Augmentation of natural killer cytotoxicity by alpha or gamma natural and recombinant interferons and interferon inducers. Effect of monocytes

We investigated the effect of human peripheral blood monocytes on the augmentation of natural killer cytotoxicity by alpha or gamma natural and recombinant interferons (IFN) and certain interferon inducers. We observed that: (1) in the majority of the donors examined (75%) human peripheral blood monocytes do not affect natural killer cytotoxicity, determined by a 4-hour chromium-51 release assay, against target cells from hemopoietic human tumor cell lines. (2) Monocytes are not required and do not affect the augmentation of natural killer cytotoxicity by Escherichia coli-derived IFN-gamma, natural human IFN-gamma, E. Coli-derived IFN-alpha 2 or natural human IFN-alpha. E. Coli-derived IFN-gamma and natural human IFN-gamma have been reported to activate monocyte cytotoxicity determined in 72-hour assay. (3) Monocytes are not required for the augmentation of natural killer cytotoxicity against target cells from hemopoietic tumor cell lines by polyinosinic acid-polycytidylic acid or staphylococcal enterotoxin A.     

Activation of natural killer cells via the Fc gamma RIII (CD16) requires initial tyrosine phosphorylation.

Triggering of the Fc gamma RIII (CD16) on natural killer (NK) cells by monoclonal antibodies or antibody-coated target cells stimulates a rapid phospholipase C (PLC)-mediated hydrolysis of inositol phospholipids and results in subsequent delivery of the lytic hit. The role of initial tyrosine phosphorylation in these events was investigated with a tyrosine protein kinase (TPK) inhibitor, genistein. At doses that inhibited CD16-triggered tyrosine phosphorylation of substrates in intact cells, genistein did not influence serine/threonine phosphorylation or target cell binding but prevented PLC activation, cell-mediated cytotoxicity and antibody-dependent cellular cytotoxicity. These findings indicate that tyrosine phosphorylation is an early and critical event during receptor-mediated activation of the lytic machinery.     

Anti-Gal IgG potentiates natural killer cell migration across porcine endothelium via endothelial cell activation and increased natural killer cell motility triggered by CD16 cross-linking

Xenoreactive antibodies (Ab) are important for the development of acute vascular rejection (AVR) of xenografts characterized by monocytes, natural killer (NK) cells and neutrophils infiltrating the graft. The mechanisms by which anti-galactose alpha 1,3galactose (alpha-Gal) IgG influence NK cell migration across porcine aortic endothelium (PAEC) were investigated. NK cell migration across PAEC increased in the presence of anti-alpha-Gal IgG. Anti-alpha-Gal IgG exposure activated PAEC as shown by an increased expression of CD62E and CD106. NK cells adhered, spread and showed motile forms on plastic surfaces coated with human IgG, IgG Fc and on mAb against CD16, but not on mouse IgG or BSA, suggesting that CD16 cross-linking can mediate increased adhesiveness. Increased NK cell motility was observed on Boyden filters coated with human IgG, IgG Fc, and mAb against CD16 and the alpha 4, alpha 5, alpha L, beta 1 and beta 2 integrin chains. No motile response was seen on mouse IgGor CD7, CD56 and alpha 6 integrin mAb. NK cell migration on human IgG and anti-CD16 Ab was blocked by anti-CD16 or anti-beta 2, but not anti-beta 1 Ab, implying that the motile response triggered by CD16 cross-linking is mediated via beta 2 integrins. Preformed or induced anti-alpha-Gal IgG may therefore contribute to AVR by stimulating innate immune cell infiltration of the graft.     

Functions of uterine natural killer cells are mediated by interferon gamma production during murine pregnancy.

The dominant lymphocytes in healthy human and murine implantation sites are pregnancy-associated uterine natural killer (uNK) cells. These cells produce 90% of pregnancy-induced, uterine interferon (IFN)- gamma, a cytokine that regulates expression of more than 0.5% of the mouse genome. Implantation sites in uNK cell-deficient and IFN- gamma -signal-disrupted mice display anomalies in decidua and its spiral arteries. Reconstitution of uNK cell-deficient females with bone marrow containing normal NK cell progenitors, establishes uNK cells and reverses the anomalies. Grafts from IFN- gamma(-/-)mice are restored uNK cells, but the uNK cells did not reverse the phenotypes. This review focuses on the functions of uNK cell-derived IFN- gamma and the genes that it may regulate in the pregnant uterus.     

Patients with condyloma acuminatum exhibit decreased interleukin-2 and interferon gamma production and depressed natural killer activity

Peripheral blood mononuclear cells were obtained from 20 untreated condyloma acuminatum patients and from an equal number of sex- and age-matched controls and assayed for cell surface antigen expression, natural killer activity, and lymphokine production. Patient peripheral blood mononuclear cells had significantly lower helper-to-suppressor T-cell ratios (Leu3/Leu2) (p<0.05) and significantly higher percentages of Leu 2+ Tac+ cells (activated suppressor/cytotoxic cells) (P<0.05) and Leu 2+ OKM1+ cells (suppressor cells) (P<0.01). Natural killer activity of condyloma acuminatum patients was significantly lower (P<0.05) than that of controls. Production of interleukin-2 and interferon gamma, but not interferon alpha, was significantly (P<0.01) decreased in condyloma acuminatum patients. There was an inverse correlation between thein vitro production of interleukin-2 and interferon gamma and the percentage of Leu 2+ OKM1+ cells (suppressor) (P<0.01). Thus, patients with condyloma acuminatum differ from controls by demonstrating (1) decreased natural killer-cell activity, (2) decreased production of lymphokines which enhance natural killer-cell activity (i.e., interferon gamma and interleukin-2), and (3) an increased proportion of T cells with a suppressor phenotype.     

Early gamma interferon production by natural killer cells is important in defense against murine listeriosis.

A spot enzyme-linked immunosorbent assay was used to show that subcutaneous inoculation of a sublethal number of Listeria monocytogenes resulted in the early appearance of gamma interferon (IFN-gamma)-producing cells in the draining lymph nodes. In contrast, inoculation of UV-killed L. monocytogenes failed to cause the appearance of IFN-gamma-producing cells. The appearance of IFN-gamma-secreting cells in response to the living organisms peaked at 24 h of infection and then declined. The draining lymph node cells responsible for secreting IFN-gamma belonged to a cell population that was positive for the NK1.1, asialo-GM1, and Thy-1 markers but negative for the CD4 and CD8 T cell subset markers. Early elimination of natural killer (NK) cells by treatment with anti-NK cell antibodies resulted in severe exacerbation of infection, as did early neutralization of endogenous IFN-gamma by treatment with a rat anti-murine IFN-gamma monoclonal antibody. In contrast, depletion of CD4+ and CD8+ T cells failed to exacerbate infection. The results serve to show that the early production of IFN-gamma by NK cells, rather than by T cells, is an essential event in resistance to listeriosis.     

CD40-CD154 interactions between macrophages and natural killer cells during sepsis are critical for macrophage activation and are not interferon gamma dependent

Natural killer (NK) cell interactions with macrophages have been shown to be important during bacterial sepsis in activating macrophages to improve bacterial clearance. The mechanism for this increased activation, however, is unclear. This study determines the relative roles of interferon (IFN)-gamma and CD40/CD154 direct cell interactions on macrophage and NK cell activation in an experimental model of sepsis. Splenic NK cells and peritoneal macrophages were isolated and cultured alone or in coculture, with and without LPS. CD69 expression on NK cells, phagocytosis ability of macrophages, and cell cytokine production was assessed at 24 and 48 h. Coculture of NK cells and macrophages significantly increased activation levels of both cell types, and through experiments culturing NK cells with supernatants from stimulated macrophages and macrophages with supernatants from stimulated NK cells, this activation was determined to be cell-contact-dependent. Similar experiments were conducted using NK cells from IFN-gamma deficient (-/-) mice, as well as anti-IFN-gamma neutralizing antibody. These experiments determined that IFN-gamma is not required for NK or macrophage activation, although it did augment activation levels. Experiments were again repeated using peritoneal macrophages from CD40-/- mice or splenic NK cells from CD154-/- mice. CD40/CD154 interactions were important in the ingestion of bacteria by macrophages, but did not affect NK cell activation at 24 h. There was, however, a protective effect of CD40/CD154 interactions on NK cell activation-induced cell death that occurred at 48 h. CD40/CD154 interactions between macrophages and NK cells are therefore important in macrophage phagocytosis, and are not dependent on IFN-gamma.     

Contact of lymphocytes with Helicobacter pylori augments natural killer cell activity and induces production of gamma interferon.

We studied the capacity of glutaraldehyde-fixed Helicobacter pylori to stimulate natural killer (NK) cell activity. Bacteria were incubated overnight with peripheral blood lymphocytes enriched for large granular lymphocytes (LGL), the mediators of non-major histocompatibility complex-restricted cellular cytotoxicity. Then, the cytolytic activity of LGL was tested against various tumor target cells. We observed that efficient cytolytic activity was generated against resistant and nonresistant tumor target cell lines. Nine local clinical isolates of H. pylori and the reference strain NCTC 11637 were tested, and they all were equally effective in inducing NK cell activity. However, flagellin antigen, glycine extract, urease, and lipopolysaccharide prepared from H. pylori NCTC 11637 all failed to induce significant NK cell activity. The supernatants which were collected after coincubation of bacteria with LGL contained a factor(s) which could activate resting LGL into efficient cytolytic activity. The supernatants were also analyzed for interferon (IFN) activity. We observed that high titers of IFN were produced and that IFN activity was neutralized with anti-gamma interferon (IFN-gamma) antiserum, but not with anti-IFN-alpha antiserum. Thus, contact of lymphocytes with H. pylori leads to efficient stimulation of NK cell activity and the production of IFN-gamma.     

Interleukin 2 enhances natural killer cell activity through induction of gamma interferon

Highly purified interleukin 2 (IL 2), free of interferon activity, enhanced natural killer (NK) cell activity against tumor cells in mouse spleen cell cultures and in human peripheral lymphocyte cultures in a manner similar to that of interferon (IFN). We determined that IL 2 enhanced NK activity indirectly in a cascade manner by the induction of gamma IFN (IFN-gamma) in the cultures, which actually mediated the enhanced killing. Accordingly, lymphocyte cultures treated with IL 2 alone produced 10 to 100 U of IFN per ml in 6 to 24 h of culture. The IFN was typed as IFN-gamma by specific antibodies. Specific antibodies either to natural IFN-gamma or to a synthetic peptide corresponding to the human IFN-gamma N-terminal amino acids, when added to cultures treated with IL 2, completely blocked IL 2 enhancement of NK cell activity for both the mouse and human systems. IL 2-induced proliferation was not affected by the antibodies. Thus, the enhancement of NK cell activity by IL 2 is completely mediated by IL 2-induced IFN-gamma. The findings clearly indicate a cascade effect whereby one lymphokine (IL 2) induces the production of another. The latter lymphokine (IFN-gamma) then mediates an important biological effect (natural killing).     

Cytokine production by CD16 CD56bright natural killer cells in the human early pregnancy decidua

Using a cell sorter, CD16^–CD56^bright natural killer (NK)cells were sorted from decidual mononuclear cells at an earlystage of pregnancy. These cells were examined by the reversetranscrlptase-polymerase chain reaction (RT-PCR) method fortheir expression of mRNA coding for the following 12 cytokines:IL-1ß, IL-2, IL-3, IL-4, IL-5, IL-6, granulocyte colony-stimulatingfactor (G-CSF), granulocyte-macrophage colony-stimulating factor(GM-CSF), macrophage colonystimulating factor (M-CSF), tumornecrosis factor- (TNF-), interferond- (IFN-), and leukemia inhibitoryfactor (LIF). Although mRNA coding for every cytokine was detectedin decidual mononuclear cells, mRNAs coding for only G-CSF,GM-CSF, M-CSF, TNF-, IFN-, and LIF were detected In CD16^–CD56^brightNK cells. Also, the supernatant of CD16^–CD56^bright NKcell cultures was found to contain G-CSF, GM-CSF, M-CSF, TNF-,IFN-, and LIF. These findings indicate that CD16^–CD56^brightNK cells produce many different cytokines and that these cytokinesmay play an important role in a successful pregnancy.     

Enhanced gamma interferon production through activation of Valpha14(+) natural killer T cells by alpha-galactosylceramide in interleukin-18-deficient mice with systemic cryptococcosis

We showed recently that activation of Valpha14(+) natural killer T cells (NKT cells) by alpha-galactosylceramide (alpha-GalCer) resulted in increased gamma interferon (IFN-gamma) production and host resistance to intravenous infection with Cryptococcus neoformans. In other studies, interleukin-18 (IL-18) activated NKT cells in collaboration with IL-12, suggesting the possible contribution of this cytokine to alpha-GalCer-induced IFN-gamma synthesis. Here we examined the role of IL-18 in alpha-GalCer-induced Th1 response by using IL-18KO mice with this infection. In these mice, levels of IFN-gamma in serum and its synthesis in vitro by spleen cells stimulated with live organisms were not reduced, but rather enhanced, compared to those in wild-type (WT) mice, while such production was completely absent in IL-12KO mice. The enhanced production of IFN-gamma correlated with increased IL-12 synthesis but not with reduced production of IL-4, which was rather increased. IFN-gamma synthesis in IL-18KO mice was abolished by neutralizing anti-IL-12 antibody and significantly inhibited by neutralization of endogenous IL-4 with a specific monoclonal antibody. In addition, administration of recombinant IL-4 significantly enhanced the production of IFN-gamma in WT mice. Finally, the enhanced production of IFN-gamma in IL-18KO mice correlated with increased host defense against cryptococcal infection, as indicated by enhancement in alpha-GalCer-related clearance of microorganisms. Our results indicated that in IL-18KO mice, IFN-gamma synthesis was enhanced through overproduction of IL-12 and IL-4 after intravenous infection with C. neoformans and a ligand-specific activation of Valpha14(+) NKT cells.     

Effect of interferon alpha on natural killer cell cytotoxicity in kidney transplant recipients

Natural killer (NK) cell cytotoxicity was monitored in 32 kidney transplant recipients during a double-blind, placebo-controlled trial of interferon alpha prophylaxis. During the first month following transplantation, NK cytotoxicity diminished in both interferon and placebo-treated patients. Thereafter, mean values for NK cytotoxicity decreased further in interferon recipients, whereas they returned toward baseline in placebo recipients. Paradoxically, interferon alpha consistently augmented NK cytotoxicity of all patients in vitro. Various immunosuppressive regimens were employed in these patients. NK cytotoxicity was slightly diminished in patients receiving prednisone and azathioprine, moderately depressed in those who received prednisone, azathioprine, and OKT3 antibody. Three or four placebo recipients with clinically severe cytomegalovirus (CMV) infections had diminished NK cell function. These studies indicate that a variety of factors including exogenous interferon, immunosuppressive regimens, and CMV infections can influence NK cytotoxicity in renal transplant recipients.     

CXCL16 regulates cell-mediated immunity to Salmonella enterica serovar Enteritidis via promotion of gamma interferon production

CXCL16 is a recently discovered multifaceted chemokine that has been shown not only to recruit activated T lymphocytes but also to play a direct role in the binding and phagocytosis of bacteria by professional antigen-presenting cells. In this study, we investigated the role of CXCL16 in vivo in the regulation of the immune response using a murine model of Salmonella enterica serovar Enteritidis infection. The expression of CXCL16 was strongly upregulated in the spleens and livers of animals developing an immune response to a primary acute infection but not in the Peyer's patches. Animals developing a secondary response after reexposure to the bacteria displayed a similar pattern of expression. During the primary response, prior treatment with neutralizing antibodies to CXCL16 induced a significant increase in bacterial burden in the spleen and liver. The production of gamma interferon (IFN-gamma) by the lymphocytes in the spleen was decreased by anti-CXCL16 treatment. In comparison, during the secondary response, anti-CXCL16 treatment also significantly increased bacterial burden in both the spleen and liver but had no effect on IFN-gamma production. No role was found for CXCL16 in the production of antibody against SefA, a major surface antigen of S. enteritidis. Together, these results demonstrate a role for CXCL16 in the control of bacterial colonization of target organs and, more specifically, in the regulation of the cell-mediated arm of the primary response to S. enteritidis.     

Expression of the CD80 and CD86 molecules enhances cytotoxicity by human natural killer cells

In contrast to the inhibitory pathway of NK cell regulation, much less is known about stimulatory or activation signals in NK cells. Both CD80 and CD86 function as costimulatory molecules in T-cell cytotoxicity. Several previous reports, most of them in the murine system, have indirectly or directly indicated the possible role of B7 molecules (CD80 and CD86) triggering NK cell-mediated cytotoxicity in vitro. Nevertheless, only little is known about the role of these molecules on human target cells. Therefore, anti-CD80 and anti-CD86 mAbs were used in blocking experiments and both were shown to inhibit lysis by human NK cells. The degree of inhibition observed was variable. 64% of these NK clones were strongly inhibited by both anti-CD80 and anti-CD86 (Type 1). A small number (19%) were only moderately inhibited by both of these antibodies (Type 2), and 17% of these NK clones were inhibited strongly by anti-CD86 but weakly or not at all by anti-CD80 (Type 3). To further examine the importance of these proteins, B7.1 (CD80) and B7.2 (CD86) genes were transfected into the mouse mastocytoma P815 cell line that could not be killed by the human NK cells. These transfectant cell lines were then tested in cytotoxicity assays using a number of human NK lines. Expression of the CD80 and CD86 molecules resulted in enhanced lysis of P815 by most of the NK lines tested. Thus, both CD80 and CD86 molecules are involved in triggering of human NK cells.     

Association of decreased T-cell-mediated natural cytotoxicity and interferon production in Down's syndrome

Total peripheral blood lymphocytes (PBL) and isolated subpopulations from children with Down's Syndrome (DS) and age-matched healthy controls were investigated for their (1) natural killer (NK) and antibody-dependent cellular cytotoxic activities, (2) interleukin 2 (IL-2)-induced augmentation of NK activity, (3) lectin-dependent cellular cytotoxicity (LDCC), (4) ability of serum- and culture-derived soluble suppressor factor(s) to inhibit NK activity of normal lymphocytes, and (5) capacity to produce interferon (IFN) against tumor targets in vitro. T lymphocytes from DS patients demonstrated significantly decreased NK activity against K562 target cells compared to controls. DS lymphocytes also demonstrated a significant reduction in LDCC activity and IL-2-induced enhancement of NK activity. Furthermore, the ability of DS lymphocytes to produce IFN in vitro against K562 target cells was also significantly lower than that for normal PBL. Although sera from DS patients showed a significantly greater inhibitory effect on the NK activity of allogeneic normal PBL than normal sera, culture supernates from DS lymphocytes demonstrated suppressive effects comparable to culture supernates from normal PBL. These studies suggest an association between the decreased NK activity of T-cell subpopulations and lower IFN production by PBL from patients with DS.     

Phenotypical and functional profiles of natural killer cells exhibiting matrix metalloproteinase-mediated CD16 cleavage after anti-HIV antibody-dependent activation

Natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC) has been linked to protection from HIV infection and slower progression towards AIDS. However, antibody-dependent activation of NK cells results in phenotypical alterations similar to those observed on NK cells from individuals with progressive HIV infection. Activation of NK cells induces matrix metalloproteinase (MMP)-mediated cleavage of cell surface CD16. In the present study we assessed the phenotype and functional profile of NK cells exhibiting post-activation MMP-mediated CD16 cleavage. We found that NK cells achieving the highest levels of activation during stimulation exhibit the most profound decreases in CD16 expression. Further, we observed that educated KIR3DL1⁺ NK cells from human leucocyte antigen (HLA)-Bw4-carrying donors exhibit larger decreases in CD16 expression post-activation than the KIR3DL1⁻ NK cell subset containing cells educated via other inhibitory receptor/ligand combinations and non-educated NK cells. Lastly, we assessed the ex-vivo expression of CD16 on educated KIR3DL1⁺ NK cells and the KIR3DL1⁻ NK cell subset from HLA-Bw4-carrying HIV-uninfected and HIV-infected donors. Suggestive of in-vivo activation of KIR3DL1⁺ NK cells during HIV infection, CD16 expression was higher on KIR3DL1⁺ than KIR3DL1⁻ NK cells in uninfected donors but similar on both subsets in HIV-infected donors. These results are discussed in the context of how they may assist with understanding HIV disease progression and the design of immunotherapies that utilize antibody-dependent NK cell responses.