Activating Ly-49 NK receptors: central role in cytokine and chemokine production

In an attempt to understand potential novel functions of receptors in vivo, we evaluated gene expression after cross-linking the activating Ly-49D mouse NK receptor. Gene expression was evaluated using a mouse GEM 2 microarray chip (Incyte Genomics, St. Louis, MO). Each chip displays a total of 8734 elements. The strongly induced genes fell into two categories: 1) soluble factors and 2) apoptotic genes. The majority of the strongly induced mRNAs as analyzed by microarray hybridization were chemokine genes. RNase protection assays and chemokine protein production analysis validated the microarray results, as cross-linking the Ly-49D mouse NK receptor induced high levels of IFN-gamma, lymphotactin, macrophage-inflammatory protein (MIP)1alpha, and MIP1beta. This gene expression was specific because other chemokines were not induced by anti-Ly-49D receptors. In addition, a series of pharmacological inhibitors were used to identify the key signaling pathways involved in the cellular response. The primary Ly-49D signaling for IFN-gamma production is predominantly mediated through Src kinase pathways involving membrane proximal events, whereas MIP1alpha and MIP1beta gene induction is more complex and may involve multiple biochemical pathways. Thus, we conclude that a primary role for the activating NK receptors in vivo may be to trigger soluble factor production and regulation of the immune response. This would place NK cells and their activating Ly-49 receptors as important initiators of microbial immunity and key elements of the innate immune system.     

Structure/function relationship of activating Ly-49D and inhibitory Ly-49G2 NK receptors.

Murine NK cells express Ly-49 family receptors capable of either inhibiting or activating lytic function. The overlapping patterns of expression of the various receptors have complicated their precise biochemical characterization. Here we describe the use of the Jurkat T cell line as the model for the study of Ly-49s. We demonstrate that Ly-49D is capable of delivering activation signals to Jurkat T cells even in the absence of the recently described Ly-49D-associated chain, DAP-12. Ly-49D signaling in Jurkat leads to tyrosine phosphorylation of TCRzeta and requires Syk/Zap70 family kinases and arginine 54 of Ly-49D, suggesting that Ly-49D signals via association with TCRzeta. Coexpression studies in 293-T cells confirmed the ability of Ly-49D to associate with TCRzeta. In addition, we have used this model to study the functional interactions between an inhibitory Ly-49 (Ly-49G2) and an activating Ly-49 (Ly-49D). Ly-49G2 blocks activation mediated by Ly-49D in an immunoreceptor tyrosine-based inhibitory motif (ITIM)-dependent manner. In contrast, Ly-49G2 was incapable of inhibiting activation by the TCR even though human killer cell inhibitory receptor (KIR) (KIR3DL2(GL183)) effectively inhibits TCR. Both the ability of Ly-49G2 to block Ly-49D activation and the failure of Ly-49G2 to inhibit TCR signaling were confirmed in primary murine NK cells and NK/T cells, respectively. These data demonstrate the dominant effects of the inhibitory receptors over those that activate and suggest an inability of the Ly-49 type II inhibitory receptors to efficiently inhibit type I transmembrane receptor signaling in T cells and NK cells.     

Mouse CD94 participates in Qa-1-mediated self recognition by NK cells and delivers inhibitory signals independent of Ly-49

Inhibitory receptors expressed on NK cells recognize MHC class I molecules and transduce negative signals to prevent the lysis of healthy autologous cells. The lectin-like CD94/NKG2 heterodimer has been studied extensively as a human inhibitory receptor. In contrast, in mice, another lectin-like receptor, Ly-49, was the only known inhibitory receptor until the recent discovery of CD94/NKG2 homologues in mice. Here we describe the expression and function of mouse CD94 analyzed by a newly established mAb. CD94 was detected on essentially all NK and NK T cells as well as small fractions of T cells in all mouse strains tested. Two distinct populations were identified among NK and NK T cells, CD94(bright) and CD94(dull) cells, independent of Ly-49 expression. The anti-CD94 mAb completely abrogated the inhibition of target killing mediated by NK recognition of Qa-1/Qdm peptide on target cells. Importantly, CD94(bright) but not CD94(dull) cells were found to be functional in the Qa-1/Qdm-mediated inhibition. In the presence of the mAb, activated NK cells showed substantial cytotoxicity against autologous target cells as well as enhanced cytotoxicity against allogeneic and "missing self" target cells. These results suggest that mouse CD94 participates in the protection of self cells from NK cytotoxicity through the Qa-1 recognition, independent of inhibitory receptors for classical MHC class I such as Ly-49.     

CD98 induces LFA-1-mediated cell adhesion in lymphoid cells via activation of Rap1

CD98 is a multifunctional heterodimeric membrane protein involved in the regulation of cell adhesion as well as amino acid transport. We show that CD98 cross-linking persistently activates Rap1 GTPase in a LFA-1-dependent manner and induces LFA-1/ICAM-1-mediated cell adhesion in lymphocytes. Specific phosphatidylinositol-3-kinase (PI3K) inhibitors suppressed both LFA-1 activation and Rap1GTP generation, and abrogation of Rap1GTP by retroviral over-expression of a specific Rap1 GTPase activating protein, SPA-1, totally inhibited the LFA-1/ICAM-1-mediated cell adhesion. These results suggest that CD98 cross-linking activates LFA-1 via the PI3K signaling pathway and induces accumulation of Rap1GTP in a LFA-1-dependent manner, which in turn mediates the cytoskeleton-dependent cell adhesion process.     

Interferon-gamma induction of LFA-1-mediated homotypic adhesion of human monocytes

Cell-cell adhesion plays an important role in monocyte function. To investigate the molecular basis for monocyte adhesion, we used recombinant interferon-gamma to induce the formation of homotypic monocyte adhesions. The induction of homotypic adhesions correlated with the increased expression of the LFA-1 membrane molecule. LFA-1 surface expression was increased twofold, whereas expression levels of other monocyte surface molecules including CR3 and p150,95 were unchanged. The direct involvement of LFA-1 in monocyte adhesion was addressed by anti-LFA-1 monoclonal antibody inhibition of homotypic adhesions. Two monoclonal antibodies to distinct epitopes on the LFA-1 alpha-chain completely inhibited homotypic adhesions. Antibodies to a variety of other monocyte surface molecules, often present at higher cell surface density than LFA-1, did not inhibit homotypic adhesion. A panel of monoclonal antibodies that recognized different functional epitopes on the LFA-1 alpha-chain inhibited homotypic monocyte in a hierarchy identical to that observed in previous studies of cell-mediated cytotoxicity. These findings suggest that LFA-1 serves an adhesive function for human mononuclear phagocytes. In addition to providing a molecular basis for homotypic monocyte adhesions, the results suggest a more general role for LFA-1 in monocyte adhesion reactions.     

Expansion of NK cells with reduction of their inhibitory Ly-49A, Ly-49C, and Ly-49G2 receptor-expressing subsets in a murine helminth infection: contribution to parasite control

Natural killer cell-associated direct cytotoxicity and cytokine production are crucial mechanisms for early innate host resistance against viruses, bacteria, or protozoa. The engagement of inhibitory NK cell receptors can influence host responses to viruses. However, these receptors have not been investigated to date in parasitic infections, and little is known about the role of NK cells in the defense against helminths. Therefore, we have correlated the frequencies of cells expressing the pan-NK marker DX5 and subsets bearing inhibitory Ly-49 receptors with worm survival and cytokine production during infection with Litomosoides sigmodontis in BALB/c mice (H2(d)), the only fully permissive model of filariasis. A marked influx of DX5(+)/CD3(-) NK cells and DX5(+)/CD3(+) T cells into the pleural cavity, where the parasites were located, was observed. The frequency of pleural NK cells expressing the H2(d)-reactive inhibitory receptors Ly-49A, Ly-49C, or Ly-49G2 declined most strongly compared with spleen and blood. In the peripheral blood, longitudinal analysis revealed an early and stable reduction of Ly-49C(+) and Ly-49G2(+) NK cells, a subsequent significant increase of the entire NK cell and DX5(+)/CD3(+) T cell populations, and a reduction in the Ly-49A(+) subset. The in vivo depletion of NK cells strongly enhanced the worm load and influenced IL-4 and IL-5 plasma levels. These data demonstrate a new role for NK cells in the host defense against filariae and, for the first time, alterations of Ly-49 receptor-expressing NK cell subsets in a parasitic infection.     

NK cell activation by dendritic cells is dependent on LFA-1-mediated induction of calcium-calmodulin kinase II: inhibition by HIV-1 Tat C-terminal domain.

In this study, we show that binding to autologous dendritic cells (DC) induces a calcium influx in NK cells, followed by activation of the calcium-calmodulin kinase II (CAMKII), release of perforin and granzymes, and IFN-gamma secretion. CAMKII is induced via LFA-1: indeed, oligomerization of LFA-1 leads to CAMKII induction in NK cells. Moreover, release of lytic enzymes and cytotoxic activity is strongly reduced by masking LFA-1 or by adding CAMKII inhibitors such as KN62 and KN93, at variance with the inactive compound KN92. NK cell-mediated lysis of DC and IFN-gamma release by NK cells upon NK/DC contact are inhibited by exogenous HIV-1 Tat: the protein blocks calcium influx and impairs CAMKII activation elicited via LFA-1 in NK cells, eventually inhibiting degranulation. Experiments performed with synthetic, overlapping Tat-derived peptides showed that the C-terminal domain of the protein is responsible for inhibition. Finally, both KN62 and Tat reduced the extension of NK/DC contacts, possibly affecting NK cell granule polarization toward the target. These data provide evidence that exogenous Tat inhibits NK cell activation occurring upon contact with DC: this mechanism might contribute to the impairment of natural immunity in HIV-1 infection.     

Inhibition of ICAM-1/LFA-1-mediated heterotypic T-cell adhesion to epithelial cells: design of ICAM-1 cyclic peptides

In this work, we have designed cyclic peptides (cIBL, cIBR, cIBC, CH4 and CH7) derived from the parent IB peptide (ICAM-1(1-21)) that are inhibitors of ICAM-1/LFA-1-mediated T-cell adhesion to Caco-2 cell monolayers. Cyclic peptide cIBR has the best activity of any of the peptides evaluated. The active ICAM-1 peptides have a common Pro-Arg-Gly sequence that may be important for binding to LFA-1.     

Ly-15.2 and Ly-21.2 are distinct polymorphisms of the LFA-1 heavy chain

Address correspondence and offprint requests to: I. F. C. McKenzie.     

Cutting edge: a small molecule antagonist of LFA-1-mediated cell adhesion.

LFA-1 (CD18,CD11a) is a cell-adhesion molecule that mediates critical immunological processes. In this paper we report the discovery and characterization of (R)-5-(4-bromobenzyl)-3-(3, 5-dichlorophenyl)-1,5-dimethylimidazolidine-2,4-dione (BIRT 377), an orally bioavailable small molecule that interacts specifically with LFA-1 via noncovalent binding to the CD11a chain and prevents LFA-1 from binding to its ligand, ICAM-1. BIRT 377 inhibits lymphocyte activity both in vitro and in vivo, in functional assays that require LFA-1-mediated cell adhesion. These results demonstrate that LFA-1-mediated leukocyte adhesion can be antagonized with noncharged, low m.w. molecules and suggest that the potential therapeutic value of adhesion inhibitors can be attained with a small, orally bioavailable compound.     

Molecular events associated with CD4-mediated Down-regulation of LFA-1-dependent adhesion.

We have previously shown that CD4 ligand binding inhibits LFA-1-dependent adhesion between CD4+ T cells and B cells in a p56(lck)- and phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner. In this work, downstream events associated with adhesion inhibition have been investigated. By using HUT78 T cell lines, CD4 ligands were shown to induce a dissociation of LFA-1 from cytohesin, a cytoplasmic protein known to bind LFA-1 and to enhance the affinity/avidity of LFA-1 for its ligand ICAM-1. A dissociation of PI3-kinase from cytohesin is also observed. In parallel, we have found that CD4 ligand binding induced a redistribution of PI3-kinase and of the tyrosine phosphatase SHP-2 to the membrane and induced a transient formation of protein interactions including PI3-kinase; an adaptor protein, Gab2; SHP-2; and a SH2 domain-containing inositol phosphatase, SHIP. By using antisense oligonucleotides or transfection of transdominant mutants, down-regulation of adhesion was shown to require the Gab2/PI3-kinase association and the expression of SHIP and SHP-2. We therefore propose that CD4 ligands, by inducing these molecular associations, lead to sustained local high levels of D-3 phospholipids and possibly regulate the cytohesin/LFA-1 association.     

A ligand for the murine NK activation receptor Ly-49D: activation of tolerized NK cells from beta 2-microglobulin-deficient mice

Mouse NK cells express inhibitory NK receptors that recognize target cell MHC class I molecules and activation receptors that are less well defined. The Ly-49D activation receptor on C57BL/6 NK cells recognizes Chinese hamster ovary cells and triggers natural killing. In this study, we demonstrate that a Chinese hamster classical MHC class I molecule is the ligand for Ly-49D in a reporter gene assay system as well as in NK cell killing assays. Ly-49D recognizes the Chinese hamster class I molecule better when it is expressed with Chinese hamster beta(2)-microglobulin (beta(2)m) than murine beta(2)m. However, it is still controversial that Ly-49D recognizes H-2D(d), as we were unable to demonstrate the specificity previously reported. Using this one ligand-one receptor recognition system, function of an NK activation receptor was, for the first time, investigated in NK cells that are tolerized in beta(2)m-deficient mice. Surprisingly, Ly-49D-killing activity against ligand-expressing targets was observed with beta(2)m-deficient mouse NK cells, albeit reduced, even though "tolerized" function of Ly-49D was expected. These results indicate that Ly-49D specifically recognizes the Chinese hamster MHC class I molecule associated with Chinese hamster beta(2)m, and indicate that the Ly-49D NK cell activation receptor is not tolerized in beta(2)m deficiency.     

Regulation of locomotion and cell-cell contact area by the LFA-1 and ICAM-1 adhesion receptors.

We demonstrate complementary differences in the behavior of B lymphoblastoid cells adhering to LFA-1 or its counter-receptor ICAM-1. The interaction of B lymphoblastoid cells with glass-supported planar bilayers bearing LFA-1 or ICAM-1 was observed by time-lapse video microscopy, and the distribution of adhesion receptors on cells interacting with the planar bilayers was studied by immunofluorescence microscopy. B lymphoblasts formed a large contact area and crawled rapidly (up to 25 microns/min) on planar bilayers bearing ICAM-1. In contrast, these cells attached to planar bilayers bearing LFA-1 through a fixed point about which the cells actively pivoted, using a single stalk-like projection. Phorbol ester-stimulated lymphoblasts, which adhere more strongly to ICAM-1-bearing substrates than unstimulated lymphoblasts, were still capable of locomotion on ICAM-1. Phorbol ester stimulation of B lymphoblasts on planar bilayers bearing LFA-1 promoted a rapid conversion from "stalk" attachment to symmetrical spreading of the cell on the substrate. Cellular LFA-1 remained uniformly distributed on the cell surface during interaction with bilayers bearing purified ICAM-1 as determined by immunofluorescence. In contrast, ICAM-1 was concentrated in the stalk-like structure through which the unstimulated B lymphoblasts adhered to LFA-1 in planar bilayers, but ICAM-1 immunofluorescence became more uniformly distributed over the cell surface within minutes of phorbol ester addition. Neither LFA-1 or ICAM-1 colocalized with the prominent staining of filamentous actin in the ruffling membrane regions. Interaction through cell surface LFA-1 and ICAM-1, 2, or 3 promotes different cellular morphologies and behaviors, the correlation of which with previously observed patterns of lymphocyte interaction with different cell types is discussed.     

Ly-49CB6 NK inhibitory receptor recognizes peptide-receptive H-2Kb.

NK-mediated cytotoxicity involves two families of receptors: activating receptors that trigger lysis of the target cells being recognized and inhibitory receptors specific primarily for MHC I on the target cell surface that can override the activating signal. MHC I molecules on the cell surface can be classified into molecules made stable by the binding of peptide with high affinity or unstable molecules potentially capable of binding high affinity peptide (hence, peptide receptive) and being converted into stable molecules. It has been previously shown that the Ly-49A inhibitory receptor recognizes stable Dd molecules. We show in this study that the inhibitory receptor Ly-49CB6 recognizes peptide-receptive Kb molecules, but does not recognize Kb molecules once they have bound high affinity peptide.     

The SH2-containing inositol-5'-phosphatase enhances LFA-1-mediated cell adhesion and defines two signaling pathways for LFA-1 activation

The inside-out signaling involved in the activation of LFA-1-mediated cell adhesion is still poorly understood. Here we examined the role of the SH2-containing inositol phosphatase (SHIP), a major negative regulator of intracellular signaling, in this process. Wild-type SHIP and a phosphatase-deficient mutant SHIP were overexpressed in the murine myeloid cell line, DA-ER, and the effects on LFA-1-mediated cell adhesion to ICAM-1 (CD54) were tested. Overexpression of wild-type SHIP significantly enhanced cell adhesion to immobilized ICAM-1, and PMA, IL-3, or erythropoietin further augmented this adhesion. In contrast, phosphatase dead SHIP had no enhancing effects. Furthermore, PMA-induced activation of LFA-1 on DA-ER cells overexpressing wild-type SHIP was dependent on protein kinase C but independent of mitogen-activated protein kinase activation, whereas cytokine-induced activation was independent of protein kinase C and mitogen-activated protein kinase activation but required phosphatidylinositol-3 kinase activation. These results suggest that SHIP may regulate two distinct inside-out signaling pathways and that the phosphatase activity of SHIP is essential for both of them.     

Keratinocyte acetylcholine receptors regulate cell adhesion

We investigated the mechanism mediating cholinergic control of cell-to-cell adhesion of human epidermal keratinocytes (KC) by non-neuronal acetylcholine produced by KC themselves. We first measured cholinergic effects on the expression of desmoglein (Dsg) 1 and 3 in KC using the semi-quantitative immunofluorescence and Western blot assays. Monolayers of KC were treated overnight with 0.25 mM of the cholinergic agonist carbachol (CCh) or the acetylcholinesterase inhibitor pyridostigmine bromide (PBr). Both CCh and PBr increased the relative amounts of Dsg 1 and Dsg 3. To determine the role for cholinergic receptor-mediated phosphorylation of Dsg molecules in assembly/disassembly of keratinocyte desmosomes, we tested the effects of a cholinergic antagonist on keratinocyte adhesion and Dsg phosphorylation status in DJM-1 cell line. Atropine (Atr), 0.02 mM, induced rapid detachment of cells from each other (acantholysis), and also increased phosphorylation of Dsg 3 by 33%. The Atr-dependent phosphorylation of Dsg 3 was inhibited in the presence of 0.5 mM CCh. Thus, keratinocyte cholinergic receptors regulate desmosomal adhesion of KC by altering the level of expression of both Dsg 1 and Dsg 3 and the phosphorylation status of Dsg 3.