Differentiation of Ly49s-positive or -negative natural killer cells is inhibited by anti-H-2b monoclonal antibodies acting at the level of bone marrow progenitors from B6 mice.

To investigate the role of MHC class I on in vitro differentiation of natural killer (NK) cells, a CD44low/-CD2-classlow population was isolated from mouse bone marrow. This population, which lacked expression of NK-1.1, Ly49A, Ly49C/I, and Ly49G, generated populations of NK-1.1+ NK cells expressing Ly49A, Ly49C/I, or Ly49G when cocultured for 13 days with syngeneic supportive stromal cells in the presence of interleukin 2. Ly49A and Ly49C/I were absent on the progeny of progenitors tested after 7 days of culture but were expressed as a late event together with low-level expression of NK-1.1, from day 8 of culture. The addition of anti-H-2b monoclonal antibody to cultures at day 0 inhibited proliferation of progenitors supported by either syngeneic, allogeneic, or H-2b-deficient stromal cells, thus suggesting that the effect was not exerted on stromal cells. Additional analyses demonstrated that class Ilow progenitors generated class I+ cells on which the anti-H-2b monoclonal antibody exerted its inhibitory effect.     

Anti-CTLA-4 antibody treatment triggers determinant spreading and enhances murine myasthenia gravis

CTLA-4 appears to be a negative regulator of T cell activation and is implicated in T cell-mediated autoimmune diseases. Experimental autoimmune myasthenia gravis (EAMG), induced by immunization of C57BL/6 mice with acetylcholine receptor (AChR) in adjuvant, is an autoantibody-mediated disease model for human myasthenia gravis (MG). The production of anti-AChR Abs in MG and EAMG is T cell dependent. In the present study, we demonstrate that anti-CTLA-4 Ab treatment enhances T cell responses to AChR, increases anti-AChR Ab production, and provokes a rapid onset and severe EAMG. To address possible mechanisms underlying the enhanced autoreactive T cell responses after anti-CTLA-4 Ab treatment, mice were immunized with the immunodominant peptide alpha(146-162) representing an extracellular sequence of the ACHR: Anti-CTLA-4 Ab, but not control Ab, treatment subsequent to peptide immunization results in clinical EAMG with diversification of the autoantibody repertoire as well as enhanced T cell proliferation against not only the immunizing alpha(146-162) peptide, but also against other subdominant epitopes. Thus, treatment with anti-CTLA-4 Ab appears to induce determinant spreading, diversify the autoantibody repertoire, and enhance B cell-mediated autoimmune disease in this murine model of MG.     

Direct NK cell-mediated lysis of syngenic dorsal root ganglia neurons in vitro

In contrast to extensive studies on the role of T and B lymphocytes in the pathogenesis of autoimmune diseases of the nervous system, little is known about NK cells and their potential role in the destruction of neural tissue. NK cells have been implicated in the selective death of sympathetic neurons resident in the superior cervical ganglia of rats after exposure to the drug guanethidine. This observation suggests that NK cells may function as principle effectors in immunological diseases of the nervous system. However, the direct mechanism of action of NK cells in this model is not known. In particular, it is not known whether NK cells can kill autologous neurons directly. The aim of the present study was to examine whether NK cells can kill directly dorsal root ganglia neurons cultured in vitro. We demonstrate that C57BL/6 (B6)-derived dorsal root ganglia neurons can be killed directly by syngenic IL-2-activated NK cells, and that this nerve cell lysis is dependent on the expression of perforin in the NK cells. NK cells were less effective in destroying neurons grown in the presence of glial cells. These observations indicate a potential role for NK cells in nerve cell degeneration in inflammatory diseases of the nervous system.     

NK cells stimulate proliferation of T and NK cells through 2B4/CD48 interactions

Few studies have addressed the consequences of physical interactions between NK and T cells, as well as physical interactions among NK cells themselves. We show in this study that NK cells can enhance T cell activation and proliferation in response to CD3 cross-linking and specific Ag through interactions between 2B4 (CD244) on NK cells and CD48 on T cells. Furthermore, 2B4/CD48 interactions between NK cells also enhanced proliferation of NK cells in response to IL-2. Overall, these results suggest that NK cells augment the proliferation of neighboring T and NK cells through direct cell-cell contact. These results provide new insights into NK cell-mediated control of innate and adaptive immunity and demonstrate that receptor/ligand-specific cross talk between lymphocytes may occur in settings other than T-B cell or T-T cell interactions.     

Memory CD8+ T cells provide an early source of IFN-gamma

During the non-Ag-specific early phase of infection, IFN-gamma is believed to be primarily provided by NK and NKT cells in response to pathogen-derived inflammatory mediators. To test whether other cell types were involved in early IFN-gamma release, IFN-gamma-producing cells were visualized in spleens and lymph nodes of LPS-injected mice. In addition to NK and NKT cells, IFN-gamma was also detected in a significant fraction of CD8(+) T cells. CD8(+) T cells represented the second major population of IFN-gamma-producing cells in the spleen ( approximately 30%) and the majority of IFN-gamma(+) cells in the lymph nodes ( approximately 70%). LPS-induced IFN-gamma production by CD8(+) T cells was MHC class I independent and was restricted to CD44(high) (memory phenotype) cells. Experiments performed with C3H/HeJ (LPS-nonresponder) mice suggested that CD8(+) T cells responded to LPS indirectly through macrophage/dendritic cell-derived IFN-alpha/beta, IL-12, and IL-18. IFN-gamma was also detected in memory CD8(+) T cells from mice injected with type I IFN or with poly(I:C), a synthetic dsRNA that mimics early activation by RNA viruses. Taken together, these results suggest that in response to bacterial and viral products, memory T cells may contribute to innate immunity by providing an early non-Ag-specific source of IFN-gamma.     

Processing and degradation of exogenous prion protein by CD11c(+) myeloid dendritic cells in vitro

The immune system plays an important role in facilitating the spread of prion infections from the periphery to the central nervous system. CD11c(+) myeloid dendritic cells (DC) could, due to their subepithelial location and their migratory capacity, be early targets for prion infection and contribute to the spread of infection. In order to analyze mechanisms by which these cells may affect prion propagation, we studied in vitro the effect of exposing such DC to scrapie-infected GT1-1 cells, which produce the scrapie prion protein PrP(Sc). In this system, the DC efficiently engulfed the infected GT1-1 cells. Unexpectedly, PrP(Sc), which is generally resistant to protease digestion, was processed and rapidly degraded. Based on this observation we speculate that CD11c(+) DC may play a dual role in prion infections: on one hand they may facilitate neuroinvasion by transfer of the infectious agent as suggested from in vivo studies, but on the other hand they may protect against the infection by causing an efficient degradation of PrP(Sc). Thus, the migrating and highly proteolytic CD11c(+) myeloid DC may affect the balance between propagation and clearance of PrP(Sc) in the organism.     

2B4/CD48-mediated regulation of lymphocyte activation and function

2B4 (CD244) is a member of the CD2 subset of the Ig superfamily. This molecule is expressed on innate immune cells, including NK cells, and on subsets of T cells. The 2B4 molecule interacts with CD48, which is widely expressed on hemopoietic cells. Although earlier reports demonstrated a role for 2B4 as an activating receptor in both mice and humans, recent studies of 2B4-deficient mice have suggested that 2B4 functions predominantly as an inhibitory receptor in mice. In addition, 2B4 may also act as a costimulatory ligand for cells expressing CD48. Thus, the 2B4 molecule is more multifunctional than previously understood. In this study, we delineate the current view of 2B4-CD48 interactions among lymphocytes and other cells.     

Targeting of human dendritic cells by autologous NK cells

NK cells have the capacity to spontaneously kill tumor cell lines, in particular cell lines of hemopoietic origin. In contrast, they do not generally kill nontransformed autologous cells. However, here we demonstrate that short-term activated polyclonal human NK cells, as well as human NK cell lines, efficiently lyse autologous dendritic cells (DC) derived from peripheral blood monocytes as well as Langerhans-like cells derived from CD34+ stem cells isolated from umbilical cord blood. Lysis of autologous DC by short-term activated NK cells and NK cell lines was dependent on granule exocytosis, since total abrogation of lysis was observed in the presence of EGTA. Induction of DC maturation by LPS, monocyte conditioned media (MCM), or stimulation through CD40 ligand (CD40L) rendered the DC less susceptible to lysis by NK cells. Infection of DC with influenza virus was likewise associated with a reduced susceptibility to lysis by NK cells. Thus, susceptibility to lysis by autologous NK cells is a particular property of immature DC. The present results are discussed in relation to the ability of DC to interact with NK cells and to the ability of NK cells to regulate development of specific immunity.     

CHANGES IN CARBOHYDRATE SUBSTRATES, AMINO ACIDS AND AMMONIA IN THE BRAIN DURING INSULIN-INDUCED HYPOGLYCEMIA

—The influence of insulin-induced hypoglycemia upon carbohydrate substrates, amino acids and ammonia in the brain was studied in lightly anaesthetized rats, and the changes observed were related to the blood glucose concentration and to the EEG. Calculations from glucose concentrations in tissue, CSF and blood indicated the presence of appreciable amounts of free intracellular glucose at blood glucose concentrations above 3 μmol/g. When the blood glucose concentration fell below 3 μmol/g, there was no calculated intracellular glucose and decreases in the concentrations of glycogen, G-6-P, pyruvate, lactate and of citric acid cycle intermediates were observed. At blood glucose levels of below 1 μmol/g the tissue was virtually depleted of glycogen, G-6-P, pyruvate and lactate. When the blood glucose concentration was reduced below about 2·5 μmol/g there were progressive increases in aspartate and progressive decreases in alanine, GABA, glutamine and glutamate, and at blood glucose concentrations below 2 μmol/g the ammonia concentration increased. It is suggested that most of the changes observed can be explained as a result of a decreased availability of pyruvate and of NADH. The decrease in the concentration of free NADH was reflected in reductions of the lactate/pyruvate and malate/oxaloacetate ratios at an unchanged intracellular pH. Slow wave activity appeared in the EEG when the hypoglycemia gave rise to reduction of the intracellular glucose concentration to zero. Convulsive activity continued until carbohydrate stores in the form of glycogen and G-6-P were depleted. When this occurred the EEG became isoelectric. In all convulsive animals the concentration of the nervous system activity inhibitor, GABA, was decreased and stimulant, aspartate, was increased.