T-cell accumulation in arthritic joints is dependent on CXCR4 expression in T cells. (a) CXCR4 was expressed on CD3+ T cells in arthritic joints. Cryostat sections from limbs of normal mice or arthritic mice were analyzed with immunohistochemistry. Red, CD3; green, CXCR4; and blue, nucleus. CD3+CXCR4- cells are indicated with white arrows, and CD3+CXCR4+ cells with yellow ones. White bars indicate 10 μm. Representative data from sections of three normal and three arthritic mice are shown. (b) The expression level of SDF-1 in joints was analyzed with real-time PCR. Normal, joints from nontreated DBA/1J mice (n = 10); arthritic, affected joints from CIA-induced mice (n = 12). Averages and SDs are shown. (c) CXCR4 expression in CD3+ cells was summarized from the immunohistochemical analyses of three normal and three arthritic mice (shown in Figures 4c, 5a, and 6a), and the averages and SDs are presented. Sections of the thymus from a DBA/1J mouse and a Cxcr4flox/flox/Lck-Cre mouse were analyzed as the controls (indicated as Thymus WT or Cxcr4flox/flox/Lck-Cre). (d) In vivo migration of T cells into inflammatory sites during development of CIA was dependent on CXCR4 expression. T cells from IIC/CFA-immunized Cxcr4+/+/Lck-Cre mice (n = 4) and Cxcr4flox/flox/Lck-Cre mice (n = 4) were purified and differentially labeled with 111In and 51Cr, as described in Materials and methods. Cell suspension containing equal numbers of cells from each genotype was injected into CIA-induced recipient mice. After 20 hours, the radioactivities in limbs or LNs were measured with a gamma counter. From the percentages of radioactivities distributed in LNs or joints, the ratio of Cxcr4flox/flox/Lck-Cre mouse-derived T cells versus Cxcr4+/+/Lck-Cre mouse-derived T cells was calculated. Data from two similar experiments were combined, and averages and SDs of five recipient mice are presented.