Nongranular proteolytic enzymes of rat IL-2–activated natural killer cells. II. Purification and identification of rat A-NKP 1 and A-NKP 2 as constituents of the multicatalytic proteinase (proteasome) complex

We have recently described nongranular, cytosolic, high-molecular-weight trypsin-like (A-NKP 1) and chymotrypsin-like (A-NKP 2) proteases of interleukin-2–activated rat natural killer (A-NK) cells. A functional correlation between the inactivation of A-NKP 2 and the inhibition of rat A-NK cell-mediated cytotoxicity was found. Herein we describe the 6,000-fold purification of A-NKP 2 to apparent homogeneity following: isopycnic sucrose gradient fractionation of postnuclear supernatants, molecular sieve chromatography, and heparin-Sepharose® chromatography. We also report the novel finding that A-NKP 2 as well as A-NKP 1, derived from either rat A-NK cells or the rat NK leukemic cell line CRNK-16, are constituents of the multicatalytic proteinase (MCP/proteasome) complexes of these cells. Characteristic biochemical, biophysical, and electron microscopic/ultrastructural similarity to the rat liver proteasome was observed. However, Western blot analysis using polyclonal antibodies to the rat liver proteasome clearly indicated differences in the rat hepatic proteasome and the CRNK-16–derived proteasomal subunits. The identification, characterization, and purification of A-NKP 1 and A-NKP 2, described herein, now allow for further investigation of the potential role of these proteasome components in NK cell function. Moreover, the proteasome of NK and A-NK cells can now be compared and contrasted to the granzymes of lytic granules with respect to their role in cell-mediated cytotoxicity. © 1994 Wiley-Liss, Inc.     

Cancer immunotherapy with interleukin-2-activated natural killer cells

Natural killer (NK) cells are a subset of lymphocytes with a distinct morphologic appearance (large granular lymphocytes [LGL]) and the ability to kill virally infected and tumor targets but to spare most normal cells. NK cells respond to a variety of biologic agents, such as interleukin-2 (IL-2), or interferons, by upregulation of cytolytic, secretory, and proliferative functions. In cancer-bearing hosts, NK cells have been considered to be the major component of antitumor immunity responsible for rapid elimination of malignant cells from the blood. More recently, however, studies have demonstrated the ability of adoptively transferred, IL-2-activated NK cells to selectively localize into solid tumors tissue and to eliminate established tumors. While these findings indicate a role for NK cells in cancer immunotherapy, additional studies are needed in both animal models and in humans to optimize clinical protocols of cancer therapy based on these cells.     

Participation of the CD27 antigen in the regulation of IL-2-activated human natural killer cells.

The CD27 Ag is expressed by the majority of resting T lymphocytes and appears to play a crucial role in T cell activation. We found that some resting peripheral blood NK cells also express CD27. Furthermore, CD27 expression was up-regulated on NK cells stimulated by IL-2. The cytolytic activity of IL-2-activated, but not resting, NK cells was inhibited by an anti-CD27 mAb (anti-1A4). However, anti-1A4 did not affect conjugate formation between IL-2-activated NK cells and tumor cell targets. In contrast, anti-1A4 inhibited CD2-mediated calcium mobilization and the serine esterase activity of NK cell granules. These inhibitory effects could be mediated in part by increase in intracellular cAMP levels induced by anti-1A4. Our results suggest that the CD27 Ag plays an important role in the regulation of activated NK cells.     

Lysis of mycobacteria-infected monocytes by IL-2-activated killer cells: role of LFA-1

Mycobacterium avium-intracellulare (MAI) is a ubiquitous soil contaminant that rarely causes disseminated disease in adults regardless of immunological status. In AIDS patients, however, this organism invades virtually every tissue and organ, and most conventional chemotherapeutic agents are usually ineffective against MAI. We report here that monocytes, in which MAI has established an intracellular parasitic stage, are under the control of natural killer (NK) cells. Autologous large granular lymphocytes (LGL), purified from human peripheral blood leukocytes, were capable of efficiently lysing autologous MAI-infected monocytes in a 5-hr 51Cr release assay. More importantly, interleukin 2 (IL-2) was able to activate the LGL to a higher degree of lysis of infected monocytes. LGL cultured in medium alone could not kill normal monocytes, but showed some degree of lysis of MAI-infected cells. IL-2 activated killer (LAK) cells, on the other hand, lysed normal monocytes to a moderate degree and this activity was makedly enhanced if the monocytes were infected with MAI. The sensitivity of monocytes was directly proportional to the inoculating number of bacteria, indicating that increased bacterial burden would enhance susceptibility to LAK-mediated lysis. Finally, the addition of monoclonal antibodies to LFA-1 (both alpha and beta chains), but not LFA-2 or LFA-3, blocked lysis of both infected and uninfected monocytes when added directly to the cytotoxicity assays, indicating that this adhesion protein is involved in the lysis of autologous, infected monocytes. Thus, NK/LAK cells may be important in containment of infection by lysis of infected monocytes before the bacteria can multiply and spread to other sites.     

IL-2 induces expression of serine protease enzymes and genes in natural killer and nonspecific T killer cells

The expression of serine protease genes was examined in murine NK cells that were purified by panning spleen cells with PMA. Although unstimulated NK cells were cytolytic, they were found not to express the C11 (chymotrypsin-like) mRNA. Culturing these cells in IL-2 (500 to 800 U/ml) for 5 to 7 days induced both the lytic activities and the protease enzymes by 20- to 30-fold. Concomitant to these activation events, the total steady state mRNA of both C11 and HF (trypsin-like) genes were also elevated. The activation of lysis, serine protease enzymes, and C11 and HF mRNA all peaked around day 5 in culture and was dose dependent. In order to exclude the possibility that PMA synergizes with IL-2 in this system, spleen cells from SCID mice, which contained mainly NK cells, were cultured under the same conditions (800 U/ml IL-2, with or without PMA) and PMA did not appear to enhance the expression of these mRNA. Similarly, IL-2 also induced the lytic activities, enzyme levels, and mRNA in the non-Ag-specific T killer cells isolated from spleens of normal mice. Lytic activity of T killer cells was not as high as the NK cells, however, the addition of PHA into the lytic assay resulted in enhanced lysis comparable to that of NK cells. These results showed that lytic activity increased along with protease enzyme levels and mRNA expression in both NK and resting T cells. Therefore, elevated levels of the protease enzymes could be one mechanism involved in optimal lytic activity of IL-2-induced lymphokine activated killer cells.     

Adhesion characteristics of human interleukin 2-activated natural killer cells

Culture of human monocyte-depleted peripheral blood mononuclear cells with recombinant IL2 (rIL2) induced adherence to plastic by 24 hr and subsequent proliferation in a subpopulation of lymphocytes with phenotypic and functional characteristics of activated natural killer (NK) cells. Purified human NK cells activated in the presence of IL2 for 24 hr upregulated the expression of the CD11c (p150.95) and CD11a antigen but not other cellular adhesion molecules (CAM). After further incubation with IL2, NK cells displayed upregulation of all of the antigens in the CD11/CD18 family of CAM. The process of adhesion was strictly dependent on culture in the presence of IL2, divalent cations, and active cellular metabolism. Adhesion also was dependent on expression of CAM on the cell surface, since monoclonal antibodies to CAM inhibited adhesion of activated NK cells to varying degrees (from 50 to 80%). An antibody (LeuM5) to the CD11c antigen (p150.95) gave the highest level of inhibition, and anti-CD11a (LFA-1) also was inhibitory, while anti-CD56 (NKH1) or anti-CD11b did not interfere with adhesion to plastic. Anti-CD11c was also the most effective in initiating the detachment of adherent-phase NK cells. Antibodies to CD18 or CD2 antigen also inhibited binding of NK cells to plastic. The blocking effects of anti-CD2 and anti-CD11a were additive in this system. On the surface of plastic-adherent and motile NK cells, all CAM except the CD56 antigen had a polar or bipolar distribution, as determined by staining with anti-CAM antibodies. Surface antigens CD11b, CD11c, CD2, and CD18 on nonadherent NK cells were clustered at the cellular poles by both immunofluorescence and immunogold electron microscopy, whereas CD11a (LFA-1) and CD56 antigens were distributed diffusely. CAM, especially CD11c, were also detected in cytoplasmic granules by immunostaining in IL2-activated NK cells. Thus, CAM may be stored in granules, allowing for their rapid transfer to the cell membrane in response to activation. Our results indicate that CAM are upregulated in IL2-activated NK cells and that some of these molecules (e.g., CD11c) play an important role in the development of plastic adherence by a subpopulation of these cells.     

The role of IL-4 in proliferation and differentiation of human natural killer cells. Study of an IL-4-dependent versus an IL-2-dependent natural killer cell clone

The role of IL-4 in proliferation and differentiation of human NK cells was studied using newly established sublines of an IL-4-dependent NK cell clone (IL4d-NK cells) and an IL-2-dependent NK cell clone (IL2d-NK cells) derived from a parental conditioned medium-dependent NK cell clone (CM-NK cells). IL-4 induced the higher proliferation of CM-NK cells, but abolished their NK activity and decreased CD16 and CD56 Ag expression. In contrast, IL-2 induced the higher NK activity and increased CD16 and CD56 Ag expression. Addition of anti-IL-4 antibody to the culture of CM-NK cells with CM inhibited the proliferation, but slightly increased NK activity, and largely increased CD56 Ag expression. Addition of anti-IL-2 antibody to the culture of CM-NK cells with CM inhibited both proliferation and cytotoxicity. Proliferation of IL4d-NK cells, which is totally dependent on rIL-4, is greater than that of IL2d-NK cells, which was greater than parental CM-NK cells. Morphologically, IL4d-NK cells are small and round, whereas IL2d-NK cells are large and elongated. Anti-IL-4 antibody inhibited proliferation of IL4d-NK but not IL2d-NK cells, whereas anti-IL-2 antibody inhibited that of IL2d-NK but not IL4d-NK cells. IL-2 was not detected in the supernatant from IL4d-NK cells, nor was IL-2-mRNA expressed in IL4d-NK cells. In contrast, IFN-gamma production and protein expression in IL4d- and IL2d-NK cells were detected. NK cell activation markers (CD16 and CD56) were expressed on IL2d-NK cells but not IL4d-NK cells. IL4d-NK cells were not cytotoxic to any tumor cells tested, whereas IL2d-NK cells displayed potent NK activity and lymphokine-activated killer activity. IL4d-NK cells failed to bind K562 tumor cells, whereas one-third of the IL2d-NK cells did. IL4d-NK cells responded to rIL-2, proliferated, and differentiated into cytotoxic NK cells, whereas IL2d-NK cells failed to respond to rIL-4 and died. These results raise a possibility that IL4d-NK cells or IL2d-NK cells primarily represent the immunologic properties of immature or activated types of human NK cells, respectively. Our results provide the first evidence of the capability of IL-4 to support continuous proliferation of a lymphocyte clone with immature NK cell characteristics and to stimulate IFN-gamma production in the clone. IL-4 is suggested as a potential growth factor for certain types of human NK cell progenitors.     

Tumor-induced apoptosis of human IL-2-activated NK cells: role of natural cytotoxicity receptors

We provide evidence that tumor cells can induce apoptosis of NK cells by engaging the natural cytotoxicity receptors (NCR) NKp30, NKp44, and NKp46. Indeed, the binding between NCR on NK cells and their putative ligands on tumor target cells led to NK cell apoptosis, and this event was abolished by blocking NCR/NCR-ligand interaction by anti-NCR-specific mAbs. The engagement of NCR induced up-regulation of Fas ligand (FasL) mRNA, FasL protein synthesis, and release. In turn, FasL interacting with Fas at NK cell surface causes NK cell suicide, as apoptosis of NK cells was inhibited by blocking FasL/Fas interaction with specific mAbs. Interestingly, NK cell apoptosis, but not killing of tumor target cells, is inhibited by cyclosporin A, suggesting that apoptosis and cytolysis are regulated by different biochemical pathways. These findings indicate that NCR are not only triggering molecules essential for antitumor activity, but also surface receptors involved in NK cell suicide.     

Subcellular localization and partial characterization of insulin proteolytic activity in rat liver

Using (A14-125I)-insulin as a tracer, insulin proteolytic activity in rat liver was found to be localized both to the cytosol and the endoplasmic reticulum. The membrane-associated activity was highly latent (70-80%). Both cytosolic and particulate activities had similar Km values and Mr of approx. 300 000 by gel filtration. Both were strongly inhibited by diamide (90%), but were unaffected by leupeptin or pepstatin. A comparison of the subcellular distributions with various 125I-isomers of insulin as tracers showed that both particulate and cytosolic activities were highest with (A14-125I)-insulin.     

Interferon-gamma upregulates the susceptibility of human neuroblastoma cells to interleukin-2-activated natural killer cells

The influence of interferon-gamma on the susceptibility of neuroblastoma cells in cell-mediated killing was investigated. Neuroblastoma cells were only weakly susceptible targets for peripheral mononuclear cells. However, enrichment of natural killer (NK) cells or activation of NK cells with interleukin-2 resulted in a considerable increase of neuroblastoma cell lysis. Pretreatment of neuroblastoma targets with interferon-gamma additionally increased the susceptibility to enriched NK cells as well as to interleukin-2-activated NK cells. The conjugate formation between enriched NK cells and the neuroblastoma targets was not affected by the pretreatment of the targets with interferon-gamma. Concomitantly, treatment of the neuroblastoma targets with interferon-gamma resulted in a strong induction of otherwise poorly expressed major histocompatibility complex (MHC) class I antigen expression. These results suggest that the increased expression of MHC class I antigens on target cells is not always correlated with decreased sensitivity for NK cells but can also be followed by an increased susceptibility for NK cells.     

Long-term killing of natural killer-resistant target cells by interferon-alpha-, interferon-gamma-, and interleukin-2-activated natural killer cells

The sensitivity of target cells to natural killer (NK) cell-mediated cytotoxicity was investigated. Five target cell lines were examined for susceptibility to killing by activated NK cells in a 4-hour cytotoxicity assay: one of them (K562) was highly sensitive, while the other four were resistant. However, the four NK-resistant target cell lines were fully susceptible to lysis when the assay was extended to 24 h. The cytotoxic cells that killed the NK-resistant target cells in a 24-hour assay were plastic- and nylon wool-nonadherent human peripheral blood mononuclear cells (PBMC) and their cytotoxicity was increased by interferon-alpha, interferon-gamma, and interleukin-2. Further, the cytotoxic activity of PBMC in the long-term assay was associated with large granular lymphocytes purified on a Percoll gradient, that killed the NK-sensitive cell line K562 in a 4-hour assay. All of the above are general criteria to qualify the cytotoxic cells as NK cells. Thus, the NK-resistant phenotype may not reflect absolute immunity to NK-mediated lysis, but it may reflect the different rates at which various target cell lines can be killed.     

Ligation of ICAM-1 molecules inhibits target cell-induced granule exocytosis of IL-12-activated natural killer cells

The importance of cell adhesion molecules such as ICAM-1 is emphasized in cell-to-cell interactions that are critical in the generation of effective immune reactions. In this study, the involvement of ICAM-1 in natural killer (NK) cell activities was characterized in IL-12-activated human NK cells. To address the question of whether ligation of ICAM-1 molecules can modulate NK cell cytolytic activities, a 4-h (51)Cr-release assay was performed after pretreatment of NK cells with R6.5 mAb (anti-human ICAM-1 mAb). Ligation of membrane ICAM-1 molecules significantly inhibited IL-12-enhanced NK cytotoxicity against K562, and the pretreatment of neutralizing soluble ICAM-1 with R6.5 mAb blocked this inhibitory effect. The involvement of Ca(2+)-dependent granular exocytosis was evaluated. BLT esterase assay demonstrated that the ligation of ICAM-1 molecules inhibited granular exocytosis of NK cells. Additionally, the ICAM-1-mediated inhibition of Ca(2+) flux in NK cells was detected using Fluo-3AM, while the pretreatment of NK cells with R6.5 mAb did not affect conjugate formation between NK and K562 cells. Collectively, these results suggest that the signals transduced from ICAM-1 molecules might be sufficient to induce inhibitory effects on NK cells.     

Subcellular localization of enzymes oxidizing citrate in the rat brain

¹RSA was calculated as the ratio: percentage of total recovered activity found in fraction to percentage of total protein found in the same fraction.     

Generation and characterization of IL-2-activated veto cells.

The regulation of in vivo cytolytic response is important in a model of murine graft-vs-host disease induced by the injection of parental splenocytes into unirradiated B6D2F1 recipients. Injection of C57BL/6J spleen cells into B6D2F1 recipients results in an acute form of graft-vs-host disease that is characterized by the presence of CTL and suppressor cells, runting, and occasionally death. In contrast, injection of DBA/2J spleen cells into B6D2F1 recipients results in a chronic form of graft-vs-host disease that is characterized by the lack of in vivo CTL and hyperproduction of Ig and autoantibodies that results in an SLE-like syndrome. One reason for the lack of donor antirecipient CTL after injection of DBA/2J donor cells is that B6D2F1 recipient cells functionally inactivate the donor DBA/2J CTL precursor cells by expressing veto activity. These B6D2F1 veto cells are radiosensitive, inhibited by anti-CD8 antibodies, found primarily in lymph nodes, and were further characterized by testing the response of these inhibitory cells to lymphokines. These studies indicate that IL-2 can potentiate the activity of the veto cells induced in vivo and veto cells with a similar phenotype can be generated by in vitro incubation of naive lymph node cells with IL-2. These cells have been designated as IL-2-activated veto cells or LAV cells. IL-2 did not increase inhibitory activity by increasing the number of CD8+ cells or the number of CD8 molecules on the LAV cell surface but by altering the activation state of the LAV cell. The inhibitory capabilities of antibodies binding various cell surface molecules indicated that CD2 and intercellular adhesion molecule-1 molecules in addition to CD8 molecules played a role in the function of LAV cells.     

Promonocytes have the functional characteristics of natural killer cells.

Promonocytes isolated from a 5-day-old L-fibroblast-conditioned liquid bone marrow culture show strong NK cell cytotoxicity. They kill YAC target cells in a short-term 125IUDR-release assay whereas P815 targets are unaffected. This NK-like cytotoxicity is enhanced in the presence of interferon preparations. Morphologically, these promonocytes resemble a medium size lymphocyte with a high nucleus to cytoplasma ratio, they are nonadherent, nonphagocytic, and negative in nonspecific esterase staining. Promonocytes are precursor cells from macrophages, which have not yet developed the typical macrophage criteria. Within 24 to 48 hr they mature to adherent macrophages. We have shown previously, that the same promonocytes have the capacity to perform K cell killing of antibody-coated tumor target cells. The cytotoxic effector functions of promonocytes are abolished when the cells are treated with the alloantimacrophage serum Mphi 1.2 plus rabbit C. The relationship or similarity between K cells, NK cells and promonocytes is discussed.     

Role of cytokines in the adoptive immunotherapy of an experimental model of human head and neck cancer by human IL-2-activated natural killer cells.

Peritumoral injection of human IL-2-activated natural killer cells into nude mice consistently induced regression of xenografts of human squamous cell carcinoma of the head and neck (SCCHN). To determine the mechanisms responsible for the tumor regression, the lymphoid cells infiltrating the tumor stroma at 24 to 48 h after adoptive immunotherapy were examined by in situ hybridization for the presence of mRNA for cytokines or IL-2R. Numerous lymphoid cells expressing cytokine or IL-2R genes were observed in these tumors, whereas the cultured IL-2-activated NK cells used for therapy were negative. Thus, it appeared that the transferred NK cells became activated in situ after coming into proximity with the tumor cells. To analyze this phenomenon, fresh or cultured human NK cells were coincubated in vitro with irradiated human SCCHN cell line, PCI-1, with or without the presence of IL-2. Expression of mRNA for IL-2R, perforin, and various cytokines was observed within 5 h. Contact with the tumor cells stimulated NK cells to proliferate, secrete IFN-gamma, TNF-alpha, and soluble IL-2R, up-regulate cell surface expression of IL2R p55 and p75 as well as CD16 Ag, and mediate higher levels of antitumor activity in 51Cr-release assays. In addition, supernatants of in vitro-activated NK cells significantly inhibited proliferation of SCCHN cell lines. By examining the effects of neutralizing mAb to various cytokines, this inhibitory activity was shown to be partially attributable to IFN-gamma. To determine the possible in vivo role of soluble factors produced by activated human NK cells, the supernatants (0.2 ml) or rIFN-gamma (10(5) U) were injected perilesionally each day for 2 wk into 3-day SCCHN established in immunosuppressed nude mice. These treatments caused significant (p less than 0.02) inhibition of tumor growth. The results of our studies indicate that human NK cells are strongly activated by SCCHN cells and that the consequent release of cytokines contribute to the regression of SCCHN growing in nude mice.     

Effects of IL-7 and IL-2 on highly enriched CD56+ natural killer cells. A comparative study.

IL-7 has been shown to induce low levels of lymphokine-activated killer cell (LAK) activity in bulk PBMC populations. We report here that immunomagnetically purified CD56+ cells from peripheral blood generated high LAK activity in response to IL-7. The LAK activity induced by IL-7 was comparable to, or slightly lower than, the LAK activity induced by IL-2. When analyzing cells from the same donor, no detectable LAK-generating effect of IL-7 was registered in the PBMC population, in contrast to a substantial effect in the CD56+ population. IL-2 induced 8- to 15-fold higher proliferative activity in CD56+ cells, relative to IL-7. At suboptimal concentrations of IL-2, IL-7 had a synergistic effect on the proliferation. IL-2-neutralizing antibodies did not abrogate the IL-7-induced proliferation or LAK generation. Both IL-7 and IL-2 induced comparable levels of 75-kDa TNFR expression, whereas IL-2R alpha expression was higher in IL-7-stimulated CD56+ cells. Low levels of TNF were produced in response to IL-7 at day 5, as opposed to a 50-fold higher TNF production in response to IL-2. No IL-2 or IL-6 production was detected. Our data indicate that IL-7 has profound and direct effects on CD56+ cells.     

Subcellular localization of sanguinarine biosynthetic enzymes in cultured opium poppy cells

Benzylisoquinoline alkaloids are a large and structurally diverse group of natural plant products that includes many compounds with potent biological activities, including the antimicrobial agent sanguinarine. The putative subcellular localization of the sanguinarine pathway was determined using in-frame N-terminal fusions between cDNAs encoding nine consecutive biosynthetic enzymes and the gene encoding the green fluorescent protein (GFP). Expression constructs were introduced into cultured opium poppy cells by particle bombardment, and the localization of fusion proteins was visualized using epifluorescence microscopy. GFP fusions with two O-methyltransferases and two N-methyltransferases in the sanguinarine pathway all produced non-targeted fluorescence in the cytosol and nucleus. Interspersed between these soluble proteins are five membrane-bound cytochromes P450. Corresponding cDNAs are available for three P450s, all of which produced fluorescence when fused to GFP in association with the endoplasmic reticulum (ER). Two enzymes with suggested or known N-terminal signal peptides were initially associated with the ER, but were subsequently transported to the central vacuole suggesting their occurrence in the ER lumen. The alternating localization of these biosynthetic enzymes to three subcellular compartments indicates extensive trafficking of pathway intermediates across the endomembranes and suggests a key role for compartmentalization in the regulation of sanguinarine metabolism.