The term chemokine
, a linguistic contraction derived from chemo
, describes a large family of mostly secreted small molecules involved in numerous physiological and pathophysiological processes. Although their nominal function, the control of directed cell migration, constitutes a defining attribute, chemokines exert a host of additional activities that modulate many fundamental properties of cellular function (1
). With the completion of the human and murine genomes, the rapid discovery of novel chemokines has come to an apparent conclusion, and the chemokines may in fact be one of the first mammalian superfamilies known in its entirety (14
). Based on a defining tetracysteine motif, chemokines can be divided into 4 distinct subfamilies (15
): CC chemokines (CCL1–CCL28) contain 2 adjacent cysteine residues near their amino terminus that are separated by a single nonconserved amino acid in the CXC chemokines (CXCL1–CXCL17) and by 3 amino acids in the sole CX3C member, CX3
CL1; the C chemokines (XCL1/2 in humans, only XCL1 in mouse) lack 1 of the first 2 cysteine residues found in the other subfamilies. In general, this subdivision limits chemokine binding to members of certain chemokine receptor subgroups. However, among the CC and CXC subfamilies, a degree of redundancy and promiscuity exists, with some receptors able to bind multiple chemokines and some chemokines capable of engaging multiple receptors. It should be noted that the structure-based taxonomy of chemokines does not supersede, but rather complements, an older classification according to functional properties (homeostatic, inflammatory, and dual-use chemokines) or the alternative clustering based on genomic organization (14
The extraordinary complexity of the chemokine system emerges from the confluence of several factors. Excluding pseudogenes and multiple copy numbers for some chemokine genes, the murine genome contains 40 distinct genes that give rise to 39 unique chemokine proteins (14
), as the Ccl21b
gene products are identical (Table ). In addition to the large number of chemokine family members (at least 46 in humans), the presence of splice variants and extensive posttranslational modifications, existence of promiscuous receptor binding and receptor-independent binding, formation of hetero-oligomeric chemokine complexes, dynamic expression patterns and functional diversity combine to generate an exceedingly broad spectrum of possible chemokine activities (1
). Although the transcriptional expression patterns of many chemokines have been detailed in various experimental and clinical settings, analytical access to specific chemokine-secreting cell types has remained somewhat limited, given methodological approaches preferentially reliant on immunoblots, ELISA assays, and/or immunohistochemistry (IHC).
Chemokine nomenclature and antibodies
The analytical method of choice for the detection of chemokine proteins in defined cellular subsets is flow cytometry (FC), which allows for multiparametric analysis of individual chemokine-producing cells within larger cell populations of interest. Here, the preferred tools are chemokine-specific mAbs conjugated to fluorochromes; however, although the list of FC-approved mAbs is growing, no such reagents are available for the majority of murine chemokines (Table ). Polyclonal Abs (pAbs) constitute an appropriate alternative, and indeed have been used for the flow cytometric detection of selected murine chemokines in a variety of immune cell subsets, such as T cells, NK cells, NKT cells, DCs, monocyte/macrophages (Mo/Mϕ), granulocytes, and others (18
). However, not all studies have rigorously excluded potential crossreactivities of these reagents, and, to our knowledge, direct visualization by means of FC has not been reported for most murine chemokines.
The use of pAbs rather than mAbs for detection of intracellular antigens offers a number of challenges and some advantages that have to be addressed in order to assure their reliable usage for FC (see Methods). With the aim to develop comprehensive analytical access to all known murine chemokines, we have selected, tested, and validated a panel of commercially available affinity-purified pAbs specific for 37 of 39 murine chemokines for use in FC (Table ). To demonstrate the principal utility of our approach to chemokine FC, we applied this methodology to an identification of homeostatic chemokines and the principal hematopoietic cell subsets in the spleen involved in their expression (Table ). In addition, we have delineated the complete chemokine profiles of NK and B cells in response to major stimuli and defined the DC chemokine response to infection (Table ).
Summary of chemokine expression patterns and cellular subsets