Development of an In Vitro Model of Inflammatory Cytokine Influences on Intervertebral Disk Cells in 3D Cell Culture Using Activated Macrophage-Like THP-1 Cells

Bulletin of Experimental Biology and Medicine - We developed a new model for evaluation of the influence of proinflammatory cytokines on intervertebral disc cells in a 3D culture based on...     

The Role of NK Cells and NK Cell Receptors inAutoimmune Disease

Natural killer (NK) cells are the first line of defense against infection and transformation. Additionally, NK cells can play seemingly opposite roles in autoimmune disease. Here, we summarize the functions of NK cells as both regulators and inducers of autoimmune disease. The role NK cells play depends on which cells become targets for NK cell attack. The activity of NK cells is controlled by inhibitory receptors specific for MHC Class I molecules, and by activating receptors with diverse specificities. The ligands for both activating and inhibitory receptors are present on potential target cells. It is the balance in expression of these different ligands that determines NK cell activation and therefore whether the cell becomes a target for NK cell-mediated killing. We further discuss the roles of NK cell receptors and their ligands in autoimmune disease.     

Mice with Asthma Are More Resistant to Influenza Virus Infection and NK Cells Activated by the Induction of Asthma Have Potentially Protective Effects

Purpose  

This study was conducted in order to investigate whether the virulence of the influenza virus infection is affected by asthma in mice.     

Elevated NK Cell Cytotoxicity,CD158a Expression in NK Cells and Activated T Lymphocytes in Peripheral Blood of Women with IVF Failures

Citation Chernyshov VP, Sudoma IO, Dons’koi BV, Kostyuchyk AA, Masliy YV. Elevated NK cell cytotoxicity, CD158a expression in NK cells and activated T lymphocytes in peripheral blood of women with IVF failures. Am J Reprod Immunol 2010; 64: 58–67 Problem The aim of this study was to evaluate the role of elevated natural killer cytotoxicity (NKc) in women with multiple implantation failures (IF) in vitro fertilization–embryo transfer (IVF–ET) cycles. Methods of study Seventy‐nine antiphospholipid antibodies‐negative women with IF including 33 women with elevated NKc were selected for investigation. K‐562 cell line was used to evaluate NKc. Lymphocyte subsets, intracellular cytokines [interferon (IFN)‐γ, interleukin (IL)‐4, tumour necrosis factor, IL‐10], expression of activating markers [CD69, human leukocyte antigen (HLA)‐DR], CD8, KIR (CD158a), CD95, and chemokine receptors (CXCR3, CCR4) were estimated by flow cytometry. Results In women with IF, levels of NKc were higher than in IVF successful women. IF was associated with higher expression of CD8, CD158a, and HLA‐DR in NK cells, activating markers in T lymphocytes, and lower levels of CCR4+ and IL‐4+ T lymphocyte subsets. Predictive value of single elevated NKc for IVF success was 0.85, but addition of two other abnormal parameters resulted in its decrease to <0.39. Conclusions Elevated NKc is negative factor, though not critical for implantation in IVF cycles. Immune mechanism of IVF failure includes not only elevated NKc but also some other factors, such as elevated expression of CD8 and CD158a on NK cells, T lymphocyte activation, and diminished T helper 2 parameters.     

Ag-Specific Type 1 CD8 Effector Cells Enhance Methotrexate-Mediated Antitumor Responses by Modulating Endogenous CD49b-Expressing CD4 and CD8 T Effector Cell Subpopulations Producing IL-10

The chemotherapeutic agent methotrexate is widely used in the treatment of breast cancer. Although its mechanism-of-action has been defined, less is known about its interaction with Ag-specific T cell-mediated antitumor responses. Type 1 CD8 T cell-mediated immune responses (Tc1) are cytolytic, produce IFN-gamma and are associated with effective antitumor responses. Using a murine transgenic TCR tumor model, we show that single-dose-treatment with methotrexate enhanced CD8-mediated type 1 antitumor responses when administered three days prior to Tc1 effector cell transfer. Co-treatment with methotrexate not only enhanced donor Tc1 cell accumulation and persistence at sites of primary tumor growth, but also promoted elevated levels of activated CD25⁺ expressing donor TIL cells. This correlated with a marked decrease in the appearance of endogenous differentiated (CD44^High) CD3/CD8/CD49b and CD3/CD4/CD49b tumor-infiltrating effector T cells at both early (Days 1–8) and late (Days 12–20) stages following treatment when compared to that of corresponding groups receiving either MTX or Tc1 cell transfer alone. Moreover, such cellular response kinetics appeared to further correlate with the down-regulation of endogenous CD4/CD44^High/CD49b effector T cells producing IL-10 and delays in tumor growth in vivo. This suggested that Ag-specific Tc1 cell transfer, in combination with chemotherapy, can enhance antitumor responses by modulating select CD49b-expressing T effector/memory cell subpopulations involved in homeostasis and immune tolerance within the tumor environment. These studies offer insight into mechanisms that enhance T cell-based immunotherapy in cancer. Supplementary materials are available for this article. Go to the publisher's online edition of Immunological Investigations for the following free supplemental resource(s): Addendum 1.     

Ovarian Stimulation Affects the Levels of Regulatory Endometrial NK Cells and Angiogenic Cytokine VEGF

Citation Junovich G, Mayer Y, Azpiroz A, Daher S, Iglesias A, Zylverstein C, Gentile T, Pasqualini S, Markert UR, Gutiérrez G. Ovarian stimulation affects the levels of regulatory endometrial NK cells and angiogenic cytokine VEGF. Am J Reprod Immunol 2011; 65: 146–153 Problem Endometrial NK cells play a critical role in uterine vascularization producing angiogenic factors. Impact of ovarian stimulation on endometrial expression of NK cells and VEGF in normal fertile oocyte donors and the effect of endometrial injury treatment on these parameters have been investigated. Method of study Endometrial tissue was obtained from oocyte donors during natural and stimulated cycles. NK cell subsets were measured by flow cytometry. VEGF was determined by ELISA and flow cytometry. Endometrial angiogenic parameters were determined by ultrasound Doppler. Local injury was induced by scratching endometrial tissue previous to implantation window. Results Ovarian stimulation decreased endometrial levels of NK cells and vascularization index but increased VEGF levels. Local injury normalized only the CD56⁺ NK cell count. Conclusion Hormonal therapy for ovarian stimulation may be associated with poor endometrial vascularization. Local injury before the implantation window seems not to influence endometrial angiogenic parameters altered by ovarian stimulation.     

Regulation of Effector Functions of Human K-Cells and Monocytes by Antigen Density of the Target Cells

Antibody-dependent cytolysis and phagocytosis mediated by human K-cells or monocytes against chicken erythrocytes (ChRBC) were studied. The antigen density of the target cells was varied by coating the cells with different amounts of 3H-labelled dinitrophenyl (DNP) hapten. The degree of antigenicity thus acquired by the target cells was assessed on the basis of their uptake of the isotope. Anti-DNP serum was used to induce lysis or phagocytosis. Below 500 antigenic determinants per ChRBC the target cells were not affected. However, at the density, lysis and/or phagocytosis was seen when the antibody concentration was high (2 X 10(-9) M). With less antibody present (2 X 10(-11) M) only monocyte-mediated phagocytosis was induced. The estimated lowest number of target-cell-bound antibodies required for K-cell-mediated lysis was approximately 50. The corresponding number for monocyte-mediated phagocytosis was approximately 20 IgG per ChRBC. The result suggests that interaction of several Fc receptors on the effector cells with IgG molecules bound to adjacent sites on the target cell membrane is an important factor in the regulation of these antibody-dependent cell-mediated effector functions.     

CD38brightCD8+ T Cells Associated with the Development of Acute GVHD Are Activated,Proliferating, and Cytotoxic Trafficking Cells

We have previously reported that a peripheral blood absolute CD38^brightCD8⁺ effector memory T cell (TEM) population expansion of >35 cells/µL predicts the development of acute graft-versus-host disease (GVHD). We hypothesized that these T cells are activated, proliferating, and cytotoxic trafficking cells that are not a response to viral reactivation and may be involved in acute GVHD. We characterized peripheral blood T cell populations at the time of maximum CD38^brightCD8⁺ TEM expansion in patients from our originally reported pediatric allogeneic hematopoietic cell transplantation recipient cohort. Samples were incubated with fluorochrome-conjugated antibodies directed against CD3, CD8, CD38, HLA-DR (T cell activation), Ki-67 (T cell proliferation), granzyme B (marker of cytotoxic T cells), CLA (skin trafficking), CCR5 (visceral trafficking), and CXCR6 (liver trafficking). We also incubated samples with Epstein-Barr virus (EBV) and cytomegalovirus (CMV) peptide pools and measured IFN-γ production by flow cytometry and performed EBV and CMV tetramer staining. Higher median proportions of cell expression of HLA-DR, Ki-67, granzyme B, CLA, CCR5, and CXCR6 were observed for CD38^brightCD8⁺ T cells compared with CD38^nonbrightCD8⁺ T cells in patients with acute GVHD (P < .05) but not in patients without acute GVHD (P not significant). No IFN-γ production was observed after incubation with CMV and EBV peptide pools. EBV-specific tetramer populations of 6.85% and 3.17% were detected in 2 patients with acute GVHD, whereas a CMV-specific tetramer population of 3.77% was detected in 1 patient with acute GVHD. No EBV- or CMV-specific tetramer populations were detected in any patient without acute GVHD. We conclude that CD38^brightCD8⁺ T cells associated with the development of acute GVHD are activated, proliferating, and cytotoxic trafficking cells that do not appear to respond to CMV or EBV reactivation. Further studies are needed to determine whether these cells are directly involved in acute GVHD pathogenesis.     

Effect of Acute Pain on Splenic NK Cell Activity,Lymphocyte Proliferation and Cytokine Production Activities

Various types of physical and physiological stress in animals have been shown to affect their humoral and cell-mediated immune responses. The present study was designed to investigate the possible influence of acute pain on the immune system. BALB/c mice were exposed to an increasing number of heat shocks using a Tail Flick apparatus; an equal number of control mice received no shock treatments. After each of the regimens was completed, the spleen of each mouse was recovered and various cell populations isolated to assess: the proliferative response to phytohemagglutinin by lymphocytes; cytotoxic activities of natural killer (NK) cells; and, the production of select important cytokines by splenic lymphocytes. The results indicated that NK cell activity and proliferation of lymphocytes were significantly (p < 0.001) increased due to the shock regimens after only a single day's rounds of stimulation (i.e., 3 rounds of ≈12 equally time-spaced shocks/hr with 30–45 min gap between rounds). After 2 and 3 days' rounds of stimulations, no significant changes were detected in the proliferative response of isolated lymphocytes; conversely, the activity of NK cells remained significantly elevated compared to the controls hosts' cells, even on the second day of stimulation but not on the third. Regarding effects on cytokines, no significant changes were detected in the amount of Interferon-γ (IFNγ) and Interlukin-10 (IL-10) produced by lymphocytes obtained from the spleens of any of the shocked mice. These results could suggest that certain acute stressors might actually strengthen a host's immunological reactivity and, possibly, result in an enhanced capacity to resist pathogens that might infect the body.     

Primary B-CLL Resistance to NK Cell Cytotoxicity can be Overcome In Vitro and In Vivo by Priming NK Cells and Monoclonal Antibody Therapy

Despite recent advances with monoclonal antibody therapy, chronic lymphocytic leukemia (CLL) remains incurable. Natural killer (NK) cells are potent antitumoral effectors, particularly against hematological malignancies. Defective recognition of B-CLL leukemic cells by NK cells has been previously described. Here, we deciphered the mechanisms that hamper NK cell-mediated clearance of B-CLL and evaluated the potential of NK cells as therapeutic tools for treatment of CLL. First of all, leukemic B cells resemble to normal B cells with a weak expression of ligands for NK receptors. Conversely, NK cells from B-CLL patients were functionally and phenotypically competent, despite a decrease of expression of the activating receptor NKp30. Consequently, resting allogeneic NK cells were unable to kill leukemic B cells in vitro. These data suggest that patients' NK cells cannot initiate a proper immune reaction due to a lack of leukemic cell recognition. We next set up a xenotransplantation mouse model to study NK-CLL cell interactions. Together with our in vitro studies, in vivo data revealed that activation of NK cells is required in order to control B-CLL and that activated NK cells synergize to enhance rituximab effect on tumor load. This study points out the requirements for immune system manipulation for treatment of B-CLL in combination with monoclonal antibody therapy.     

Two Novel Functions of Hyaluronidase from Streptococcus agalactiae Are Enhanced Intracellular Survival and Inhibition of Proinflammatory Cytokine Expression

Streptococcus agalactiae is the causative agent of septicemia and meningitis in fish. Previous studies have shown that hyaluronidase (Hyl) is an important virulence factor in many Gram-positive bacteria. To investigate the role of S. agalactiae Hyl during interaction with macrophages, we inactivated the gene encoding extracellular hyaluronidase, hylB, in a clinical Hyl⁺ isolate. The isogenic hylb mutant (Δhylb) displayed reduced survival in macrophages compared to the wild type and stimulated a significantly higher release of proinflammatory cytokines, such as interleukin-1β (IL-1β), IL-6, and tumor necrosis factor alpha (TNF-α), than the wild type in macrophages as well as in mice. Furthermore, only Hyl⁺ strains could grow utilizing hyaluronic acid (HA) as the sole carbon source, suggesting that Hyl permits the organism to utilize host HA as an energy source. Fifty percent lethal dose (LD₅₀) determinations in zebrafish demonstrated that the hylb mutant was highly attenuated relative to the wild-type strain. Experimental infection of BALB/c mice revealed that bacterial loads in the blood, spleen, and brain at 16 h postinfection were significantly reduced in the ΔhylB mutant compared to those in wild-type-infected mice. In conclusion, hyaluronidase has a strong influence on the intracellular survival of S. agalactiae and proinflammatory cytokine expression, suggesting that it plays a key role in S. agalactiae pathogenicity.     

Effects of X-RAY Irradiation on Natural Killer (NK) Cell System. L Elevation of Sensitivity of Tumor Cells and Lytic Function of NK Cells

The in vitro effect of X-ray irradiation on the human natural killer (NK) system was studied. When K562 cells were irradiated with X-rays and cultured for 18 hours, they showed increased sensitivity to lysis by blood lymphocytes and purified large granular lymphocytes (LGL). The X-ray-induced augmentation was observed as little as 2 Gy irradiation, reaching maximum at 5 to 20 Gy. The doses of X-rays did not influence the viability and spontaneous release of the target cells. On the other hand, irradiation with X-rays of NK cells at 5 to 15 Gy resulted in a transient increase in NK activity at 1 hour, and then the activity declined and was completely lost after 24 hours. However, when LGL were cultured with interferon immediately after irradiation, they maintained elevated NK activity. These results suggest the possible use of low doses of X-ray irradiation in combination with biological response modifiers for treatment of cancer.     

Effects of X-RAY Irradiation on Natural Killer (NK) Cell System. L Elevation of Sensitivity of Tumor Cells and Lytic Function of NK Cells

Abstract

The in vitro effect of X-ray irradiation on the human natural killer (NK) system was studied. When K562 cells were irradiated with X-rays and cultured for 18 hours, they showed increased sensitivity to lysis by blood lymphocytes and purified large granular lymphocytes (LGL). The X-ray-induced augmentation was observed as little as 2 Gy irradiation, reaching maximum at 5 to 20 Gy. The doses of X-rays did not influence the viability and spontaneous release of the target cells. On the other hand, irradiation with X-rays of NK cells at 5 to 15 Gy resulted in a transient increase in NK activity at 1 hour, and then the activity declined and was completely lost after 24 hours. However, when LGL were cultured with interferon immediately after irradiation, they maintained elevated NK activity. These results suggest the possible use of low doses of X-ray irradiation in combination with biological response modifiers for treatment of cancer.     

Comparison of the Effector Cells in Human Spontaneous Cellular Cytotoxicity and Antibody-Dependent Cellular Cytotoxicity: Differential Sensitivity of Effector Cells to in Vivo and in Vitro Corticosteroids

The effector cells mediating antibody dependent cellular cytotoxicity (ADCC) and spontaneous cellular cytotoxicity (SCC) in humans have been reported to possess similar characteristics. Multiple cell separation techniques were employed in an attempt to physically separate and distinguish the effector cells in these two types of cellular cytotoxicity. Subpopulations of mononuclear cells obtained by a variety of fractionation procedures which either enriched or depleted monocytes, lymphocytes bearing a receptor for sheep erythrocytes (SRBC), a receptor for complement (CRL) or an Fc receptor for IgG always had similar effects on both ADCC and SCC. Aggregated gamma globulin blockade of Fc receptors produced similar dose-dependent depressions of ADCC and SCC. Despite our inability to physically separate the effector cells of ADCC and SCC, administration of in vivo dexamethasone caused a relative increase in ADCC but a profound decrease in SCC. Furthermore, in vitro dexamethasone in pharmacologic and suprapharmacologic concentrations caused no change in ADCC but significantly decreased SCC. This study demonstrates that although the effector cells cannot be physically separated, ADCC and SCC are differentially sensitive to corticosteroids and are hence functionally distinct either on the basis of different subsets of effector cells with similar surface markers or different mechanisms of cytotoxicity by the same effector cell.     

A Monoclonal Antibody, DL10, Which Recognizes a Sugar Moiety of MHC Class I Antigens Expressed on NK Cells, NK+ T Cells, and Granulocytes in Humans

One mAb, DL10, was established from mice injected with dolphin lymphocytes. In addition to its reactivity against all dolphin lymphocytes, it reacted with some human leukocytes, including NK cells, NK⁺ T cells, and granulocytes. When its reactivity was examined in various animals, bovine, ovine, and equine leukocytes were DL10⁺. Murine, rat, and canine leukocytes were DL10^–. Although the reactivity of DL10⁺ was similar to those of CD56 and CD57 antigens in humans, the actual molecules it recognized were different. Thus, all reactivity of DL10 disappeared after treatment of cells with glycopeptidase or after culture of cells with tunicamycin. Furthermore, the immunoprecipitation method revealed that DL10 indirectly recognized the heavy chain (45kD) of MHC class I antigen in humans and animals. Considering data from analysis of the N-terminal amino acid sequence of the DL10 molecule and the HLA typing of reactive cells, DUO recognized a sugar moiety of some monomorphic MHC antigens and polymorphic MHC antigens such as HLA-B60 and -B61. If the donors are HLA-B60^– and -B61^– (>80% in Japan and >95% in the United States), DL10 would appear to be a very useful agent for the detection of pan-NK⁺ T cells.     

A Comparative Analysis of Cell Surface Markers on Murine NK Cells and CTL Target-Effector Conjugates

The rosetting of sheep erythrocytes (SRBC) coated with non-haemagglutinating monoclonal antibodies rather than conventional haemagglutinating antisera revealed readily detectable FcR on most splenic natural killer (NK) cells since 76% of splenic lymphocytes forming conjugates with YAC also resetted with SRBC coated with high concentrations of monoclonal anti-SRBC antibody of the IgG2b subclass and since Ficoll depletion or enrichment of splenic lymphocytes rosetting with IgG2b-coated SRBC resulted in a corresponding 4-fold decrease or increase in conjugate-forming cells and a 10-fold decrease or increase in NK cytolytic acttvity. NK cells bound much less readily to monoclonal IgG2a and not at all to monoclonal IgGI or IgM, but the degree of binding was directly proportional to the amount of antibody on the erythrocytes and was not isotype-restricted. In addition, immunofluorescent studies revealed that YAC-1-conjugated lymphocytes were Lyt-1^-, Lyt-2^-, partially Thy-1⁺ (60%), asiato-GMI ⁺ (80%), Qa-4⁺ (77%), Qa-5⁺ (79%), and Ly-5⁺ (94%). In comparison, a proportion (39%) of alloimmune peritoneal exudate cells which conjugated with P815–2 also siained by immunofluorescence with anti-asialo GM1 antisera. Most (>90%) P815- conjugated cells were Thy-1⁺, Lyt-2⁺. and a subpopulation of Lyt-l⁺2⁺ conjugates was observed (25 %). Qa-5 and Ly-5 were also expressed on most (two-thirds) cytolytic T lymphocytes (CTL) conjugates, whereas Qa-4 and FcR for IgG2b were not detected. The best phenotypic distinctions between NK cells and CTL were therefore based on the presence or absence of Lyt-2, Qa-4, and FcR for IgG2b on most effector cells. Anti-asialo-GMl or monoclonal anti-Qa-4 and complement treatment greatly diminished both the frequency of NK conjugates and the percentage of conjugates with detectable IgG2b FcR or asialo-GM1. These results confirm that NK cells co-express asialo-GMI and Fc receptors, at the single-celt level, and provide a simple method for greatly enriching NK populations at least 10-fold.