No abstract

NK cells respond to various chemokines, suggesting that they express receptors for these chemokines. In this paper, we show that IL-2-activated NK (IANK) cells express CC chemokine receptor 4 (CCR4) and CCR8, as determined by flow cytometric, immunoblot, and RNase protection assays. Macrophage-derived chemokine (MDC), the ligand for CCR4, induces the phosphorylation of CCR4 within 0.5 min of activating IANK cells with this ligand. This is corroborated with the recruitment of G protein-coupled receptor kinases 2 and 3 and their association with CCR4 in IANK cell membranes. Also, CCR4 is internalized between 5 and 45 min but reappears in the membranes after 60 min of stimulation with MDC. MDC, thymus and activation-regulated chemokine (TARC), and I-309 induce the chemotaxis of IANK cells, an activity that is inhibited upon pretreatment of these cells with pertussis toxin, suggesting that receptors for these chemokines are coupled to pertussis toxin-sensitive G proteins. In the calcium release assay, cross-desensitization experiments showed that TARC completely desensitizes the calcium flux response induced by MDC or I-309, whereas both MDC and I-309 partially desensitize the calcium flux response induced by TARC. These results suggest that TARC utilizes CCR4 and CCR8. Our results are the first to show that IL-2-activated NK cells express CCR4 and CCR8, suggesting that these receptors are not exclusive for Th2 cells.

The proximal region of the NK gene complex encodes the NKR-P1 family of killer cell lectin-like receptors which in mice bind members of the genetically linked C-type lectin-related family, while the distal region encodes Ly49 receptors for polymorphic MHC class I molecules. Although certain members of the NKR-P1 family are expressed by all NK cells, we have identified a novel inhibitory rat NKR-P1 molecule termed NKR-P1C that is selectively expressed by a Ly49-negative NK subset with unique functional characteristics. NKR-P1C+ NK cells efficiently lyse certain tumor target cells, secrete cytokines upon stimulation, and functionally recognize a nonpolymorphic ligand on Con A-activated lymphoblasts. However, they specifically fail to kill MHC-mismatched lymphoblast target cells. The NKR-P1C+ NK cell subset also appears earlier during development and shows a tissue distribution distinct from its complementary Ly49s3+ subset, which expresses a wide range of Ly49 receptors. These data suggest the existence of two major, functionally distinct populations of rat NK cells possessing very different killer cell lectin-like receptor repertoires.

IntroductionA superfamily of chemotactic cytokines, known as chemokines, induces the migration of leukocytes by promoting endothelial cell adhesion and transmigration toward a chemokine concentration gradient. Chemokines are small, secreted proteins of 8 to 10 kd and are subdivided into 4 families based upon a structurally related cystein motif in the amino terminal end. These are CXC (␣), CC (␤), C (␥), and CX 3 C (␦). Chemokines bind to receptors that are members of the 7 transmembrane-spanning domain receptor family that use heterotrimeric G proteins to transduce signals inside the cell. Receptor activation induces the release of G␤␥ subunits from G proteins, which triggers a series of signaling events leading to cell movement. 1 Stromal cell-derived factor 1␣ (CXCL12) (also known as SDF-1␣) is a CXC chemokine and is the only known ligand for the chemokine receptor CXCR4. 2 CXCL12 induces the chemotaxis of CD34 ϩ hematopoietic progenitor cells, T cells, B cells, natural killer (NK) cells, and monocytes, but not neutrophils. [3][4][5][6] The Src family of tyrosine kinases is a closely related group of nonreceptor tyrosine kinases that modulate a variety of cellular functions. 7 Most Src kinases consist of a unique N-terminal domain followed by an Src homology-3 (SH3), an SH2, and a catalytic domain. 7 The Src kinase Lck is negatively regulated by phosphorylation of the conserved C-terminal tyrosine residue Tyr505 by the C-terminal Src kinase (Csk), while its kinase activity is promoted by autophosphorylation of Tyr394. 8 The Src kinase Lck is confined to the lymphoid lineage, 9 where it plays an important role in antigen receptor signaling. A physical coupling between Lck and CD4 has been found necessary for T-cell chemotaxis induced by the CD4 ligand interleukin 16 (IL-16). 10,11 Moreover, IL-16 has been shown to induce an Lck-dependent inhibitory signal for CXCR4 chemotaxis, requiring the presence of the SH3 domain of Lck. 11 Also, direct stimulation of the T-cell receptor inhibits CXCL12-induced chemotaxis, 12 indicating a cross-talk between the T-cell receptor and CXCR4.Lck is a promising candidate for orchestrating the signaling pathways necessary for chemotaxis. A recent report demonstrates that ZAP-70, which is a substrate for Lck, regulates chemotaxis induced by CXCL12, 13 indicating a possible role for Lck in the signaling pathway leading to chemotaxis. ZAP-70 activation is suggested to be a necessary link to further downstream effectors such as the phosphatidylinositol-3 kinases (PI3 kinases), 14 which are also implicated in the migrational response. 15,16 It has been demonstrated that both the SH2 and the SH3 domains of Lck associate with a number of proteins implicated in the actin cytoskeletal reorganization events necessary for a migrational response, in particular the PI3 kinase, c-Cbl, and Vav. [17][18][19] Also, Lck has been shown to be necessary for both CXCR4-and CD3-mediated activation of ␤1 and ␤2 integrins, suggesting an important role for Lck in mediating cell adhesion. 20,21 Different...