129X1/SvJ Mouse Strain Has a Novel Defect in Inflammatory Cell Recruitment 1 Peter White, 2 * Stephen A. Liebhaber,* † and Nancy E. Cooke 3 * ‡ Vitamin D-binding protein (DBP) has been reported to contribute to innate immunity. To verify prior in vitro and cell-based observations supporting this role, we assessed the ability of a recently developed DBP-null mouse line to recruit neutrophils and macrophages to a site of chemical inflammation. The interrupted DBP allele had been generated by homologous recombination in 129X1/SvJ embryonic stem cells and these cells were subsequently used to generate a line of DBP / (null) mice. Initial studies revealed a marked defect in the ability of these DBP / mice to recruit cells to the peritoneum after localized thioglycolate injection. However, progressive outcrossing of the DBP / mice to the C57BL/6J strain, conducted to provide a uniform genetic background for comparison of DBP-null and control mice, resulted in a progressive increase in cell recruitment by the DBP / mice and a loss in their apparent recruitment defect when compared with the DPB wild-type controls. These data suggested that the observed recruitment phenotype initially attributed to the absence of DBP was not linked to the DBP locus, but instead reflected the underlying genetic composition of the 129X1/SvJ ES cells used for the initial DBP gene disruption. A profound cell recruitment defect was confirmed in the 129X1/SvJ mice by direct analysis. Each of three commonly used inbred lines was discovered to have a distinct level of cell recruitment to a uniform stimulus (C57BL/6J > BALB/c > CD1 > 129X1/SvJ). Thus, this study failed to support a unique role for DBP in cellular recruitment during a model inflammatory response. Instead, the data revealed a novel and profound defect of cell recruitment in 129X1/SvJ mice, the strain most commonly used for gene deletion studies. The Journal of Immunology, 2002, 168: 869 – 874. T he use of inbred or “isogenic” mouse strains has facili- tated numerous advances in biology (1). Due to the gen- eralized homozygosity in their genomes, physical differ- ences among these mouse strains can be more directly related to underlying genetic determinants than would be the case for mice with mixed genetic constitution. However, unappreciated pheno- typic differences between isogenic strains may complicate the in- terpretation of phenotypes resulting from the rapidly expanding array of targeted inactivated genes generated by homologous re- combination (“knockout” mice; Refs. 2 and 3). Gene knockout studies inactivate specific loci in the mouse genome and set the stage for subsequent assessment of the impact of the specific gene mutation on phenotype. These studies are routinely conducted in embryo stem (ES) 4 cells derived from the 129X1/Sv mouse strain due to the highly efficient germline colonizing ability of its derived ES cells (4). In addition, the use of ES cells from an isogenic strain generally increases the efficiency of homologous recombination when matched with targeting vectors containing homology (tar- geting) arms derived from the same genome (5). Thus, knockout studies, although powerful in linking genes to function, have the potential to be confounded by the distinct genetic makeup of the isogenic strain, most frequently 129X1/Sv, from which the origi- nating ES cells are derived. Vitamin D-binding protein (DBP) is a highly expressed, poly- morphic serum protein with functions ranging from the transport of vitamin D metabolites to roles in the immune system, host de- fense, and neutrophil chemotaxis (reviewed in Refs. 6 – 8). Recent studies from our laboratory have addressed central issues of DBP in the transport, metabolism, and function of vitamin D using a DBP-deficient (DBP / ) mouse model (9). In addition to these well-documented vitamin D-related functions, a body of literature suggests that DBP also plays a pivotal role in innate immunity. In particular, DBP has been reported to augment chemotaxis of neu- trophils, monocytes, and fibroblasts when studied ex vivo (10 –12). This effect has been attributed to a C5a cochemotactic activity of DBP that requires binding of DBP to chondroitin sulfate proteo- glycans on the cell surface of leukocytes (13–16). In light of these reports, we set out to test the role of DBP in recruitment of in- flammatory cells in vivo using the DBP / mouse model. Localized injection of thioglycollate medium (TG) into the peri- toneum of test animals has been established as a straightforward and effective approach in examining inflammatory cell recruitment and for isolation of peritoneal macrophages (17, 18) and neutro- phils (19). When TG is injected into the peritoneal cavity of mice or rats it induces localized sterile inflammation with subsequent recruitment of leukocytes in a characteristic, cell-specific pattern. Thermal “aging” of TG has been reported to increase its inflam- matory effect due to the formation of advanced glycation end prod- ucts that can accentuate cell recruitment (20). In contrast to other inflammatory agents such as LPS, TG can recruit large numbers of peritoneal leukocytes without activating microbicidal activity (21). The cell recruitment triggered by TG appears to operate via a com- plement-independent pathway, although the exact mechanism(s) of *Departments of Medicine and Genetics and † Howard Hughes Medical Institute, Uni- versity of Pennsylvania, Philadelphia, PA 19104; and ‡ Division of Endocrinology, Diabetes and Metabolism, Departments of Medicine and Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104 Received for publication August 29, 2001. Accepted for publication November 6, 2001. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by National Institutes of Health Grant NIH R01 GM32035 (to N.E.C.). 2 Current address: RHeoGene LLC, P.O. Box 949, Spring House, PA 19477-0949. 3 Address correspondence and reprint requests to Dr. Nancy E. Cooke, Division of En- docrinology, Diabetes and Metabolism, Departments of Medicine and Genetics, Univer- sity of Pennsylvania School of Medicine, 778 Clinical Research Building, 415 Curie Boulevard, Philadelphia, PA 19104-6149. E-mail address: necooke@mail.med.upenn.edu 4 Abbreviations used in this paper: ES, embryo stem; DBP, vitamin D-binding pro- tein; TG, thioglycolate medium; DPBS, Dulbecco’s PBS; WT, wild type. Copyright © 2002 by The American Association of Immunologists 0022-1767/02/$02.00