Reduction by OK-432 of the monolayer contact-mediated inhibition of human natural killer cell activity

In the present study we investigated the effect of OK-432, a streptococcus preparation, on the contact-mediated inhibition of human NK activity by primary cultures of monolayer cells. Either peripheral blood lymphocytes (PBL) or large granular lymphocytes (LGL) were incubated (2 x 10(6) cells/ml, total volume 2 ml) on confluent monolayer cells (uvea-derived fibroblasts, uvea-derived melanoma cells, or renal carcinoma cells) for 18 h in 24-well plates, washed twice, and tested for cytotoxicity against K562, a human myelogenous leukemia cell line, in a 4 h 51Cr-release assay. After contact with monolayer cells, NK activity of both PBL and LGL was significantly reduced. When these effector cells were preincubated with 0.1 U/ml of OK-432 for 18 h and then tested for the sensitivity to contact-mediated inhibition, the inhibition was significantly reduced. The pretreatment of monolayer cells with OK-432 or the addition of OK-432 into the coculture wells (of effector cells and monolayer cells) also significantly reduced the contact-mediated inhibition. Moreover, OK-432 (0.1 U/ml) reestablished the inhibited NK activity of PBL. These results suggest that OK-432 might enable NK cells to escape from the contact-mediated inhibition by monolayer cells and thus provide an additional potential mechanism for the observed clinical effectiveness of OK-432 reported by many groups.     

In vitro augmentation of natural killing activity by OK-432

OK-432, a streptococcal preparation, augmented the natural killing (NK) activity of peripheral blood lymphocytes of normal donors and cancer patients against both NK sensitive and resistant human target cells in vitro. The enhancement of NK activity was evident after 4 h pretreatment and maximum by 16-24 h. The manifestation of OK-432 induced augmentation required active cell metabolism, RNA and protein synthesis but no DNA synthesis of lymphocytes. The supernatants produced by OK-432 stimulated lymphocyte cultures had no enhancing substance nor interferon. Anti-interferon antibodies did not inhibit boosting activity of OK-432. Large granular lymphocytes were involved in both spontaneous and OK-432 induced cytotoxic activity. The proportion of lymphocytes conjugating to target cells was not changed by OK-432. These results suggest that OK-432 augments cytotoxic activity of large granular lymphocytes having ability to recognize target cells independent of interferon induction.     

Up-regulation of natural killer activity of human immunodeficiency virus-infected patients by in vitro-differentiated macrophages

Patients with acquired immunodeficiency syndrome (AIDS)-related complex and asymptomatic individuals seropositive for human immunodeficiency virus (HIV) have depressed natural killer (NK) activity. Normal human macrophages cultured for 3-7 days significantly up-regulated the NK activity of mononuclear cells obtained from the blood of asymptomatic HIV-seropositive individuals and patients with AIDS. Following a 4-hr incubation of patients' cells with in vitro-differentiated macrophages, the greatest augmentation of NK activity was seen in asymptomatic HIV-seropositive individuals who were receiving treatment with azidothymidine. Stimulation of macrophage immunoregulatory activities or adoptive immunotherapy with ex vivo-activated monocytes may be beneficial in HIV-infected patients.     

Biological response modifier enhances the activity of natural killer cell against human cytomegalovirus-infected cells

Peripheral blood lymphocytes (PBL) from two human cytomegalovirus (CMV)-seronegative donors and eight CMV-seropositive donors were cultured for 3 days with or without the biological response modifier OK-432 and examined for lysis of K562 cells and CMV-infected MRC-5 cells. OK-432-stimulated PBL exhibited significantly greater natural killer (NK) activity than did unstimulated PBL. There was no difference in activity of NK cells in PBL prepared from CMV-seronegative and -seropositive donors. Antibody-complement depletion studies suggested that OK-432-stimulated NK activity was associated with Leu-7-positive cells. The ability of OK-432 to sustain the NK activity in PBL was decreased when the CD4-positive population of lymphocytes was eliminated by antibody-complement depletion prior to OK-432 stimulation. The ability of OK-432 to sustain the NK activity of PBL was also significantly decreased in the presence of monoclonal antibody against recombinant human interleukin-2. The results suggest that the activity of human NK cells against K562 and CMV-infected MRC-5 target cells can be sustained in vitro by OK-432-stimulated T-helper cells and that the effect of the T-helper cells is mediated, at least in part, by interleukin-2.     

Augmentation of natural killer (NK) cell activity by a streptococcal preparation,OK-432, in patients with malignant tumors

Recently, a streptococcal preparation, OK-432 has been used successfully as an immunopotentiator for immunotherapy in patients with malignant tumors in Japan. In this paper, we report that the administration of OK-432 augments the cytotoxic activity of peripheral blood lymphoid cells against a natural killer (NK) cell-sensitive erythroleukemic cell line, K562, in tumor patients. In patients before or after surgery, sufficient amounts of OK-432 strongly augmented the cytotoxic activity within 3 days after the initial administration of OK-432. Thereafter the levels of cytotoxicity declined rapidly. The administration of a lower dose of OK-432 gave a lower increase in cytotoxicity. Enhanced cytotoxicity occurred with the reintroduction of OK-432 but remained at lower levels of activity. Characterization and fractionation of OK-432-induced effector cells revealed that the augmented cytotoxicity seemed to be carried mainly by NK cells. A low titer of interferon was detected in 3 of 10 patients within 72 hr after the first inoculation of the agent. Furthermore, we discuss the potency of OK-432 for the induction of interferon in detail.     

Enhancement of natural killer cell activity in vitro against human tumor cells by some plants from Jordan

The effect of different concentrations (0, 0.01, 0.1, and 0.5 mg/ml) of plant aqueous extracts on the anti-tumor activity of natural killer (NK) cells isolated from human blood was examined. Plant extracts induced significant enhancement of (26.6-67.7%) of NK cell activity against K562 tumor cells. This increase in NK cell cytotoxicity was found to be due to the enhancement of NK cell production of interferon-gamma (87-337%), and on tumor necrosis factor-alpha (60-200%). Furthermore, the release of both granzyme A and N-acetyl-beta-D-glucosaminidase was increased significantly when compared with controls. Activation of granzyme A and N-acetyl-beta-D-glucosaminidase was clearly observed ranging from 24.2-106.4% to 26.8-110.7%, respectively. Lastly, in the absence of IL-2, plant extracts caused a significant increase in NK-cell-induced cytotoxicity (256%) against K562 tumor cells, and in the presence of IL-2 stimulated cells plant extracts caused an increase in NK cell-cytotoxicity (112%).     

Enhancement of natural killer cell activity by Marek's disease vaccines

Vaccination against Marek's disease with herpesvirus of turkeys (HVT) has been reported to cause increased natural killer (NK) cell activity as detected in vitro against LSCC-RP9 target cells. The effect of vaccination with SB-1 (a nononcogenic chicken herpesvirus), HVT and the HVT/SB-1 combination on NK cell activation was compared in Marek's disease susceptible (P-2) and resistant (N-2) chickens. Birds were vaccinated at 7 days of age and NK cell activity was measured between 3 and 42 days after vaccination. Both SB-1 and HVT caused a significant increase in NK cell-induced specific release. The increase was similar in N-2 and P-2 chickens for either HVT or SB-1, while the combined vaccine induced a significantly higher increase in N-2 compared to P-2 birds. The maximal effect of vaccination on NK cells was detected at 7 days after vaccination. In contrast with the results in young birds, vaccination of birds between 31 and 45 days of age caused either no effect or a suppression rather than an enhancement in NK cell activity.     

Regulation in vitro of human natural killer (NK) cell activity

The natural killer cell activity of PBL from epidemic polyarthritis patients was depressed early after onset of symptoms but returned to normal as the patient recovered. This study found that the in vitro culture of Ross River virus, the agent responsible for epidemic polyarthritis, with PBL resulted inenhanced rather than depressed NK cell activity. Evidence was also obtained that NK cell activity could be suppressed by suppressor-T lymphocytes generated by culture of PBL with high concentrations of Concanavalin A. This suppressive activity was not due to release of a soluble mediator(s) by the suppressor T cells.     

Modulation of human natural killer cell activity by pharmacological mediators.

We have studied the effects of various pharmacological mediators on human NK cell activity. Prostaglandin E2 (PGE2) inhibits NK cell activity in a dose-dependent fashion, whereas PGF2 alpha has no significant effect over the same concentration range. Histamine at high doses (10(-4) M) induced a small but significant inhibition of NK cell activity which was mimicked by both H1 and H2 specific histamine receptor agonists. Inhibition of endogenous prostaglandin production by indomethacin did not alter NK cell activity. Inhibition of NK activity by cAMP but not cGMP analogues, together with other data presented suggests that the mechanism of PGE2-induced inhibition of NK cell activity is not due to impairment of effector cell movement or effector: target cell interaction, but through the adenylate cyclase system which modulates the killing process.     

The chemiluminescence response of human natural killer cells. II. Association of a decreased response with low natural killer activity

Low natural killer (NK) responders selected from a panel of 600 normal, healthy volunteers exhibited 5- to 10-fold less cytotoxicity against the human erythroleukemic cell line K562, compared with high NK responders. Antibody-dependent cellular cytotoxicity against tumor cells, which is mediated by similar or identical effectors, was also depressed in low NK donors whereas lectin-dependent T cell killing and monocyte-mediated cytolysis of tumor cells was normal. Low NK donors exhibited normal frequencies of cells expressing the HNK-1 marker of human NK cells and highly enriched NK fractions were not impaired in their ability to recognize and bind to NK-sensitive target cells. Interferon partially activated low responder NK cells but did not restore the response to normal levels. The burst of chemiluminescence that is generated by NK cells within seconds of target cell contact was markedly impaired in low NK responder donors. We have previously shown that chemiluminescence detects reactive oxygen intermediates which are necessary but not sufficient for the activation of the NK cytolytic pathway.     

In vitro inhibition of natural killer cell activity by doxycycline

The effect of four tetracyclines, tetracycline, rolitetracycline, oxytetracycline and doxycycline, on human natural killer (NK) cell-mediated cytotoxicity was examined in vitro. Doxycycline was the only tetracycline which inhibited the NK cell activity. At concentrations of 10 micrograms/ml the drug caused approximately 50% inhibition of NK cell mediated cytotoxicity against K562 target. The inhibition was not a result of a toxic effect of the drug on NK cells. These results support the previous findings that doxycycline shows immunosuppressive properties.     

经链球菌菌体制剂OK-432刺激的树突状细胞对自然杀伤细胞增殖及功能的影响

目的:探讨经链球菌菌体制剂OK-432刺激的树突状细胞(Dendritic cells,DC)对自体自然杀伤细胞(Naturalkiller cells,NK)体外扩增和功能的影响。方法:将DC分为未成熟DC组和OK-432刺激的DC组,48小时后MTT法检测DC增殖情况,FCM检测DC表型CD80、CD83、CD86的表达情况;后将未成熟DC组和OK-432刺激的DC组分别与NK细胞以1∶1、1∶5、1∶10、1∶20、1∶40比例混合继续培养。0、2、4、6天时计算NK细胞扩增倍数;FCM检测NK细胞PFP、GraB、CD107a的表达;LDH释放法检测NK对HepG2细胞的杀伤活性。结果:OK-432(5μg/ml)使DC增殖达到最佳(30%),且能显著增强DC表型表达(P<0.05)。1∶5(OK-DC)组NK细胞扩增倍数达最大(P<0.05);1∶5(OK-DC)组能显著促进NK释放穿孔素(Pore-forming protein,PFP)、颗粒酶(Granzyme B,GraB)、CD107a(P<0.05);1∶5(OK-DC)组对HepG2细胞的杀伤活性(67.12±5.36)%达到最大(P<0.05)。结论:OK-432(终浓度5μg/ml)能促进DC增殖,并促进DC成熟;OK-DC与NK细胞共培养能以剂量依赖的方式增强NK细胞杀伤HepG2细胞功能,可能与增加NK扩增倍数和PFP、GraB、CD107a的表达有关。     

Restoration of P-glycoprotein function is involved in the increase of natural killer activity with exogenous interleukin-15 in human immunodeficiency virus-infected individuals

A depressed level of natural killer (NK) activity is one of the various immunologic abnormalities in human immunodeficiency virus (HIV) infection. Interleukin-15 (IL-15), an immunotherapeutic candidate in HIV infection, increases NK activity and induces the excretion of CC-chemokines from divergent immune cells, but the mechanisms of NK activity enhancement by IL-15 stimulation is not clearly established in HIV infection. This study examined whether CC-chemokines, which are known to increase NK activity, are secreted adequately in HIV-infected individuals, and also investigated whether P-glycoprotein is involved in NK activity enhancement after IL-15 administration. NK activity increased with IL-15 stimulation in NK cells of HIV-infected individuals, as it does in normal NK cells. IL-15 stimulates NK cells to secrete CC-chemokines, such as, macrophage inflammatory protein-1alpha (MIP-1alpha), macrophage chemotactic protein-1alpha (MCP-1alpha) and regulated upon activation, normal T cells expressed and secreted (RANTES) in both HIV-infected individuals and controls with no significant difference. P-glycoprotein expression and function is decreased in HIV-infected individuals and restored only in NK cells of HIV-infected individuals after IL-15 stimulation. P-glycoprotein may play a role in the mechanism of increased NK cell activity in HIV-infected individuals after IL-15 stimulation.     

Inhibition of human natural killer cell activity by polyclonal IgM

Pretreatment of effector cells with normal human IgM induced strong dose-dependent inhibition of NK activity. The degree of inhibition by normal IgM was stronger than that induced by monomeric IgG, which has previously been reported to be a negative regulator of NK activity. For 100% inhibition, 1.1 × 10⁻⁶ M of IgM was required, whereas 66.6 × 10⁻⁶ M of IgG was needed to abolish NK activity. This inhibitory property of polyclonal IgM appeared to be localized in the Fc region of the molecule, and also was significantly reduced upon mild reduction of disulfide bonds. Monoclonal IgM purified from sera of five patients with Waldenström's macroglobulinemia and tested in parallel with normal IgM lacked or had a decreased capacity to inhibit the cytotoxic reaction. As with IgG, IgM interfered mainly with the lytic event, after binding of effector cells to target cells. The inhibition by IgM appeared to be a direct effect on NK cells, since similar effects were observed with purified large granular lymphocytes as with non-adherent lymphocytes. These results indicate a new mechanism for negative regulation of NK cells and suggest the presence of Fcμ receptors on these effector cells.     

Enhancement of natural killer cell activity and interferon production by manganese in young mice

The effect that MnCl2 has on murine splenic natural killer (NK) cell activity was investigated in infant (10 days old), pre-weanling (18 days old) and weanling (24 days old) C57BL/6J mice. A single intraperitoneal injection of 10, 20 or 40 micrograms MnCl2/g body weight caused a significant enhancement in NK activity, as determined by the in vitro 51Cr release assay. Comparable enhancement of NK activity was observed for age-matched mice injected intraperitoneally with polyinosinic polycytidylic acid (Poly I:C). Both MnCl2 and Poly I:C caused elevations in serum interferon levels. Time-course studies revealed that interferon levels returned to normal within 48 hours following injection with either MnCl2 or Poly I:C; however enhanced NK activity persisted for up to 48 hours in Poly I:C-injected mice and 72 hours in MnCl2-injected mice. The administration of rabbit anti-asialo GMl to MnCl2-injected mice completely abrogated the enhanced NK activity. In addition, the injection of rabbit anti-mouse interferon alpha, beta but not gamma completely abrogated the enhanced NK activity. In addition, the injection of rabbit anti-mouse interferon alpha, beta but not gamma completely abrogated the enhancement of NK activity by MnCl2 and to a lesser extent the enhancement of NK activity by Poly I:C. These results indicate that despite low levels of NK activity in pre-weanling mice, MnCl2 is capable of enhancing this activity by 8-9 fold. Furthermore, Mn-enhanced NK activity in these young mice appears to be mediated by the production of interferon alpha, beta.     

Cells with natural killer activity in human rabies.

Cytotoxic lymphocyte function in 13 patients with rabies was studied by counting the number of CD56 cells and assessing natural killer (NK) cell activity. There was no significant difference in the number of killer cells between rabies patients and 31 normal controls (P greater than 0.05). Two of six non-fatal encephalitic patients due to causes other than rabies had reduced CD56 numbers. Base-line NK cell responses versus K562 cell targets did not differ among the normal control and rabies groups (P greater than 0.05). Study of the non-rabies encephalitis group showed heterogeneous results with wide variation. Significant enhancement of NK activity was seen in four rabies patients and in 10 normal control subjects tested after interferon-alpha (IFN-alpha) and IL-2. None of the four patients with encephalitis due to causes other than rabies showed such enhancement. Our results suggest that NK cells of rabies patients are not fully stimulated and that this might contribute to the virulence of rabies. The cause of this phenomenon remains unknown.     

Neuroimmunomodulation with enkephalins: enhancement of human natural killer (NK) cell activity in vitro

To further define the effects of enkephalins on immune function, the effect of methionine-enkephalin and leucine-enkephalin on natural killer cell (NK) activity in isolated human peripheral blood lymphocytes was investigated. Incubation of lymphocytes with either enkephalin resulted in significant increases in natural killer cell activity. At effector:target cell ratios of 11:1 methionine-enkephalin significantly (P less than 0.05) enhanced NK activity at dilutions of 10(-6), 10(-8), 10(-10), and 10(-14) mg/ml, while leucine-enkephalin significantly (P less than 0.05) enhanced NK activity at dilutions of 10(-4), 10(-6), 10(-8), 10(-10), and 10(-14) mg/ml. Cells from individuals with low NK activity showed greater percentage increases in NK activity following enkephalin than did cells from individuals with high NK activity.     

Natural killer cell infection and inactivation in vitro by the human immunodeficiency virus

Cytolytic activity of human mononuclear peripheral blood leukocytes from healthy donors, cultured in interleukin-2 conditioned medium, was abrogated by in vitro infection with the lymphadenopathy associated virus (LAV) isolate of the human immunodeficiency virus (HIV). Although viral antigens are not expressed in cultured cells until 14 days postinfection, cytolytic activity was lost as early as 3 days after infection. Loss of cytolytic function was not a result of the release of suppressive factors from either infected cells or uninfected CEM cells since supernatants from neither infected cultures nor CEM cell cultures had any inhibitory effects on the function of uninfected cells. Cultured lymphocytes expressing Leu 11b were also shown to express HIV antigens via immunofluorescence after 14 days in culture. These results suggest that natural killer (NK) cells, as defined by expression of Leu 11b, were infected by HIV in vitro and the loss of lytic function was likely a direct consequence of that infection.