Bovine natural killer cells acquire cytotoxic/effector activity following activation with IL-12/15 and reduce Mycobacterium bovis BCG in infected macrophages

Abstract

Bovine natural killer (NK) cells were recently identified by positive selection of a NK cell-activating receptor p46 (NKp46)+ CD3- lymphocyte population, which expresses CD25 and CD8 and lyses tumor cell lines following stimulation with recombinant interleukin-2. In the current work, we characterize the cytotoxic/effector potential of a CD3(-)CD8(-)CD11b- population isolated through negative selection of bovine peripheral blood leukocytes. This population is CD25(lo)CD62(hi) when isolated and becomes CD25hiCD62L(lo) following cytokine stimulation. Activated bovine NK cells increase expression of granulysin, interferon-gamma, and perforin and have cytotoxic activity against human tumor cells and Mycobacterium bovis bacillus Calmette-Guerin-infected alveolar and monocyte-derived macrophages. Expression of a bovine homologue of the CD56 neural adhesion molecule expressed by human NK cells was detected in mRNA from brain tissue but was not detected in peripheral blood mononuclear cells or purified NK cell mRNA. Analysis of mRNA from nonstimulated peripheral blood NK cells demonstrates the constitutive expression of homologues of human NK receptors NKp46, CD244, and CD94 and the granule proteins granulysin and perforin. Phorbol ester-stimulated CD8+ T cells also expressed CD244 and CD94, and CD4+ T cells expressed CD94. These NK cell receptors bearing T lymphocytes may represent memory subsets characterized in humans. The results of these studies demonstrate that bovine NK cells may play an important role in the innate immune responses of cattle.

Rapid and Transient Activation of γδ T Cells to IFN-γ Production, NK Cell-Like Killing, and Antigen Processing during Acute Virus Infection

“…PBMC isolated from the same animals and stimulated in vitro with human IL-2 and IL-12, a method previously reported to activate NK cells (17), were included as positive controls for K562-GFP killing. Cells were incubated for 4 h at 37˚C.…”

Antibacterial Role for Natural Killer Cells in Host Defense to Bacillus anthracis

“…Following overnight incubation to allow adherence, supernatants were removed, and cells were carefully washed three times with antibiotic-free medium. MN were then infected at a 1:1 bacterium-to-MN infecting ratio with B. anthracis Ames bacilli in 50 l of antibiotic-free cRPMI per well, as we have previously described for assessing NK cell activity against mycobacteria (9). Plates were returned to a 37°C incubator for 1 h, at which time the supernatants were gently aspirated and each well was washed three times with RPMI 1640 with 10% fetal bovine serum and 50 g/ml gentamicin to remove extracellular bacilli.…”

“…Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Accuprep (Accurate Chemicals, Westbury, NY). Monocytes (MN) were positively selected by using CD14 ϩ MicroBeads (Miltenyi Biotech, Auburn, CA) with the AutoMacs system and cultured with 1,400 U/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) as we have previously described (9). NK cells were negatively selected by using an NK cell isolation kit (Miltenyi Biotech) and cultured with medium alone or recombinant human interleukin-15 (rhIL-15) (15 ng/ml; R&D Systems).…”

“…Cytotoxic NK cell activity was measured by using the K562 human leukemia cell line (ATCC 45506) as we have previously described (9). K562 cells were maintained in cRPMI, and 1 day prior to use, 10 l/ml of DIOC (3,3=-dihexyloxacarbocyanine iodide; Sigma) was added to fluorescently label K562 cells as previously described (9). NK cells were treated with NT, PA, LT, ET, or LT plus ET for 1 h, and 15 ng/ml of IL-15 or carrier (medium) was then added.…”

“…More recently, the role and function(s) of NK cells in response to various bacterial infections have begun to be defined. NK cells (which are potent producers of gamma interferon [IFN-␥] and cytotoxic granule proteins) have antibacterial activity and reduce the level of bacteremia or sepsis in bacterially infected hosts (9,14,16,29). Following infection, NK cell action in the innate immune response to tumors or virus occurs through lytic activity against infected or altered host cells.…”