Purified glycoproteins of influenza virus stimulate cell-mediated cytotoxicity in vivo

Previously, we reported that purified surface influenza viral glycoproteins can induce cell-mediated cytotoxicity (CMC) in vitro. Both neuraminidase (NA) and hemagglutinin (HA) were equally good stimulators, on an equimolar basis. In order to broaden the scope of these observations, we examined whether these glycoproteins stimulate natural killer (NK) activity in vivo. Biologically active preparations of glycoproteins NA and HA were purified from virus A/USSR/90/77 (H1N1) and recombinant virus A/USSR/92/77 (H1) x A/Prague/1/56 (N7), respectively. The studies were carried out using the optimal doses of NA and HA. In a 4-hour NK assay, using NK-sensitive YAC-1 cells as targets, both viral glycoproteins stimulated the NK activity of splenocytes of BALB/c and C3H mice. This stimulation was independent of the route of administration (intravenous or intraperitoneal) of the antigen. The observed NK activity was viral antigen-specific and could be modulated to levels comparable to those observed with the standard stimulator, polyinosinic acid-polycytidylic acid, by the use of an appropriate synthetic adjuvant, stearyl tyrosinate. Direct and indirect evidences suggest that the enhanced CMC is due to NK cells. These observations imply that enhancement of NK activity is the intrinsic property of influenza NA and HA.     

Tuftsin: a naturally occurring immunopotentiating factor. I. In vitro enhancement of murine natural cell-mediated cytotoxicity

Tuftsin is a physiologic tetrapeptide, which has recently been shown to possess immunoadjuvant properties including the stimulation of macrophage and granulocyte phagocytosis, migration, bactericidal, and tumoricidal activities. Tuftsin has also been reported to possess in vivo immunologically mediated anti-tumor potential. To determine the potential role of tuftsin as an antineoplastic immunoadjuvant, the in vitro effects of tuftsin on murine natural cell-mediated cytotoxicity were studied. We observed that in vitro treatment of mouse splenic effector cells with synthetic tuftsin induced a pronounced enhancement of natural killer cell (NKC) cytotoxicity against the T cell lymphoma Yac-1. The magnitude of NKC enhancement was directly dependent upon the concentration of tuftsin employed, with maximum NKC stimulation observed at tuftsin concentrations of 50 to 100 microgram/ml. The tuftsin induced enhancement of NKC activity was not strain specific, since equivalent stimulation was seen in CBA/J, C56BL/10, and DBA/2 mice. Elimination of macrophages, monocytes, T cells, and immunoglobulin-bearing cells had no effect on the dose-dependent tuftsin stimulation of natural cell-mediated cytotoxicity; thus the characteristics of the effector cells activated by tuftsin were consistent with those reported for NKC. We also observed that treatment of splenic effector cells with tuftsin prolonged the cytotoxic capabilities of these cells beyond 18 hr.     

In vitro enhancement of natural cytotoxicity by atrial natriuretic peptide fragment 4-28.

The presence of atrial natriuretic peptide (ANP) binding sites in the thymic cortex, medulla, and splenic white pulp suggests that this peptide may have immunoregulatory activity. We examined the effect of ANP on human natural killer (NK) cell activity. ANP significantly augmented NK cell cytotoxicity after twenty-four hours of incubation but had no effect on NK activity after short-term incubations of one hour. In addition, atrial natriuretic peptide did not effect the expression of natural killer or T cell surface markers. This study demonstrates that atrial natriuretic fragment 4-28 enhances natural killer cell activity.     

In vitro enhancement of natural and antibody-dependent lymphocyte-mediated cytotoxicity against tumor target cells by interferon.

Interferon derived from virus-infected human leukocytes or fibroblasts was found to enhance spontaneous and antibody-dependent lymphocyte cytotoxicity against human target cell lines in vitro. The greater enhancement occurred with spontaneous lymphocyte cytotoxicity. Interferon exerted its effect directly on lymphocytes; no effect on target cells was seen. The mechanism of enhancement was unclear: It did not reflect antibody production or lymphocyte proliferation. Enhancement appeared to be immunologically nonspecific, but clarification of this effect awaits further study.     

Augmentation of human natural cell-mediated cytotoxicity by recombinant human interleukin 2

Human peripheral blood mononuclear cells (PBMC) demonstrated increased natural cell-mediated cytotoxicity (NCMC) activity after only 5 min of exposure to purified recombinant human IL 2 or interferon (IFN)-gamma. The mechanism of NCMC augmentation by treatment with IL 2 is not entirely dependent on IFN-gamma production because: a) IL 2 was found to augment NCMC activity at levels which did not induce detectable IFN-gamma; b) IL 2 required only 5 min of exposure to PBMC to augment NCMC activity, whereas 3 hr of contact were required to demonstrate detectable IFN-gamma levels; c) the levels of NCMC enhancement by treatment with IL 2 exceeded the amount of NCMC enhancement that could be due to IFN alone; d) anti-recombinant IFN-gamma, which totally eliminated the augmentation of NCMC enhancement by IFN-gamma, only partially reduced the augmentation of NCMC activity by IL 2; and e) combination treatment of PBMC with IL 2 and IFN-gamma resulted in a synergistic enhancement of NCMC. The results strongly support the conclusion that augmentation of NCMC by IL 2 and IFN-gamma involve overlapping mechanisms.     

Monocyte-mediated augmentation of human natural cell-mediated cytotoxicity

Normal human monocytes can significantly and rapidly augment natural cell-mediated cytotoxicity (NCMC) against K562 target cells. Approximately 50% augmentation was observed after direct mixture of monocytes with autologous null cells in the 4-hr chromium-release assay. This effect was dependent on the number of monocytes, and B cells and granulocytes were not effective. Coculture of null cells with monocytes and subsequent recovery of null cells for use as effector cells also produced significantly elevated cytolytic activity. This effect was dependent upon the number of monocytes, the length of time of coculture, and the cell donor. Augmentation of NK activity was rapid and observed after 0.5-12 hr of coculture, but suppression was observed after 36 hr; augmentation was observed with high monocyte:null cell (1:1, 1:2) ratios, and no effect was generally observed with lower ratios (1:8). At the single-cell level, the augmentation was associated with an increase in the proportion of target-binding cells which were lytically active. The augmentation of NK activity by monocytes required close cellular proximity, was mediated by a factor which was active or induced only in close proximity of the effector and producer cells, and/or was mediated by a soluble factor with a molecular weight greater than 50,000. This new demonstration that monocytes can augment as well as suppress NCMC may represent another avenue by which NK cell activity may be modulated in vivo.     

Human influenza viral neuraminidases augment cell-mediated cytotoxicity in vitro

Previously, we reported that influenza virus-induced cell-mediated cytotoxicity (CMC) was largely due to its glycoproteins, hemagglutinin and neuraminidase (NA). These observations were based on the use of a single influenza virus strain, the A/Port Chalmers/3/73 (H3N2), and these were considered insufficient to generalize that all human influenza virus NAs augment CMC. Therefore, antigenically different NAs of human influenza strains were used to study whether (a) all NAs possess the potential to stimulate NK activity and (b) does the enzymatic activity of NA play a role in the CMC stimulation. Biologically active preparations of N1 subtype NA (A/USSR/90/77 (H1N1) and N2 subtype NAs (A/Aichi/2/68 (H3N2) and A/Port Chalmers) were evaluated for NK activity stimulation in an overnight radiolabeled chromium-release assay consisting of human peripheral blood lymphocytes and K562 target cells. The level of CMC stimulation was the same at equivalent protein concentrations with all the NAs tested. The addition of homologous NA-monospecific antibody almost completely reduced the CMC stimulation, while the addition of homosubtypic antibody reduced the CMC by 56-75%. However, in the presence of heterosubtypic monospecific antibody, NA-augmented CMC was reduced by 27-47% in most experiments. The results suggest that the CMC stimulation site is probably the same in all NAs tested. This putative site is thermo-resistant and is independent of the conformational change of the NA molecule. Furthermore, it is distinct from the enzymatic and probably from the antigenic sites.     

Lysis of dengue virus-infected cells by natural cell-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity

Peripheral blood mononuclear cells (PBMC) from humans without antibodies to dengue 2 virus lysed dengue 2 virus-infected Raji cells to a significantly greater degree than uninfected Raji cells. The addition of mouse anti-dengue antibody increased the lysis of dengue-infected Raji cells by PBMC. Dengue 2 immune human sera also increased lysis of dengue-infected Raji cells by PBMC. These results indicate that both PBMC-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) can cause significant lysis of dengue-infected Raji cells. The lysis of infected Raji cells in the ADCC assay correlated with the dilution of dengue-specific antibody which was added, indicating the dengue virus specificity of the lysis of dengue virus-infected Raji cells. Alpha interferon (IFN alpha) was detected in the culture supernatant of PBMC and dengue-infected Raji cells. However, enhanced lysis of dengue-infected Raji cells by PBMC may not be due to the IFN produced, because neutralization of all IFN activity with anti-IFN alpha antibody did not decrease the lysis of dengue-infected cells, and effector cells pretreated with exogenous IFN alpha also lysed dengue-infected cells to a greater degree than uninfected cells. The effector cells responsible for lysis of dengue virus-infected Raji cells in the natural killer and ADCC assays were analyzed. Nonadherent PBMC caused more lysis than did adherent cells. Characterization of nonadherent cells with monoclonal antibodies showed that the predominant responsible effector cells were contained in OKM1+ and OKT3- fraction in the natural killer and ADCC assays.     

In vitro inhibition of human influenza A virus replication by chloroquine

Inhibition of the human cytomegalovirus UL97 kinase by maribavir is thought to be responsible for the antiviral activity of this compound. Some mutations that confer resistance to maribavir map to UL97, however additional mutations that also confer resistance to the drug were mapped to UL27. These open reading frames share a low level of homology, yet the function of pUL27 remains unknown. A recombinant virus with a deletion in the UL27 open reading frame was reported previously to exhibit a slight replication deficit, but a more important function in vivo was hypothesized given its homology to the UL97 kinase. The potential for an important function in vivo was investigated by determining if these knockout viruses could replicate in human tissue implanted in SCID mice. None of the AD169 derived viruses replicated well in the implanted thymus/liver tissue, and is consistent with previous observations, although all of the viruses replicated to some degree in retinal tissue implants. Replication of the parent viruses was observed at 7 days post inoculation, whereas no replication was detected with any of the recombinant viruses with deletions in UL27. By day 14, replication was detected in two of the three knockout viruses and in all of the viruses by day 42. These data are consistent with minimal defects observed in cell culture, but are not consistent with an important role for UL27 in vivo. We conclude that UL27 is not required for viral replication in vivo.     

In vitro natural cell-mediated cytotoxicity against Candida albicans: macrophage precursors as effector cells

Bone marrow cells, cultured in L-929 CSF, consist of cells of granulocyte and macrophage lineages. Cells of the granulocyte lineage are known to be cytotoxic for Candida albicans. In this paper we report that macrophage precursor cells also display strong cell-mediated cytotoxicity against the yeast form of the dimorphic fungus C. albicans. The macrophage precursors responsible for this activity are nylon wool-nonadherent, nonphagocytic cells and lack asialo GM1 surface antigen. A purified population of macrophage precursors (greater than 95%) was obtained by means of Percoll density centrifugation. The interaction of these purified effectors with the target yeast cells was analyzed at a single cell level, and their activity was compared with that displayed by cells of the granulocytic series derived from the same bone marrow culture. Macrophage precursor cells proved to be more effective in binding the target cells and showed the same killing ability as the granulocytes: macrophage precursors were not damaged by contact with the target, in contrast to that which happened with granulocytes. In a long-term colony-forming unit assay, in fact, granulocytic cells showed a decrease over time in their ability to inhibit the growth of C. albicans, probably due to cell damage and death after the interaction with the target. In contrast, no loss of activity was observed with the macrophage precursor fraction. The same macrophage precursor cells also proved able to exert good natural killer activity against YAC-1 lymphoma cells, but not against P815 mastocytoma cells, as reported previously. The macrophage precursor cells, when cultivated in vitro to mature macrophages, lost completely their natural cytotoxicity against C. albicans and YAC-1 cells. The implications of these findings, as well as the possible role in vivo of such a precursor cell population during an infection, are discussed.     

In vitro studies on cell-mediated cytotoxicity by means of a terminal labeling technique.

A modification is described of the Takasugi-Klein assay for cell-mediated cytotoxicity, based on the measurement of ⁵¹Cr uptake by viable cells at the conclusion of effector-target cell interactions. Findings showing the applicability of this method to the quantitative determination of cell-mediated cytotoxicity against syngeneic solid tumors of mice are presented. It was found that repeated washing of splenocytes from donors with large tumors often elevated appreciably the cytotoxic capacity of the effector cells, and that washing of splenocytes from normal animals reduced their apparently nonspecific toxic effects.     

Influenza viral glycoproteins induced cell-mediated cytotoxicity by an interferon-independent mechanism

Human natural killer (NK) cells exposed to the influenza surface antigen neuraminidase (NA) show high cytotoxic activity, as evaluated using chromium-labeled K562 target cells in a standard overnight cytotoxicity assay. The role of interferon (IFN) in the stimulation of NK cells was examined by using three separate approaches. The use of appropriate antibodies to check IFN- and NA-specific cell-mediated cytotoxicity (CMC) stimulation showed that antibodies to IFN (-alpha, -gamma) did not alter NA-induced CMC, and vice versa. The treatment of NK cells with actinomycin D, before or after stimulation with IFN and NA revealed that only IFN-induced CMC was inhibited (50 to 100%). However, NK cells that were stimulated with NA before their exposure to actinomycin D became susceptible to stimulation by IFN. The interaction kinetics between IFN and NA demonstrated the presence of two mechanisms of CMC stimulation. Taken together, the results clearly show that stimulation of CMC by a viral component is effected through an IFN-independent pathway, and that this mechanism is probably followed by IFN under certain conditions.     

In vitro activities of purified visna virus integrase.

Although integration generally is considered a critical step in the retrovirus life cycle, it has been reported that visna virus, which causes degenerative neurologic disease in sheep, can productively infect sheep choroid plexus cells without detectable integration. To ascertain whether the integrase (IN) of visna virus is an inherently defective enzyme and to create tools for further study of integration of the phylogenetically related human immunodeficiency virus type 1 (HIV-1), we purified visna virus IN by using a bacterial expression system and applied various in vitro oligonucleotide-based assays to studying this protein. We found that visna virus IN demonstrates the full repertoire of in vitro functions characteristic of retroviral integrases. In particular, visna virus IN exhibits site-specific endonuclease activity following the invariant CA found two nucleotides from the 3' ends of viral DNA (processing activity), joins processed oligonucleotides to various sites on other oligonucleotides (strand transfer or integration activity), and reverses the integration reaction by resolving a complex that mimics one end of viral DNA integrated into host DNA (disintegration activity). In addition, although it has been reported that purified HIV-1 IN cannot specifically nick visna virus DNA ends, purified visna virus IN does specifically process and integrate HIV-1 DNA ends.     

The role of microtubules in human natural killer cell-mediated cytotoxicity

The effect of four different microtubule (MT) inhibitors on the various stages of human natural killer (NK) cell-mediated cytotoxicity was studied. The MT-disrupting effect of the drugs was monitored by indirect immunofluorescence microscopy and transmission electron microscopy. All the drugs tested, vinblastine sulfate, demecolcine, nocodazole, and taxol, had only a slight inhibitory effect on NK activity. Cells with nonfunctional MT were capable of normal conjugate formation and polarization of actin-containing microfilaments. Natural killer cell cytotoxic factor (NKCF) activity produced by cells with nonfunctional MT was slightly diminished. MT disruption caused enlargement of Golgi cisternae, but did not, however, dissociate the overall structural organization of the Golgi complex. The results indicate that fresh human NK cells are capable of lytic activity without functional MT although MT play a small supportive role in production or secretion of NKCF and mediation of the lytic activity. Previous experiments by us and others have strongly suggested that NK cells mediate their cytolytic activity by directed secretion of toxic material. As NK cells with unfunctional microtubules are capable of close to normal secretion the results presented in this report are not inconsistent with the earlier suggested stimulus-secretion model.     

Mistletoe viscotoxins increase natural killer cell-mediated cytotoxicity.

Mistletoe extracts have immunomodulatory activity. We show that nontoxic concentrations of Viscum album extracts increase natural killer (NK) cell-mediated killing of tumor cells but spare nontarget cells from NK lysis. The compounds responsible for this bioactivity were isolated from mistletoe and characterized. They have low molecular mass and are thermostable and protease-resistant. After complete purification by HPLC, they were identified by tandem MS as viscotoxins A1, A2 and A3 (VTA1, VTA2 and VTA3, respectively). Whereas micromolar concentrations of these viscotoxins are cytotoxic to the targets, the bioactivity with respect to NK lysis is within the nanomolar range and differs between viscotoxin isoforms: VTA1 (85 nm), VTA2 (18 nm) and VTA3 (8 nm). Microphysiometry and assays of cell killing indicate that, within such nontoxic concentrations, viscotoxins do not activate NK cells, but act on cell conjugates to increase the resulting lysis.     

Murine trophoblast resists cell-mediated lysis. II. Resistance to natural cell-mediated cytotoxicity

The susceptibility of murine trophoblast cells to natural cell-mediated cytotoxicity has been assessed. Primary short-term cultures of murine trophoblast cells isolated from 14-day placentas were found to be resistant to endogenous and interferon-activated natural killer (NK) cells and natural cytotoxic cells. That the relevant target structures are expressed on the surface of trophoblast cells and accessible to the effectors was demonstrated by their ability to inhibit the lysis of NK-sensitive target cells (YAC-1) in a dose-dependent manner. The lytic resistance of trophoblast cells was unaffected by neuraminidase treatment, inhibition of protein synthesis, or extending the assay time to 12 hr. Moreover, trophoblast cells were resistant to antibody-dependent cell-mediated cytotoxicity when coated with an alloantibody capable of mediating their lysis in the presence of heterologous complement. Neither the preincubation of effector cells in concentrated trophoblast culture supernatants nor the direct exposure of effectors to monolayers of trophoblast cells inhibited their NK lytic activity, indicating that the secretion of a suppressive factor or the direct inactivation of the NK cells was not responsible for the observed resistance to lysis. These observations, together with previous results showing the resistance of trophoblast to cytotoxic T cell-mediated lysis, reveal that murine trophoblast cells possess a resistance mechanism against several forms of cell-mediated lysis. This feature of trophoblast cells at the maternal-fetal interface is likely to play an important role in protecting the fetoplacental allograft from immune rejection.