Flavone-8-acetic acid augments systemic natural killer cell activity and synergizes with IL-2 for treatment of murine renal cancer

The investigational drug flavone-8-acetic acid (FAA) potently augments NK activity in the spleen, liver, lungs, and peritoneum in a dose-dependent manner after i.v. or i.p. administration. Augmented NK activity peaks by 24 h after FAA injection and returns to normal after 6 days. Combined treatment of established murine renal cancer with FAA and rIL-2 results in up to 80% long term survival whereas FAA or rIL-2 alone were unable to induce any long term survivors. The optimal dose of rIL-2 required for use with FAA was in the range of 10,000 to 30,000 U/day. Further studies demonstrated that the regimen of FAA plus rIL-2 administration that was effective in treating established murine renal cancer also induced a more potent augmentation of NK activity than did either FAA or rIL-2 alone. Subsequent studies revealed that the therapeutic effectiveness of FAA plus rIL-2 was significantly reduced when tumor-bearing mice were treated with anti-asialo GM1 serum. These results are consistent with a role for augmented NK activity in the therapeutic effects of FAA plus rIL-2 murine renal cancer. In addition, these studies demonstrate that FAA and rIL-2 is a useful approach for cancer treatment in that subtoxic doses of rIL-2 can be used and significant anti-tumor efficacy occurs even without accompanying adoptive immunotherapy.     

Trafficking and activation of murine natural killer cells: differing roles for IFN-gamma and IL-2

The repeated ip injection of highly purified recombinant IFN-gamma or IL-2 resulted in a local increase in peritoneal NK activity. This increase in lytic activity was paralleled by increases in the number of peritoneal leukocytes reacting with a rat monoclonal antibody directed against the NK cell-associated surface antigen LGL-1. LGL-1 reacts specifically with the majority of murine NK cells in BALB/c and C57BL/6 mice. A single injection of IFN-gamma induced more peritoneal NK activity at 24 hr than IL-2 on a protein basis. Both cytokines induced increases in the number of LGL-1+ peritoneal cells by 24 hr after injection. Simultaneous injection of suboptimal amounts of IFN-gamma (100 U) and IL-2 (10,000 U) resulted in a significant augmentation of peritoneal NK activity over that observed with either cytokine alone. Also, the peritoneal NK activity generated in response to ip injection of high doses of IL-2 (100,000 U) could be dramatically reduced by simultaneous injection of a neutralizing monoclonal antibody to IFN-gamma. Administration of IFN-gamma 1 day prior to IL-2 resulted in a significant augmentation of the NK activity above that observed with the individual cytokines. In contrast, injection of IL-2 prior to IFN-gamma did not enhance NK activity over that observed with the individual cytokines. Both cytokines must be injected ip for the complementary effects of IFN-gamma and IL-2 on peritoneal NK activity to occur. In contrast, in vitro incubation of peritoneal leukocytes with IFN-gamma resulted in neither a significant enhancement of NK lytic activity nor an increase in the number of LGL-1+ cells. In vitro treatment of peritoneal leukocytes with IL-2 always resulted in significant augmentation of NK lytic activity in the absence of any increase in the number of LGL-1+ cells. These data are consistent with the hypothesis that the local release of IFN-gamma increases peritoneal NK activity by promoting the influx of blood-borne LGL-1+ NK cells from other sites. In contrast, low doses of IL-2 augment the lytic activity of local resident NK cells, whereas high doses of this cytokine induce both an activation of local NK cells and emigration of LGL-1+ NK cells from other sites due to the endogenous generation of IFN-gamma within the peritoneal cavity. Therefore, the local release of IFN-gamma may play an important role in regulating NK cell infiltration in vivo.     

Modulation of natural cytotoxicity by alloantibodies: III. Augmentation of natural killer activity by alloantisera directed against K,D, and I regions of the murine H-2 complex

Natural killer activity of mouse spleen cells toward a human myeloid leukemia cell line, K562, can be enhanced by alloantisera directed against individual antigens in the H-2 region. By using a panel of 13 antisera (8 directed against antigens in the K and D regions and 5 directed against antigens in the I region) and four strains of mice (C57BL/6J, CBA, DBA/2, and A/J) it was found that certain antisera would stimulate target cell lysis by spleen cells only if the antisera had specificity for antigens which were a part of the haplotype represented on the spleen NK effector cells. Anti Ia antisera could stimulate the anti K562 NK activity of nude mouse spleen cells which lack mature T cells. Depletion of B cells and macrophages from nude spleen cells, by passing through a nylon-wool column also did not abolish the effect of anti-Ia antiserum. It appears likely therefore that the anti-Ia antibodies exert this effect directly on NK cells and that Ia antigens may be expressed on NK cells. Since the antisera directed against different antigens in H-2 complex irrespective of subregion specificity (K, D, or I) stimulated the NK activity of mouse spleen cells, the phenomenon offered an interesting method for testing the presence of a given alloantigen on mouse spleen cells. Log-dose response curves for the augmentation of lysis induced by appropriate alloantisera were linear over a dilution range of 1:320 to 1:5120. By using the dose-response curves, potency ratios of two preparations of antisera (directed against antigen 33 of the K region) could be successfully determined. Besides the K562 cell line, many human lymphoblastoid cell lines could also be used as target cells in this assay system.     

IL-4 regulation of murine lymphokine-activated killer activity in vitro. Effects on the IL-2-induced expansion, cytotoxicity, and phenotype of lymphokine-activated killer effectors

The in vitro incubation of B6 splenocytes with purified, mouse rIL-4 for 4 to 5 days was sufficient to generate lymphokine-activated killer (LAK) activity. In addition, rIL-4 augmented LAK cytotoxic activity when combined with rIL-2, as measured in a 4 h 51Cr-release assay against fresh, syngeneic MCA-sarcoma (MCA-102 and MCA-105) cells. Interestingly, this augmentation was not observed against the cultured YAC-1 target. LAK generation and augmentation of cytotoxicity by rIL-4 was species-specific, because human rIL-4 (up to 20,000 U/ml) failed to elicit these effects in the mouse splenocyte cultures. When 5-day B6 LAK cells (splenocytes incubated in rIL-2 at 1000 U/ml for 5 days) were split and recultured in the combination of rIL-2 plus rIL-4 for 4 additional days at least a twofold greater expansion in cell number resulted compared to similar cells cultured in either rIL-2 or rIL-4 alone. Moreover, LAK cells expanded in rIL-2 plus rIL-4 exhibited substantial increases in in vitro cytolytic activity (on a per cell basis) against MCA-102 and MCA-105 sarcoma cells, but not against YAC-1 targets. FACS analysis or negative selection using Lyt-2 or NK-1.1 mAb plus C revealed no differences in effector phenotype(s) of LAK cells expanded in rIL-2 alone compared to rIL-2 plus rIL-4 to account for the differences observed in both expansion and cytolytic activity by rIL-4. The majority of cells was Thy-1+, Lyt-2+, T3+, and ASGM-1+. However, a marked increase in the granule-associated serine esterase, BLT-E, was found only in LAK cells expanded in the combination of both lymphokines. Collectively, these studies show that rIL-4 has potent regulatory activities on splenic LAK generation, expansion, and cytotoxic function in the mouse.     

Augmentation of killer activity of murine spleen cells against allogeneic and syngeneic tumor cells by inoculation of alloantigen in vivo

After C57BL/6 (B6) mice were inoculated with BALB/c spleen cells via tail vein, kinetics of cytotoxic activities in the B6 mice against sensitizing alloantigens (H-2d) and against syngeneic antigens were investigated using, as target cells, P815 mastocytoma cells (H-2d) and B16 melanoma cells (H-2b). Cytotoxic activity against P815 in the B6 spleen cells reached a peak 3 days after alloantigen inoculation, decreased drastically on day 5 and rose again thereafter. The profile of anti-B16 cytotoxic activity was similar to that of anti-P815 activity. The cytotoxic activity against P815 was inhibited partially by cold B16, but that against B16 was not inhibited by cold P815. Surface phenotype of cytotoxic cells against P815 was Lyt2+, Thy1+, Asialo GM1+ and that of cytotoxic cells against B16 was Lyt2-, Thy1+/-, and Asialo GM1+. The results indicate that inoculation of B6 mice with allogeneic BALB/c spleen cells induce two types of cytotoxic cells; one is similar to lymphokine-activated killer (LAK) cells and the other is activated natural killer cells.     

Functional interaction between the tumor necrosis factor and interleukin 2 during induction of lymphokine activated killer cytotoxic activity

TNF has a differential effect on the induction of LAK activity which is dependent on the IL-2 concentrations used for LGL stimulation. TNF was found to synergize with low concentrations of IL-2 which are normally ineffective in inducing LAK activity. When IL-2 was used at concentrations optimal for LAK generation, TNF did not further enhance the differentiation of LGL into LAK effectors. When anti-TNF was included in the 51Cr-release assay, no effect on the cytolytic activity was observed. We also demonstrate that IL-2 induced an increase of specific TNF binding to LGL.     

Further development of local IL-2 therapy of cancer: multiple versus single IL-2 treatment of transplanted murine colon carcinoma

We have compared the effect of one and up to four local IL-2 treatments of transplanted MC38 colon carcinoma. A single IL-2 treatment prolonged the survival time (p=0.015), but no cure was obtained. One local IL-2 treatment inhibited tumor growth for about 1 week. After the start of tumor regrowth, a further IL-2 injection was given. After four IL-2 injections 6 out of 13 mice were cured. Histological studies show that IL-2 induced a local vascular leakage syndrome leading to massive peritumoral edema and subsequent necrosis of tumor tissue. IL-2 also attracted infiltrating cells, mainly macrophages. Subsequent IL-2 injections led to complete tumor regression. We believe that the combination of necrotic tumor debris and the IL-2–induced macrophage reaction enhanced a tumor-specific immune response. This local IL-2 application was not toxic.     

Changes in the host natural killer cell population in mice during tumor development. 2. The mechanism of suppression of NK activity

Our earlier studies revealed that a rapid and progressive loss of splenic NK activity in mice during the development of a number of transplanted tumors as well as of spontaneous tumors was due to an inactivation of natural killer (NK) lineage cells rather than to their disappearance. The mechanism of this inactivation have now been explored in CBA/J mice receiving transplanted Ehrlich ascites tumors and in C3H/HeJ mice bearing spontaneous mammary tumors or receiving transplants of syngeneic mammary tumor lines of recent origin. A poor activation state or maturation arrest of NK lineage cells due to a low interferon level in vivo was ruled out, since the host NK activity could not be restored after administration of either an interferon inducer poly(I:C) or interferon-alpha, although such treatments enhanced the activity in tumor-free mice by four- to eightfold. Possible presence of host suppressor cells acting on the effector or preeffector stage of NK cells was explored by mixing spleen cells from tumor bearers with normal spleen cells either during the NK assay, or for a 20-hr period of in vitro short-term culture prior to the NK assay. Mixing during the NK assay led to a reduction of NK activity explicable by a simple dilution of active NK cell concentration rather a suppression of active NK cells. On the other hand, a 20-hr coculture of the mixed population at various ratios led to a complete abrogation of the NK activity, indicating that the suppressor cells acted on the preeffector stage of the NK Lineage. A further characterization of suppressor cells revealed that they were (1) contained in the adherent fraction of the spleen of tumor bearers, (2) of monocyte/macrophage morphology, (3) capable of phagocytosing latex particles, and (4) positive for surface Mac-1 antigen, as noted from a radioautographic binding of 125I-labeled monoclonal anti-Mac-1 antibody. The mechanism of the suppression was identified, at least in part, as being mediated by prostaglandin-like molecules, since the presence of indomethacin, a prostaglandin-inhibitor, during the 20-hr coculture period completely abrogated the suppression. Indomethacin exerted no direct effect on the recruitment or killer activity of NK lineage cells in vitro. NK cell suppression may be another normal immunoregulatory mechanism which alters the host-tumor balance in favor of the tumor rather than the host.     

Synergistic induction of lymphokine (IL-2)-activated killer activity by IL-2 and the polysaccharide lentinan,and therapy of spontaneous pulmonary metastases

Summary Spleen cells of C57BL/6N mice bearing lung metastases were induced to the cytotoxic state by subcutaneous injection of recombinant human interleukin-2 (IL-2) at a minimum dose of 5×10⁴ U/mouse three times a day for 3 consecutive days. A single intraperitoneal injection of lentinan alone at concentrations of up to 10 mg/kg body weight did not render spleen cells cytotoxic to P-29 cells, but a combination of subthreshold doses of these agents (5×10⁴ U/ml IL-2 and 5 mg/kg lentinan) induced significant in vivo lymphokine-activated killer activity in spleen cells of tumor-bearing mice. Similarly, spleen cells from mice treated i.p. with lentinan became cytotoxic on in vitro treatment with IL-2. The in vitro responsiveness of spleen cells to IL-2 was maximal 3 days after i.p. injection of lentinan. Synergism between IL-2 and lentinan was also observed in mice bearing spontaneous lung micrometastases: neither IL-2 (<5×10⁴ U/mouse) nor lentinan (<2.5 mg/kg) alone had a therapeutic effect, but multiple injections of IL-2 with a single injection of lentinan resulted in significant inhibition of spontaneous pulmonary metastases. From these results we conclude that IL-2 and lentinan in combination are more effective than either one alone for inducing destruction of pulmonary metastases.     

Interleukin 2 (IL-2) activity during tumor growth: IL-2 production kinetics,absorption of and responses to exogenous IL-2

A temporal study assessed the relationship between fibrosarcoma growth and immunologic encumberance due to the inability of BALB/c mouse splenocytes to elaborate the lymphokine Interleukin 2 (IL-2). Nylon-wool fractionation and antiserum treatments suggested the existence of a mildly nylon-wool-adherent, anti-Lyt 2-sensitive tumor-induced suppressor T (T_s,) cell which significantly decreased IL-2 activity. Absorption investigations indicated that ligand-activated tumor-bearing host (TBH) spleen cells were less receptive to IL-2 than their normal counterparts. When splenocytes were antiserum treated before absorption, removal of Lyt 2⁺ (suppressor T) cells resulted in greater IL-2 absorption by the remaining cells. Purified IL-2 only partially restored suppressed TBH spleen cell mitogen- or alloantigen-induced blastogenesis; whereas, normal host reactivity was significantly augmented. The collective data suggest that TBH spleen cells were capable of producing IL-2 and of responding to the IL-2 amplification signal when tumor-induced T_s cells were depleted.     

Recognition specificities, development, and possible biological function of natural killer cells in the mouse. I. Spleen focus forming assay for natural killer activity and analysis of lectin-like recognition structures on the surface of murine natural killer cells

A number of simple sugars have been tested and found to be effective in blocking lysis of YAC-1 tumor target cells by nonimmune murine natural killer (NK) effector cells. Using a spleen fragment culture system an assay has been developed which allows us to compare the inhibition of lysis observed in replicate culture wells prepared from cells contained in one spleen fragment (less than or equal to 1 X 10(6) cells). The inhibition pattern of any well was found to fall naturally into 1 of 25 (of the total 128 possible tested) patterns. Using this panel analysis of NK activity in individual mice of the same or different strain has been compared. Our data suggest that within any given strain the inhibition pattern of NK effector cells is quite uniform. Consistent differences are seen between strains which are interpreted in terms of a genetic control of the final expression of the NK recognition repertoire. In adult F1 hybrid individuals the pattern of recognition by NK cells is best considered a result of the codominant expression of genes contributed by each parent.     

Synergistic antitumor activity of cyclophosphamide and ABPP in the treatment of established and advanced tumors in murine tumor models

Summary We have previously shown that the in vivo administration of ABPP, an interferon inducing pyrimidinone, generates activated killer cells that can lyse fresh tumor cells in vitro in 4-h ⁵¹chromium release assays. The administration of this agent however has no effect on established tumor. In this communication we show that ABPP, when used in combination with low or moderate doses of cyclophosphamide, can be quite effective against early established (day 3) tumor as well as against advanced, grossly visible (day 8–10) tumor in both the i.p. and pulmonary metastasis model. The synergistic antitumor activity of this cheoimmunotherapeutic regimen was very strong against immunogenic tumors but rather weak against nonimmunogenic tumors. Two treatment cycles were significantly more effective than one and multiple cycles even cured the majority of mice with established i.p. tumor. These experiments demonstrate the potential of chemoimmunotherapeutic regimens and highlight the efficacy of multiple treatment cycles.     

The proinflammatory cytokines IL-2, IL-15 and IL-21 modulate the repertoire of mature human natural killer cell receptors

Natural killer (NK) cells play a crucial role in the immune response to micro-organisms and tumours. Recent evidence suggests that NK cells also regulate the adaptive T-cell response and that it might be possible to exploit this ability to eliminate autoreactive T cells in autoimmune disease and alloreactive T cells in transplantation. Mature NK cells consist of a highly diverse population of cells that expresses different receptors to facilitate recognition of diseased cells and possibly pathogens themselves. Ex vivo culture of NK cells with cytokines such as IL-2 and IL-15 is an approach that permits significant expansion of the NK cell subpopulations, which are likely to have potent antitumour, antiviral, or immunomodulatory effects in autoimmunity. Our data indicate that the addition of IL-21 has a synergistic effect by increasing the numbers of NK cells on a large scale. IL-2 and IL-15 may induce the expression of killer cell immunoglobulin-like receptors (KIRs) in KIR-negative populations, the c-lectin receptor NKG2D and the natural cytotoxic receptor NKp44. The addition of IL-21 to IL-15 or IL-2 can modify the pattern of the KIR receptors and inhibit NKp44 expression by reducing the expression of the adaptor DAP-12. IL-21 also preserved the production of interferon-γ and enhanced the cytotoxic properties of NK cells. Our findings indicate that the proinflammatory cytokines IL-2, IL-15 and IL-21 can modify the peripheral repertoire of NK cells. These properties may be used to endow subpopulations of NK cells with specific phenotypes, which may be used in ex vivo cellular immunotherapy strategies.     

Delayed hypersensitivity and nonspecific cellular immunity. The cytotoxic activity of macrophages, natural and antibody-dependent killer cells

The experiment on (BALB/cXC57BL)F1 mice, showing a high level of delayed hypersensitivity (DH) when sensitized with BCG vaccine and Staphylococcus aureus strain B-243, has demonstrated the influence of such sensitization and DH reaction induced by the injection of a specific antigen (old tuberculin or staphylococcal phagolysate) into the sensitized animals on the cytotoxicity of macrophages, natural killers (NK) and antibody-dependent killers (ADK). Sensitization with BCG vaccine alone results in an insignificant rise in the activity of these effector cells, and sensitization with S. aureus produces no changes at all. The pronounced activation of the cytotoxicity of macrophages, NK and, to a lesser extent, ADK has been observed in DH reaction induced by the injection of a specific antigen into the sensitized mice. In the course of DH reaction a rise in the activity of NK and ADK not only against tumor target cells, but also against microbial ones (Candida albicans and S. aureus) has been found to occur.     

IL-2- and IFN gamma-enhanced natural cytotoxic activity: analysis of the role of different lymphoid subsets and implications for activation routes

To elucidate the immunoregulatory mechanisms by which human recombinant interleukin 2 (r.IL-2) and human recombinant interferon (IFN)-gamma influence natural cytotoxic activity of human peripheral blood lymphocytes (PBL), experiments were performed in which we studied: the kinetics of augmentation of natural cytotoxic activity by IL-2 and IFN-gamma; the phenotypes of the natural cytotoxic precursor cells acted upon by IL-2 and IFN-gamma; the role of IL-2-induced IFN-gamma in the natural cytotoxic activity enhancing effects of IL-2 and (d) the surface phenotypes of the natural cytotoxic cells activated by IL-2 or IFN-gamma. Three phenotypical distinct precursor cells can be identified. The phenotypes of the precursor cells sensitive to r.IFN-gamma are T3-, Leu 7+or-, FcR-gamma+. The phenotypes of the precursor cells sensitive to r.IL-2 are T3-, Leu 7-, FcR-gamma+or-. It appears that during prolonged culture relatively more of the FcR-gamma- precursor cells acquire natural cytotoxic activity upon stimulation with r.IL-2. Antibodies to IFN-gamma, known to neutralize both recombinant and natural IFN-gamma, completely inhibited the IFN-gamma-induced augmentation of natural cytotoxic activity but only slightly affected the IL-2-mediated augmentation. This indicates that only part of the augmenting effect of r.IL-2 is mediated through IL-2-induced IFN-gamma. This implies that r.IL-2 augments natural cytotoxic activity predominantly through an IFN-gamma-independent pathway. The phenotype of the effector cells expressing natural cytotoxic activity after a 4-day culture period with IL-2 or IFN-gamma was analyzed by depletion experiments. Virtually all the cells that exerted the IFN-gamma-enhanced natural cytotoxic activity are FcR-gamma+, whereas a portion of the cells that exerted the IFN-gamma-enhanced natural cytotoxic activity are FCR-gamma-. Both natural killer cells and lymphokine-activated killer cells contribute to natural cytotoxic activity. The relative contributions of both activities to the natural cytotoxic activity augmented by IL-2, respectively, IFN-gamma, is discussed.     

Effects of natural and recombinant IL 2 on regulation of IFN gamma production and natural killer activity: lack of involvement of the Tac antigen for these immunoregulatory effects

Highly enriched populations of human large granular lymphocytes (LGL), natural killer (NK) cells, and T cells were obtained from low and high density fractions, respectively, of discontinuous Percoll gradients. The NK cells were composed of 75 to 90% LGL, with the majority of the contaminating cells being monocytes. The T cells were greater than 95% OKT3+. The proliferative and cytotoxic progenitors in both fractions were examined by using a limiting dilution assay with interleukin 2 (IL 2) from four sources: 1) crude supernatant of a gibbon lymphoma (MLA-144), 2) purified (150,000-fold) MLA-144 IL 2, 3) partially purified human IL 2, and 4) purified recombinant human IL 2. The proliferative capacity was measured at day 7 by [3H]thymidine incorporation, whereas the progenitors of cells with NK-like activity were evaluated by assessing cytotoxic activity against K562 cells at day 8 in a 4-hr 51Cr-release assay. The frequency of proliferative progenitors among T cells was approximately 1/5 and was approximately 1/60 with LGL. Titration of the highly purified IL 2 preparation demonstrated that LGL proliferated with as little as 2 U of IL 2. The frequency of detectable cytotoxic progenitors in the LGL population, however, fell sharply when less than 40 U of IL 2 were employed. The T cells failed to demonstrate cytotoxic activity against the NK-susceptible target cells at any concentration of IL 2 tested. The IL 2 preparations also were examined for their ability to directly and rapidly enhance the cytotoxic activity of highly purified NK cells. All four preparations of IL 2 enhanced the cytotoxic activity of LGL without any detectable accessory requirement after incubation for as little as 6 hr, even though the MLA-144 IL 2 preparations were devoid of detectable interferons (IFN). These data indicate that IL 2 has dual effects on NK cells, regulating their activity was well as promoting their proliferation. Collectively, these results demonstrate that highly purified IL 2, devoid of other detectable lymphokines, is capable of supporting the growth of human NK cells and augmenting their in vitro activity. In parallel experiments, these same IL 2 preparations were quite active in causing the proliferation of T lymphocytes, clearly demonstrating a role of IL 2 in promoting the proliferation of NK cells as well as T cells. The mechanism of IL 2 boosting appears to be a direct interaction with LGL, resulting in the production of IFN gamma.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cutting edge: a murine,IL-12-independent pathway of IFN-gamma induction by gram-negative bacteria based on STAT4 activation by Type I IFN and IL-18 signaling

IFN-alphabeta is a potent immunoregulatory cytokine involved in the defense against viral and bacterial infections. In this study, we describe an as yet undefined IFN-alphabeta-dependent pathway of IFN-gamma induction in mice. This pathway is based on a synergism of IFN-alphabeta and IL-18, and is independent of IL-12 signaling yet dependent on STAT4. In contradiction to current dogma, we show further that IFN-alphabeta alone induces tyrosine phosphorylation of STAT4 in murine splenocytes of different mouse strains. This pathway participates in the induction of IFN-gamma by Gram-negative bacteria and is therefore expected to play a role whenever IFN-alpha or IFN-beta and IL-18 are produced concomitantly during bacterial, viral, or other infections.     

Cytokine production and killer activity of NK/T-NK cells derived with IL-2, IL-15, or the combination of IL-12 and IL-18

NK cell populations were derived from murine splenocytes stimulated by IL-2, IL-15, or the combination of IL-12 and IL-18. Whereas NK cells derived with the latter cytokines consisted of an homogeneous population of NK cells (DX5+CD3-), those derived with IL-2 or IL-15 belonged to two different populations, namely NK cells (DX5+CD3-) and T-NK cells (DX5+CD3+). Among NK cells, only those derived with IL-12/IL-18 produced detectable levels of cytokines, namely IFN-gamma, IL-10, and IL-13 (with the exception of IL-13 production by NK cells derived with IL-2). As for T-NK cells, IL-2-stimulated cells produced a wide range of cytokines, including IL-4, IL-5, IL-9, IL-10, and IL-13, but no IFN-gamma, whereas IL-15-derived T-NK cells failed to produce any cytokine. Switch-culture experiments indicated that T-NK cells derived in IL-2 and further stimulated with IL-12/IL-18 produced IFN-gamma and higher IL-13 levels. Next, we observed that NK/T-NK cell populations exerted distinct effects on Ig production by autologous splenocytes according to the cytokines with which they were derived. Thus, addition of NK cells derived in IL-12/IL-18 inhibited Ig production and induced strong cytotoxicity against splenocytes, whereas addition of NK or T-NK cells grown in IL-2 or IL-15 did not. Experiments performed in IFN-gammaR knockout mice demonstrated that IFN-gamma was not involved in the killer activity of IL-12/IL-18-derived NK cells. The hypothesis that their cytotoxic activity was related to the induction of target apoptosis was confirmed on murine A20 lymphoma cells. Experiments performed in MRL/lpr mice indicated that IL-12/IL-18-derived NK cells displayed their distinct killer activity through a Fas-independent pathway. Finally, perforin was much more expressed in IL-12/IL-18-derived NK cells as compared with IL-2- or IL-15-derived NK cells, an observation that might explain their unique cytotoxicity.     

Mechanisms for virus-induced liver disease: tumor necrosis factor-mediated pathology independent of natural killer and T cells during murine cytomegalovirus infection.

The contribution of endogenous NK cells and cytokines to virus-induced liver pathology was evaluated during murine cytomegalovirus infections of mice. In immunocompetent C57BL/6 mice, the virus induced a self-limited liver disease characterized by hepatitis, with focal inflammation, and large grossly visible subcapsular necrotic foci. The inflammatory foci were most numerous and contained the greatest number of cells 3 days after infection; they colocalized with areas of viral antigen expression. The largest number of necrotic foci was found 2 days after infection. Overall hepatic damage, assessed as increased expression of liver enzymes in serum, accompanied the development of inflammatory and necrotic foci. Experiments with neutralizing antibodies demonstrated that although virus-induced tumor necrosis factor (TNF) can have antiviral effects, it also mediated significant liver pathology. TNF was required for development of hepatic necrotic foci and increased levels of liver enzymes in serum but not for increased numbers of inflammatory foci. The necrotic foci and liver enzyme indications of pathology occurred independently of NK and T cells, because mice rendered NK-cell deficient by treatment with antibodies, T- and B-cell-deficient Rag-/- mice, and NK- and T-cell-deficient E26 mice all manifested both parameters of disease. Development of necrotic foci and maximally increased levels of liver enzymes in serum also were TNF dependent in NK-cell-deficient mice. Moreover, in the immunodeficient E26 mice, virus-induced liver disease was progressive, with eventual death of the host, and neutralization of TNF significantly increased longevity. These results establish conditions separating hepatitis from significant liver damage and demonstrate a cytokine-mediated component to viral pathogenesis.