Leukocyte recruitment has been recognized as an early event in inflammatory processes since the late 19th century. Accumulation and trafficking of leukocytes in tissues under physiological and pathological conditions are orderly (typically neutrophils precede mononuclear cells) and selective because, in certain states, one or more leukocyte subsets are recruited preferentially (e.g., eosinophils in allergy). The current paradigm of recruitment is that of a multistep process involving the action of chemotactic signals (1,2).
Classical chemoattractants include complement components, formyl peptides, and leukotriene B4. In addition, various cytokines are able to elicit directional migration of leukocytes. Whereas molecules, such as monocyte colony-stimulating factor (M-CSF), tumor necrosis factor, and vascular endothelial growth factor (VEGF), exert chemotactic activity, the main chemotactic cytokines are a superfamily of molecules known as chemokines (for chemotactic cytokines). However, the in vivo role of M-CSF and VEGF as chemoattractants is well established.
Several independent lines of work lead to the identification of chemokines such as monocyte chemotactic protein-1 (MCP-1) and related molecules. In the early 1970s it had already been noted that supernatants of activated blood mononuclear cells contained attractants active on monocytes and neutrophils (3). Subsequently, a chemotactic factor active on monocytes was identified in culture supernants of mouse (4) and human (5,6) tumor lines and was called tumor-derived chemotactic factor (TDCF) human (5–7). At the time, TDCF was rather unique in that it was active on monocytes but not on neutrophils (6) and had a low molecular weight (5,6). Moreover, correlative evidence suggested its involvement in the regulation of macrophage infiltration in murine and human tumors (5,6,8). A molecule with similar cellular specificity and physicochemical properties was independently identified in the culture supernatant of smooth muscle cells (SMDCF) (9). The JE gene had been identified as an immediate– early platelet-derived growth factor (PDGF)-inducible gene in fibroblasts (10,11). Thus, in the mid-1980s, a gene (JE) was in search of function and a monocytespecific attractant was waiting for molecular definition. In 1989, MCP-1 was successfully purified from supernatants of a human glioma (12), a human monocytic leukemia (13) and a human sarcoma cell line (14–16): sequencing and molecular cloning revealed its relationship with the long-known JE gene (17–19).
Here, we will focus on selected methods used to investigate chemoattractants at large, with emphasis on chemokines. In particular, classic protocols used for studying cell movement including chemotaxis will be presented, along with methods for transendothelial migration and reverse transmigration. In particular, sources of vascular endothelium and the generation of lymphatic endothelial cultures are discussed. In vivo approaches to monitor leukocyte traffic are discussed elsewhere in this volume; here, we will describe the air-pouch model as a simple in vivo recruitment system.