptors for these mediators have been identified, histamine-driven chemotaxis has not been conclusively attributed to any of the three known histamine receptor subtypes, suggesting the possibility of a 4th histamine-responsive receptor on eosinophils. We have identified and cloned a novel G protein-coupled receptor (GPCR), termed Pfi-013, from an IL-5 stimulated eosinophil cDNA library which is homologous to the human histamine H3 receptor, both at the sequence and gene structure level. Expression data indicates that Pfi-013 is predominantly expressed in peripheral blood leukocytes, with lower expression levels in spleen, testis and colon. Ligand-binding studies using Pfi-013 expressed in HEK-293Galpha15 cells, demonstrates specific binding to histamine with a Kd of 3.28 +/- 0.76 nM and possesses a unique rank order of potency against known histaminergic compounds in a competitive ligand-binding assay (histamine > clobenpropit > iodophenpropit > thioperamide > R-alpha-methylhistamine > cimetidine > pyrilamine). We have therefore termed this receptor human histamine H4. Chemotaxis studies on isolated human eosinophils have confirmed that histamine is chemotactic and that agonists of the known histamine receptors (H1, H2, and H3) do not induce such a response. Furthermore, studies employing histamine-receptor antagonists have shown an inhibition of chemotaxis only by the H3 antagonists clobenpropit and thioperamide. Since these compounds are also antagonists of hH4 we postulate that the receptor mediating histaminergic chemotaxis is this novel histamine H4 receptor.

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