Macrophage migration inhibitory factor (MIF) accounts for one of the first cytokine activities to have been described, and it has emerged recently to be an important regulator of innate and adaptive immunity. MIF is an upstream activator of monocytes/macrophages, and it is centrally involved in the pathogenesis of septic shock, arthritis, and other inflammatory conditions. The protein is encoded by a unique but highly conserved gene, and X-ray crystallography studies have shown MIF to define a new protein fold and structural superfamily. Although recent work has begun to illuminate the signal transduction pathways activated by MIF, the nature of its membrane receptor has not been known. Using expression cloning and functional analysis, we report herein that CD74, a Type II transmembrane protein, is a high-affinity binding protein for MIF. MIF binds to the extracellular domain of CD74, and CD74 is required for MIF-induced activation of the extracellular signal–regulated kinase–1/2 MAP kinase cascade, cell proliferation, and PGE₂ production. A recombinant, soluble form of CD74 binds MIF with a dissociation constant of ∼9 × 10⁻⁹ K_d, as defined by surface plasmon resonance (BIAcore analysis), and soluble CD74 inhibits MIF-mediated extracellular signal–regulated kinase activation in defined cell systems. These data provide a molecular basis for MIF's interaction with target cells and identify it as a natural ligand for CD74, which has been implicated previously in signaling and accessory functions for immune cell activation.

Fibrocytes are a distinct population of blood-borne cells that display a unique cell surface phenotype (collagen I⁺/CD11b⁺/CD13⁺/CD34⁺/CD45RO⁺/MHC class II⁺/CD86⁺) and exhibit potent immunostimulatory activities. Circulating fibrocytes rapidly enter sites of tissue injury, suggesting an important role for these cells in wound repair. However, the regulatory processes that govern the differentiation of blood-borne fibrocytes and the mechanisms that underlie the migration of these cells to wound sites are currently not known. We report herein that ex vivo cultured fibrocytes can differentiate from a CD14⁺-enriched mononuclear cell population and that this process requires contact with T cells. Furthermore, we demonstrate that TGF-β1 (1–10 ng/ml), an important fibrogenic and growth-regulating cytokine involved in wound healing, increases the differentiation and functional activity of cultured fibrocytes. Because fibrocytes home to sites of tissue injury, we examined the role of chemokine/chemokine receptor interactions in fibrocyte trafficking. We show that secondary lymphoid chemokine, a ligand of the CCR7 chemokine receptor, acts as a potent stimulus for fibrocyte chemotaxis in vitro and for the homing of injected fibrocytes to sites of cutaneous tissue injury in vivo. Finally, we demonstrate that differentiated, cultured fibrocytes express α smooth muscle actin and contract collagen gels in vitro, two characteristic features of wound-healing myofibroblasts. These data provide important insight into the control of fibrocyte differentiation and trafficking during tissue repair and significantly expand their potential role during wound healing.

Transforming growth factor-beta (TGF-beta) is a potent regulator of cellular differentiation, proliferation, migration, and protein expression. These properties have been exploited to create a variety of bioassays for detecting the mature growth factor. In this paper, we describe a highly sensitive and specific, nonradioactive quantitative bioassay for TGF-beta based on its ability to induce plasminogen activator inhibitor-1 (PAI-1) expression. Mink lung epithelial cells (MLEC) were stably transfected with an expression construct containing a truncated PAI-1 promoter fused to the firefly luciferase reporter gene. Addition of TGF-beta (0.2 to > 30 pM) to the transfectants resulted in a dose-dependent increase in luciferase activity in the cell lysates. Although responsive to TGF-beta, this promoter fragment was only minimally influenced by other known inducers of PAI-1 expression. When compared to the widely used MLEC assay, this assay demonstrated greater sensitivity and specificity, allowing quantification of TGF-beta in complex biological solutions.