The relative contribution to cytotoxicity of each of the multiple NK cell activation receptors has been difficult to assess. Using Drosophila insect cells, which express ligands of human NK cell receptors, we show that target cell lysis by resting NK cells is controlled by different receptor signals for cytolytic granule polarization and degranulation. Intercellular adhesion molecule (ICAM)-1 on insect cells was sufficient to induce polarization of granules, but not degranulation, in resting NK cells. Conversely, engagement of the Fc receptor CD16 by rabbit IgG on insect cells induced degranulation without specific polarization. Lysis by resting NK cells occurred when polarization and degranulation were induced by the combined presence of ICAM-1 and IgG on insect cells. Engagement of receptor 2B4 by CD48 on insect cells induced weak polarization and no degranulation. However, coengagement of 2B4 and CD16 by their respective ligands resulted in granule polarization and cytotoxicity in the absence of leukocyte functional antigen-1–mediated adhesion to target cells. These data show that cytotoxicity by resting NK cells is controlled tightly by separate or cooperative signals from different receptors for granule polarization and degranulation.

Cytotoxicity of human NK cells is activated by receptors that bind ligands on target cells, but the relative contribution of the many different activating and inhibitory NK cell receptors is difficult to assess. In this study, we describe an experimental system that circumvents some of the difficulties. Adhesion through β2 integrin LFA-1 is a common requirement of CTLs and NK cells for efficient lysis of target cells. However, the contribution of LFA-1 to activation signals for NK cell cytotoxicity, besides its role in adhesion, is unclear. The role of LFA-1 was evaluated by exposing NK cells to human ICAM-1 that was either expressed on a Drosophila insect cell line, or directly coupled to beads. Expression of ICAM-1 on insect cells was sufficient to induce lysis by NK cells through LFA-1. Coexpression of peptide-loaded HLA-C with ICAM-1 on insect cells blocked the LFA-1-dependent cytotoxicity of NK cells that expressed HLA-C-specific inhibitory receptors. Polarization of cytotoxic granules in NK cells toward ICAM-1- and ICAM-2-coated beads showed that engagement of LFA-1 alone is sufficient to initiate activation signals in NK cells. Thus, in contrast to T cells, in which even adhesion through LFA-1 is dependent on signals from other receptors, NK cells receive early activation signals directly through LFA-1.

Systemic lupus erythematosus (SLE) is a chronic inflammatory disease generated by deregulation of T cell-mediated B-cell activation, which results in glomerulonephritis and renal failure. Disease is treated with immunosuppressants and cytostatic agents that have numerous side effects. Here we examine the use of inhibitors of phosphoinositide 3-kinase (PI3K) gamma, a lipid kinase that regulates inflammation, in the MRL-lpr mouse model of SLE. Treatment reduced glomerulonephritis and prolonged lifespan, suggesting that P13Kgamma may be a useful target in the treatment of chronic inflammation.

Activation of the transcription factor NF-kappa B and pre-T cell receptor (pre-TCR) expression is tightly correlated during thymocyte development. Inhibition of NF-kappa B in isolated thymocytes in vitro results in spontaneous apoptosis of cells expressing the pre-TCR, whereas inhibition of NF-kappa B in transgenic mice through expression of a mutated, superrepressor form of I kappa B alpha leads to a loss of beta-selected thymocytes. In contrast, the forced activation of NF-kappa B through expression of a dominant-active I kappa B kinase allows differentiation to proceed to the CD4(+)CD8(+) stage in a Rag1(-/-) mouse that cannot assemble the pre-TCR. Therefore, signals emanating from the pre-TCR are mediated at least in part by NF-kappa B, which provides a selective survival signal for developing thymocytes with productive beta chain rearrangements.

Spatially restricted activation of signaling molecules governs critical aspects of cell migration; the mechanism by which this is achieved nonetheless remains unknown. Using time-lapse confocal microscopy, we analyzed dynamic redistribution of lipid rafts in chemoattractant-stimulated leukocytes expressing glycosyl phosphatidylinositol–anchored green fluorescent protein (GFP-GPI). Chemoattractants induced persistent GFP-GPI redistribution to the leading edge raft (L raft) and uropod rafts of Jurkat, HL60, and dimethyl sulfoxide–differentiated HL60 cells in a pertussis toxin–sensitive, actin-dependent manner. A transmembrane, nonraft GFP protein was distributed homogeneously in moving cells. A GFP-CCR5 chimera, which partitions in L rafts, accumulated at the leading edge, and CCR5 redistribution coincided with recruitment and activation of phosphatidylinositol-3 kinase γ in L rafts in polarized, moving cells. Membrane cholesterol depletion impeded raft redistribution and asymmetric recruitment of PI3K to the cell side facing the chemoattractant source. This is the first direct evidence that lipid rafts order spatial signaling in moving mammalian cells, by concentrating the gradient sensing machinery at the leading edge.

On the basis mainly of pharmacological experiments, the p38α MAP kinase isoform has been established as an important regulator of immune and inflammatory responses. However, the role of the related p38γ and p38δ kinases has remained unclear. Here, we show that deletion of p38γ and p38δ impaired the innate immune response to lipopolysaccharide (LPS), a Toll-like receptor 4 (TLR4) ligand, by blocking the extracellular signal-regulated kinase 1/2 (ERK1/2) activation in macrophages and dendritic cells. p38γ and p38δ were necessary to maintain steady-state levels of tumor progression locus 2 (TPL2), the MKK kinase that mediates ERK1/2 activation after TLR4 stimulation. TNFα, IL-1β, and IL-10 production were reduced in LPS-stimulated macrophages from p38γ/δ-null mice, whereas IL-12 and IFNβ production increased, in accordance with the known effects of TPL2/ERK1/2 signaling on the induction of these cytokines. Furthermore, p38γ/δ-deficient mice were less sensitive than controls to LPS-induced septic shock, showing lower TNFα and IL-1β levels after challenge. Together, our results establish p38γ and p38δ as key components in innate immune responses.

No abstract

The β2 integrin LFA-1 (CD11a/CD18) mediates adhesion of lymphocytes to cells expressing ICAM. The strength of this adhesion is regulated by different signals delivered by cytokines and chemokines, and by the TCR in the case of T cells. To determine the receptor-ligand interactions required for adhesion of resting NK cells, Drosophila cells expressing different combinations of ligands of human NK cell receptors were generated. Expression of ICAM-1 alone was sufficient for an adhesion of resting NK cells that was sensitive to inhibitors of src family kinase and of phosphatidylinositol 3-kinase. Binding of resting NK cells to solid-phase ICAM-1 showed similar signaling requirements. A pulse of either IL-2 or IL-15 to resting NK cells resulted in strongly enhanced, actin-dependent adhesion to insect cells expressing ICAM-1 alone. Coexpression of either LFA-3 (CD58) or CD48 with ICAM-1 resulted in strong adhesion by resting NK cells, even in the absence of cytokines. Therefore, receptors for LFA-3 and CD48 on resting NK cells strengthen the adhesion mediated by LFA-1.