Cells undergoing apoptosis are efficiently located and engulfed by phagocytes. The mechanisms by which macrophages, the professional scavenging phagocytes of apoptotic cells, are attracted to sites of apoptosis are poorly defined. Here we show that CX3CL1/fractalkine, a chemokine and intercellular adhesion molecule, is released rapidly from apoptotic lymphocytes, via caspase-and Bcl-2-regulated mechanisms, to attract macrophages. Effective chemotaxis of macrophages to apoptotic lymphocytes is dependent on macrophage fractalkine receptor, CX3CR1. CX3CR1 deficiency caused diminished recruitment of macrophages to germinal centers of lymphoid follicles, sites of high-rate B-cell apoptosis. These results provide the first demonstration of chemokine/chemokine-receptor activity in the navigation of macrophages toward apoptotic cells and identify a mechanism by which macrophage infiltration of tissues containing apoptotic lymphocytes is achieved. (Blood. 2008;112:5026-5036) IntroductionWhen apoptosis occurs at high rates in mammalian tissues, apoptotic cells are almost invariably encountered in situ in association with macrophages. 1 These professional scavengers are attracted to the dying cells and engage in their safe, nonphlogistic disposal by phagocytosis. Examples of this innate immune response to dying cells are readily apparent during normal organogenesis, in normal adult tissues, such as the germinal centers of lymphoid follicles, in inflammatory responses, and in pathologic conditions including tumors. The efficient clearance of apoptotic cells by phagocytes is a homeostatic mechanism that militates against histotoxic, proinflammatory, or immunogenic effects that may result from persistence of apoptotic cells. [1][2][3] In recent years, much progress has been made in improving our understanding of the molecular mechanisms underlying the interactions between apoptotic cells and macrophages and the immunologic implications of those interactions. [1][2][3][4][5][6] Before the tethering/ engulfment phases of macrophage-mediated apoptotic-cell clearance, phagocytes are required to navigate effectively to sites of apoptosis. Active release of chemoattractant ("find-me") signals from apoptotic cells at an early stage after engagement of the cell-death program would be predicted to underpin this process, but knowledge of the molecules involved is currently limited. Lysophosphatidylcholine (LPC) is released from apoptotic cells and functions in soluble form as a chemoattractant for mononuclear phagocytes. 7 Significantly, no chemokine family members have previously been implicated in this chemotactic process. Here we show that the chemokine and adhesion molecule CX3CL1, 8,9 also known as neurotactin or fractalkine (FKN), together with its cognate receptor CX3CR1, 10,11 plays an active role in the chemotaxis of macrophages to apoptotic cells. FKN is a type I transmembrane protein, the extracellular portion of which comprises the chemokine domain attached to a mucin stalk. Well known for its roles in inflammatory processe...

Purpose: Poly(ADP-ribose) polymerase (PARP) inhibitors selectively target homologous recombination (HR)-defective cells and show good clinical activity in hereditary breast and ovarian cancer associated with BRCA1 or BRCA2 mutations. A high proportion (up to 50%) of sporadic epithelial ovarian cancers (EOC) could be deficient in HR due to genetic or epigenetic inactivation of BRCA1/BRCA2 or other HR genes. Therefore, there is a potential for extending the use of PARP inhibitors to these patients if HR status can be identified. We developed a functional assay of HR status in primary cultures of EOCs based on Rad51 focus formation that correlates well with sensitivity to the potent PARP inhibitor AG014699.Experimental Design: Primary cultures were derived from ascitic fluid from patients with EOCs. HR status was investigated by γH2AX and Rad51 focus formation by immunofluorescence. Cytotoxicity to PARP inhibitors was tested by sulforhodamine B and survival assay.Results: Twenty-five cultures were evaluated for HR status and cytotoxicity to PARP inhibitor. Following exposure to AG014699, there was an increase in Rad51 foci (HR competent) in 9 of 24 (36%) but no increase (HR deficient) in 16 of 24 (64%) cultures. Cytotoxicity was observed in 15 of 16 (93%) HRdeficient samples but not in 9 of 9 HR-competent samples following 24-hour exposure to 10 μmol/L AG014699.Conclusion: HR status can be determined in primary cancer samples by Rad51 focus formation, and this correlates with in vitro response to PARP inhibition. Use of this assay as a biomarker now needs testing in the setting of a clinical trial.