Characterization of the stage in natural killer cell development in 14.5-day mouse fetal liver using adult bone marrow stroma.

Nonstimulated fetal liver (FL) from 14.5-day gestation mice had no natural killer (NK) cell activity and <3% expressed NK1.1. Even after short-term (3-4 day) culture of FL with the late-acting cytokines, interleukin (IL)-15 or IL-2, little or no NK activity was detected. However, longer-term (13 day) culture with IL-2 plus stroma derived from bone marrow (BM) of adult mice, resulted in extensive proliferation and differentiation to mature NK cells. Cell numbers began to increase after 4 days, and by day 13, they had increased 40-fold and 69% of the cells were NK1.1+ with high NK activity and 5%-10% were NK1.1- B220+. With stroma, but no IL-2, equivalent proliferation occurred, but differentiated cells were predominantly NK1.1- B220+, not NK cells. Culture for 13 days without stroma, but with either IL-2, IL-15, FLTK3-ligand (L) or stroma-conditioned medium, resulted in less than fivefold expansion, and minimal NK activity. Culture with combinations of FLTK3-L or ckit-L plus IL-15 or IL-2 increased both cell number and NK activity, but the increase in cell number was less than that seen with stroma plus IL-2. By limiting dilution assay on stroma plus IL-2, the precursor frequency was 1/(2660+/-292) whole FL cells and the absolute number, but not the frequency, increased during culture on stroma without IL-2. The NK cell progenitors were found in sorted NK1.1- and Sca-1+ c-kit+ lineage- subpopulations at a frequency of 1/(156+/-52.5). Together, these data suggest that the NK lineage cells in FL are primarily in early stages of development. They are highly proliferative, respond to early acting cytokines and express stem cell markers.     

Malignant granular lymphoproliferation after Epstein-Barr virus infection: partial immunologic reconstitution with interleukin-2

This report describes a patient who developed a malignant proliferation of granular lymphocytes following Epstein-Barr virus (EBV) infection. For many months, his illness resembled prolonged infectious mononucleosis with persistent fatigue, fever, leukocytosis, and serologic evidence of recent primary EBV infection. After approximately 1 year, however, he developed progressive granular lymphocytosis and extensive lymphocytic infiltration of the bone marrow and liver. Tests for EBV DNA in pre- and postmortem tissue samples using a sensitive DNA hybridization technique were negative. Southern blot analysis of DNA prepared from blood mononuclear cells demonstrated clonal T-cell antigen receptor gene rearrangement. Despite increased numbers of circulating lymphocytes with the morphology and surface phenotype of normal donor natural killer (NK) cells, the patient's NK activity was consistently depressed in a standard in vitro assay. However, in vitro incubation with interleukin-2 (IL-2), but not with alpha- or gamma-interferon, increased the NK activity of the patient's lymphocytes. Intravenous recombinant IL-2 treatment transiently increased the patient's blood NK activity and was associated with seroconversion to EBV nuclear antigens but failed to affect the progression of his disease. Our findings indicate that clonal granular lymphocytic proliferation may develop after EBV infection and confirm the utility of DNA hybridization analysis in distinguishing monoclonal from benign immunoreactive lymphoproliferation. Furthermore, our results suggest that certain functionally inert neoplastic granular lymphocytes acquire NK activity when exposed to IL-2.     

Leukemia-derived growth factor (non-interleukin-2) produced by murine lymphoma T-cell lines.

Autocrine growth factor activity was found in supernatants of AKR T-cell lymphoma lines cultured in serum-free medium. This factor was designated leukemia-derived growth factor (LDGF). Active supernatants stimulated the growth of the AKR murine T-cell lymphoma line SL 12, its cloned derivatives, and all other murine T-cell lymphoma lines tested. Growth factor activity in conditioned medium was found to be different from interleukin 2 (IL-2) and several other known growth factors. LDGF was able to stimulate growth of the human leukemia T-cells MOLT4f, and the LDGF from MOLT4f cells stimulated the mouse cells. Because mouse T-cell lymphoma lines produced and respond to this factor, it may support the continued proliferation of these cells and could be responsible for their malignant in vivo properties.     

Interleukin 18 (IL-18) in synergy with IL-2 induces lethal lung injury in mice: a potential role for cytokines, chemokines, and natural killer cells in the pathogenesis of interstitial pneumonia

Interleukin 18 (IL-18) was discovered as an interferon-gamma (IFN-gamma)-inducing factor and plays important roles in natural killer (NK) cell activation. IL-18 also induces proinflammatory cytokines; chemokines; helper T-cell 2 (T(H)2) cytokines (eg, IL-4, IL-13); and immunoglobulin E (Ig-E) and IgG1 production. The combination of IL-18 plus IL-2 or IL-12 up-regulates IFN-gamma gene expression and NK cytotoxicity and has synergistic antitumor activity in vivo and in vitro. Here it is reported that daily administration of IL-18 with IL-2, but not of IL-18 or IL-2 alone, induces lethal lung injury in normal mice, but not in IL-18 receptor alpha (IL-1 receptor-related protein)-deficient (IL-18 receptor alpha(-/-)) mice. Marked interstitial infiltration of lymphocytes, composed mainly of NK cells, was found in the lungs of IL-18/IL-2-treated mice. Increased cytokine and chemokine levels were observed in the sera and lungs of IL-18/IL-2-treated mice. Administration of IL-18/IL-2 was also lethal to mice treated with a metalloproteinase inhibitor, which inhibited tumor necrosis factor-alpha and Fas-ligand release. While IFN-gamma(-/-) mice were partially resistant to the treatment, IL-4(-/-), IL-13(-/-), IL-4/IL-13(-/-), and Stat6(-/-) mice were sensitive to IL-18/IL-2, indicating that these genes were not involved in the host response. The lethal effect by IL-18/IL-2 was completely eliminated in severe combined immunodeficient mice pretreated with antiasialo-GM1 antibody and normal mice pretreated with anti-NK1.1 but not with anti-CD4 or anti-CD8, monoclonal antibody. These results suggest that specific cytokines, chemokines, and NK cells are involved in the pathogenesis of interstitial pneumonia. These results suggest that the clinical use of this interleukin may result in unexpected physiological consequences.     

Targeting IL-2 to the endoplasmic reticulum confines autocrine growth stimulation to NK-92 cells

OBJECTIVE: Anti-tumor effects mediated by adoptively transferred natural killer (NK) cells are dependent on the presence of interleukin-2 (IL-2). IL-2 is considered to be a survival factor for NK cells and an enhancer of their cytotoxic potential. However, systemic administration of IL-2 is frequently impeded by undesirable side effects, such as high toxicity and nonlocalized administration. Genetic modification of NK cells expressing IL-2 in a localized and controlled manner could be a powerful tool for overcoming these obstacles. METHODS: Consequently, we have cloned the IL-2 gene using PCR and designed constructs that target IL-2 to specific subcellular compartments. The IL-2-dependent NK-92 cell line was used to verify the functionality of the subcellularly targeted IL-2 constructs. RESULTS: IL-2 targeted specifically to the endoplasmic reticulum (ER) was sufficient to support growth of NK-92 cells. In such cell lines, IL-2 was verified to be localized to the ER. IL-2 was not detected in the supernatant and growth of non-IL-2-modified NK-92 cells was not supported during coculturing experiments. IL-2-transduced NK-92 cell lines showed comparable functional activity and cytotoxicity to parental NK-92 cells. CONCLUSION: We demonstrate the ability of ER-retained IL-2 to provide autocrine growth stimulation to NK-92 cells, without secretion of the cytokine to the extracellular compartment. Therapy with IL-2 gene-modified autoactivating NK cells may avoid side effects imposed by exogenously administered IL-2.     

Lymphoid development in mice with a targeted deletion of the interleukin 2 receptor gamma chain.

The interleukin 2 receptor gamma chain (IL-2R gamma) is a component of the receptors for IL-2, IL-4, IL-7, and IL-15. Mutations in IL-2R gamma in man appear responsible for the X chromosome-linked immunodeficiency SCIDX1, characterized by a defect in T-cell and natural killer (NK)-cell differentiation with the presence of poorly functioning B cells. To explore at which level IL-2R gamma affects lymphoid development in vivo, we have analyzed mice derived from embryonic stem (ES) cells with mutant IL-2R gamma loci generated by Cre/loxP-mediated recombination. In the peripheral blood of chimeric animals, lymphoid cells derived from IL-2R gamma- ES cells were not detected, although control ES cells carrying an IL-2R gamma gene with embedded loxP sites gave rise to T-, B-, and NK-cell lineages. Germline IL-2R gamma-deficient male animals, however, developed some mature splenic B and T cells, although the absolute number of lymphocytes was almost 10-fold reduced. In contrast, there was a complete disappearance of NK cells (over 350-fold reduction). Development of gut-associated intraepithelial lymphocytes was also severely diminished, and Peyer's patches were not detected. In vitro mitogenic responses of thymocytes, IL-4-directed immunoglobulin class switch of splenocytes, and NK activity were defective. Thus, IL-2R gamma facilitates mainstream B- and T-cell generation and function and also appears to be essential for NK-cell development.     

Enforced expression of the Ikaros isoform IK5 decreases the numbers of extrathymic intraepithelial lymphocytes and natural killer 1.1+ T cells.

The zinc-finger protein Ikaros plays an important role in lymphoid homeostasis, and loss of Ikaros expression through germline disruption impairs lymphoid development. However, the role played by Ikaros after commitment to the T-cell lineage is unclear. To address this question, this study used the lck proximal promoter to drive the expression in T-cell progenitors of a naturally occurring short Ikaros isoform (IK5), which lacks the DNA-binding domain, reasoning that IK5 will form heterodimers with long isoforms and perturb their function. The IK5 transgene led to a selective and dramatic decrease in extrathymic intestinal intraepithelial lymphocytes (IELs) and natural killer 1.1+ T (NK T) cells with little effect on conventional alphabeta T cells, which resembles the T-cell phenotype of interleukin-15 receptor alpha chain (IL-15Ralpha) and IL-2/IL-15 receptor beta chain (IL-2Rbeta) knockout mice. The expression of IL-2Rbeta on double-negative T-cell progenitors of bi-5 was reduced, but enforced expression of IL-2Rbeta did not rescue IELs or NK T cells in bi-5 transgenic mice, suggesting that Ikaros or Ikaros family members regulate the expression of additional genes that are essential for the development of IELs and NK T cells. The study concludes that modest changes in the ratio of short to long Ikaros isoforms can substantially perturb T-cell development, and the development of IELs and NK T cells is particularly sensitive to such changes.     

Interleukin-2-activated natural killer cells can support hematopoiesis in vitro and promote marrow engraftment in vivo.

Purified natural killer (NK) cells were obtained from mice with severe combined immune deficiency (SCID) to ascertain their effect on hematopoiesis. When activated and propagated with recombinant human interleukin-2 (rhIL-2) in vitro, SCID spleen cells maintained a phenotypic and lytic spectrum consistent with a pure population of activated NK cells. When added with syngeneic bone marrow cells (BMC) in soft agar, the activated NK cells could support hematopoietic growth in vitro without the addition of exogenous hematopoietic growth factors. However, when syngeneic BMC were added along with cytokines to produce optimal growth conditions, the addition of NK cells was then inhibitory for hematopoietic colony formation. Antibodies to interferon-gamma (IFN-gamma) partially reversed the inhibitory effects. Supernatants from the NK-cell cultures could also exert these effects on hematopoiesis, although to a lesser extent. Analysis of the NK cell RNA demonstrated that activated NK cells express genes for hematopoietic growth factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte CSF (G-CSF), and IL-1 beta. The NK cells were also found to express IFN-gamma, transforming growth factor-beta 1 (TGF-beta 1), and tumor necrosis factor-alpha (TNF-alpha) mRNA. Analysis of the NK-cell supernatants using factor-dependent myeloid progenitor cell lines showed that the NK cells were producing G-CSF and growth-promoting activity that could not be attributed to IL-1, IL-3, IL-4, IL-5, IL-6, GM-CSF, G-CSF, macrophage CSF (M-CSF), or stem cell factor. The transfer of activated NK cells with BMC into lethally irradiated syngeneic mice resulted in greater BMC engraftment in the recipients. Thus, these results using a pure population of activated NK cells indicate that when activated, these cells can produce a variety of growth factors for hematopoiesis and exert significant hematopoietic growth-promoting effects in vivo.     

Natural killer cell activity of rhesus macaques against retrovirus-pulsed CD4+ target cells

Rhesus peripheral blood mononuclear cells (PBMC) fail to demonstrate natural killer (NK) activity against the human T-cell lines CEM, CEM x 174, or SUP-T1. However, these cell lines could act as NK-sensitive target cells if they were pulsed with heat-inactivated, whole simian immunodeficiency virus (SIV). The ability of these SIV-pulsed T-cell lines to act as NK-sensitive target cells was directly related to the relative density of CD4 on their surface. Target cell generation was inhibited by preincubation of cell lines with CD4 monoclonal antibody (MAb) with specificity for the SIV binding site. In addition, NK activity was seen against target cells that had been prepared with human immunodeficiency virus type 1 (HIV-1) gp120, nonglycosylated gp120, env A of feline leukemia virus (FeLV), and simian type D retrovirus (SRV). Addition of leupeptin to target cells prior to SIV pulsing did not result in a significant decrease in cytotoxic activity, suggesting that processing is not required for the generation of target cells. The cells that mediate NK activity are nonadherent, do not form rosettes with AET-treated sheep red blood cells (SRBC), and are phenotypically CD16+ and CD8+. NK activity of SIV-infected macaques was significantly decreased against both K562 cells and SIV-pulsed target cells as compared with uninfected animals. However, treatment of PBMC with interleukin-2 (IL-2) resulted in a partial restoration of NK activity.     

Interleukin-10 is a proliferation factor but not a differentiation factor for human myeloma cells

Because interleukin-10 (IL-10) is a potent differentiation factor of human B cells into mature plasma cells, we investigated its effect on human malignant plasma cells. IL-10 did not induce any differentiation and increase in Ig synthesis in four human IL-6-dependent malignant plasma cell lines. However, it stimulated the proliferation of two of four cytokine-dependent cell lines in the absence of IL-6 and IL-10- dependent myeloma cell lines have been obtained. The myeloma cell growth activity of IL-10 was unaffected by anti-IL-6 and anti-IL-6R antibodies. Similarly, IL-10 stimulated (P = .001) the proliferation of freshly-explanted myeloma cells in IL-6-deprived cultures of tumor samples from patients with active multiple myeloma (MM) and produced twice as many myeloma cells in these cultures. Again, this cytokine was unable to induce further differentiation (assessed by rate of Ig production) of fresh myeloma cells. A very sensitive enzyme-linked immunosorbent assay (ELISA; 1 pg/mL) only rarely detected IL-10 in the sera of MM patients (3 of 89). On the contrary, serum IL-10 was detected in 60% of patients with plasma cell leukemia (12 of 20). These data show that IL-10 is an IL-6-unrelated growth factor for malignant plasmablastic cells. This cytokine could be involved in the late phase of MM in vivo.     

Interleukin 2 mediates the inhibition of oligodendrocyte progenitor cell proliferation in vitro.

In the immune system, T-lymphocyte proliferation depends on interleukin 2 [IL-2 (T-cell growth factor)] interaction with specific receptors. In this study we show that IL-2 can specifically inhibit the proliferation of neonatal rat oligodendrocyte progenitor cells cultured in a serumless, chemically defined medium (oligodendrocyte-defined medium; ODM). IL-2 inhibited both [3H]thymidine incorporation and increase in cell number. Specificity was shown by precipitating IL-2 activity with anti-IL-2 antiserum. Furthermore, growth inhibition depended on the expression of Tac (an anti-IL-2 receptor monoclonal antibody)-positive receptors (IL-2 receptor). When cells were cultured in the presence of IL-2, both Tac-positive staining and growth inhibition were no longer expressed. The addition of interleukin 1 had no effect on [3H]thymidine incorporation or changes in cell number. However, when IL-1 was subsequently added together with IL-2, Tac expression and IL-2-mediated inhibition of cell proliferation was induced. This inhibitory effect was not due to a sensitive subpopulation because greater than 90% of the culture was Tac positive. Taken together, these data show that IL-2 can specifically inhibit oligodendrocyte proliferation and acts via Tac-positive receptors.     

Production of natural killer cell activity-augmenting factor (interleukin-6) by human epiphyseal chondrocytes.

OBJECTIVE. We sought to determine the capacity of human epiphyseal chondrocytes to modulate the cytotoxic activity of human natural killer (NK) cells by determining whether they release interleukin-6 (IL-6), a cytokine recently shown to stimulate NK cell activity. METHODS. Conditioned medium from human epiphyseal chondrocyte cultures (Ch-CM) was tested for IL-6 activity using the B9 cell hybridoma assay. Its NK cell-stimulating capacity in the presence of K562 (myelogenous leukemia) cells or human chondrocytes was evaluated in a 4-hour 51Cr-release assay. Ch-CM-derived IL-6/NK cell-augmenting factor activity was partially purified by high-performance liquid chromatography (HPLC) gel filtration and Western blot. RESULTS. Ch-CM contained an NK cell-augmenting factor (NKAF) which was blocked by IL-2 or IL-6 antibodies. Ch-CM did not contain detectable IL-2 activity, but it stimulated IL-2 production by human peripheral blood lymphocytes (PBL). This IL-2-inducing capacity was inhibited by IL-6 antibodies, indicating that chondrocytes release an IL-6-like activity. Ch-CM significantly enhanced the proliferation of IL-6-dependent B9 hybridoma cells, and Western blot analysis of Ch-CM revealed specific bands corresponding to those of highly purified IL-6. Upon HPLC gel filtration, chondrocyte NKAF copurified with chondrocyte IL-6. Pure IL-6 and chondrocyte IL-6 were tested for their ability to stimulate the cytotoxic activity of human PBL against chondrocytes. Both mediators significantly enhanced chondrocyte killing. Lysis of chondrocytes by PBL was mediated by NK cells, since depletion of CD16+ cells resulted in inhibition of the activity. CONCLUSION. Thus, upon stimulation, chondrocytes produce IL-6 which, through IL-2 induction, augments the activity of NK cells against K562 target cells as well as against chondrocytes.     

Evidence for a paracrine pathway of B-cell stimulation in hairy cell leukaemia

It is a well-known phenomenon that the growth of malignant B-lymphocytes, i.e. hairy cells, is regulated by cytokines. Several investigators have suggested that the stimulating cytokines are produced by the malignant B cells themselves, indicating an autocrine growth regulation. In this paper we demonstrate that T-lymphocyte clones produce soluble mediators which stimulate the growth of malignant B lymphocytes. The incidence of the growth-stimulating T-cell clones derived from peripheral blood is identical in patients with hairy cell leukaemia (HCL) and healthy controls. About 50% of the clones stimulate the growth of hairy cells, but not the growth of purified B lymphocytes of healthy donors. The stimulating activity of a single clone varies when tested on different hairy cells. Interferon alpha (IFNa), but not antibodies against tumour necrosis factor alpha (TNFQ) or interleukin-2 (IL-2), completely inhibit the growth-stimulating activity. Our results indicate that a paracrine growth regulation has to be considered in addition to the postulated autocrine loop in the growth regulation of malignant B cells.     

Early human thymocyte proliferation is regulated by an externally controlled autocrine transforming growth factor-beta 1 mechanism

Early thymocytes undergo extensive proliferation after their entry into the thymus, but cellular interactions and cytokines regulating this intrathymic step remain to be determined. We analyzed the effects of various T-cell growth factors and cellular interactions on in vitro proliferation of early CD2+CD3/TCR-CD4-CD8- (triple negative [TN]) human thymocytes. Freshly isolated TN cells were then assayed for their growth capacity after incubation with CD2I+III-monoclonal antibody (MoAb), recombinant human interleukin-2 (IL-2), IL-7, and/or IL-4. These cells displayed significant proliferative responses with IL-4, IL- 7, or CD2-MoAb+IL-2. The addition of recombinant transforming growth factor beta (TGF beta) or autologous irradiated CD3+CD8+CD4- cells to TN cell cultures dramatically decreased their growth responses to IL-2 and IL-7, whereas IL-4-induced proliferation was less sensitive to growth inhibition. We thus asked whether the CD8+ cell-derived inhibitory effect was due to TGF beta. The addition of neutralizing anti-TGF beta MoAb completely abolished CD8+ cell-derived inhibition of TN cell growth. Analysis of CD8+ cell-derived supernatants indicated that these cells had low TGF beta 1 production capacity, whereas TN cells secrete significantly high levels of TGF beta 1. Cell fixation studies showed that TN cells were the source of the TGF beta. TGF beta 1 released from TN cells was in the latent form that became the active inhibitory form through interaction of TN cells with CD8+ cells. Together, these data suggest a role for TGF beta 1 as an externally controlled, autocrine inhibitory factor for human early thymocytes, with a regulatory role in thymic T-cell output.     

Interleukin 7 receptor-deficient mice lack gammadelta T cells.

The interleukin 7 receptor (IL-7R) plays a crucial role in early B- and T-cell development. It consists of a unique a chain and a common gamma chain [IL-2 receptor gamma chain (IL-2Rgamma)]. Gene inactivation of IL-7, IL-7R, and IL-2Rgamma resulted in severe impairment of B and T lymphopoiesis in mice. In addition, IL-2Rgamma-deficient mice lack gammadelta T cells in the skin and have the impaired development of natural killer (NK) cells and intraepithelial lymphocytes. To explore the role of IL-7/IL-7R system in gammadelta T- and NK-cell development, we have generated and analyzed IL-7R-deficient mice. gammadelta T cells were absent from skin, gut, liver, and spleen in the deficient mice. In contrast, alphabeta T and B cells were detected in reduced, but certain, numbers, and NK cells developed normally. The gammadelta T-cell development in fetal and adult thymus was also completely blocked. These results clearly demonstrate that the signal from IL-7R is indispensable for gammadelta T-cell development in both thymic and extrathymic pathways. On the contrary, it is suggested that NK-cell development requires cytokine(s) other than IL-7.     

A lymphokine, provisionally designated interleukin T and produced by a human adult T-cell leukemia line, stimulates T-cell proliferation and the induction of lymphokine-activated killer cells.

In early phases of human T-cell lymphotrophic virus I-induced adult T-cell leukemia (ATL), the malignant cell proliferation is associated with an autocrine process involving coordinate expression of interleukin (IL) 2 and its receptor. However, during late-phase ATL, leukemic cells no longer produce IL-2 yet continue to express high-affinity IL-2 receptors. During studies to define pathogenic mechanisms that underlie this IL-2-independent proliferation, we demonstrated that the ATL cell line HuT-102 secretes a lymphokine, provisionally designated IL-T, that stimulates T-cell proliferation and the induction of lymphokine-activated killer cells. Conditioned medium from HuT-102, when added to the IL-2-dependent CTLL-2 line, yielded a stimulation index of 230. Since CTLL-2 was purported to be IL-2-specific, we performed a number of studies to exclude IL-2 production by HuT-102. Stimulation of CTLL-2 cells by HuT-102-conditioned medium was not meaningfully inhibited by addition of an antiserum to IL-2. Furthermore, uninduced HuT-102 cells did not express mRNA encoding IL-2 as assessed by Northern blot analysis. No biological activity on CTLL-2 cells was mediated by purified IL-1, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12, IL-13, or granulocyte/macrophage colony-stimulating factor, thus differentiating these factors from IL-T. Based on preliminary biochemical data, IL-T is a protein with a pI value of 4.5 and a molecular mass in SDS gels of 14 kDa. In addition to its action on CTLL-2 cells, 3200-fold-purified IL-T stimulated proliferation of the human cytokine-dependent T-cell line Kit-225. Furthermore, addition of IL-T enhanced cytotoxic activity of large granular lymphocytes (i.e., induced lymphokine-activated killer cells). Thus, IL-T is a lymphokine that plays a role in T-cell proliferation and induction of lymphokine-activated killer cells. Furthermore, IL-T may contribute to IL-2-independent proliferation of select ATL cells and lines.     

Production of natural killer cell activity-augmenting factor (interleukin-6) by human epiphyseal chondrocytes

Objective. We sought to determine the capacity of human epiphyseal chondrocytes to modulate the cytotoxic activity of human natural killer (NK) cells by determining whether they release interleukin-6 (IL-6), a cytokine recently shown to stimulate NK cell activity. Methods. Conditioned medium from human epiphyseal chondrocyte cultures (Ch-CM) was tested for IL-6 activity using the B9 cell hybridoma assay. Its NK cell-stimulating capacity in the presence of K562 (myelogenous leukemia) cells or human chondrocytes was evaluated in a 4-hour ⁵¹Cr-release assay. Ch-CM–derived IL-6/NK cell–augmenting factor activity was partially purified by high-performance liquid chromatography (HPLC) gel filtration and Western blot. Results. Ch-CM contained an NK cell-augmenting factor (NKAF) which was blocked by IL-2 or IL-6 antibodies. Ch-CM did not contain detectable IL-2 activity, but it stimulated IL-2 production by human peripheral blood lymphocytes (PBL). This IL-2–inducing capacity was inhibited by IL-6 antibodies, indicating that chondrocytes release an IL-6–like activity. Ch-CM significantly enhanced the proliferation of IL-6–dependent B9 hybridoma cells, and Western blot analysis of Ch-CM revealed specific bands corresponding to those of highly purified IL-6. Upon HPLC gel filtration, chondrocyte NKAF copurified with chondrocyte IL-6. Pure IL-6 and chondrocyte IL-6 were tested for their ability to stimulate the cytotoxic activity of human PBL against chondrocytes. Both mediators significantly enhanced chondrocyte killing. Lysis of chondrocytes by PBL was mediated by NK cells, since depletion of CD16+ cells resulted in inhibition of the activity. Conclusion. Thus, upon stimulation, chondrocytes produce IL-6 which, through IL-2 induction, augments the activity of NK cells against K562 target cells as well as against chondrocytes.     

Mast cell growth-enhancing activity (MEA) stimulates interleukin 6 production in a mouse bone marrow-derived mast cell line and a malignant subline

A novel mast cell growth-enhancing activity (MEA/P40/interleukin 9 [IL-9]) purified from the conditioned medium of a murine interleukin 2 (IL-2)-dependent Mlsa-specific T-cell line (MLS4.2) was tested for its capacity to induce interleukin 6 (IL-6) production in a mouse bone marrow-derived factor-dependent mast cell line (L138.8A). This interleukin 3 (IL-3)/interleukin 4 (IL-4)/MEA-responsive cell line was demonstrated recently to express IL-6 mRNA and to secrete IL-6 when cultured with IL-3/IL-4. Now we were able to show that conditioned medium from L138.8A mast cells stimulated with MEA alone contained growth factor activity for the IL-6-dependent mouse hybridoma cell line 7TD1 that was completely blocked by the monoclonal anti-IL-6 antibody 6B4. A dose-response study including IL-3, IL-4, and MEA tested either alone or in different combinations revealed that among these growth factors MEA was the most potent inducer of IL-6 in L138.8A cells. Moreover, IL-4 but not IL-3 had a strong synergistic effect on MEA-induced IL-6 production. The autonomous malignant mast cell subline L138Cauto also showed enhanced IL-6 production when stimulated with MEA. Our findings indicate that MEA (IL-9) not only provides a proliferation signal, but also leads to a marked functional activation of responsive mast cells.     

Interleukin-2-activated rat natural killer cells express inducible nitric oxide synthase that contributes to cytotoxic function and interferon-gamma production.

Natural killer (NK) cells are large granular lymphocytes capable of destroying cells infected by virus or bacteria and susceptible tumor cells without prior sensitization and restriction by major histocompatability complex (MHC) antigens. Their cytotoxic activity could be strongly enhanced by interleukin-2 (IL-2). Previous findings, even if obtained with indirect experimental approaches, have suggested a possible involvement of the inducible nitric oxide (iNOS) pathway in the NK-mediated target cell killing. The aim of the present study was first to directly examine the induction of iNOS in IL-2-activated rat NK cells isolated from peripheral blood (PB-NK) or spleen (S-NK), and second to investigate the involvement of the iNOS-derived NO in the cytotoxic function of these cells. Our findings clearly indicate the induction of iNOS expression in IL-2-activated PB-NK and S-NK cells, as evaluated either at mRNA and protein levels. Accordingly, significantly high levels of iNOS activity were shown, as detected by the L-arginine to L-citrulline conversion in appropriate assay conditions. The consequent NO generation appears to partially account for NK cell-mediated DNA fragmentation and lysis of sensitive tumor target cells. In fact, functional inhibition of iNOS through specific inhibitors, as well as the almost complete abrogation of its expression through a specific iNOS mRNA oligodeoxynucleotide antisense, significantly reduced the lytic activity of IL-2-activated NK cells. Moreover, IL-2-induced interferon-gamma production appears also to be dependent, at least in part, on iNOS induction.     

Spontaneous and glucocorticoid-induced apoptosis in human mature T lymphocytes

Glucocorticoid (GC)-induced apoptosis is a well-recognized physiologic regulator of murine T-cell number and function. We have analyzed its mechanisms in human mature T cells, which have been thought to be insensitive until recently. Peripheral blood T cells showed sensitivity to GC-induced apoptosis soon after the proliferative response to a mitogenic stimulation, and were also sensitive to spontaneous (ie, growth factor deprivation-dependent) apoptosis. CD8+ T cells were more sensitive to both forms than CD4+ T cells. Acquisition of sensitivity to GC-induced apoptosis was not associated with any change in number or affinity of GC receptors. Both spontaneous and GC-induced apoptosis were increased by the macromolecular synthesis inhibitors, cycloheximide (CHX) and puromycin. A positive correlation between the degree of protein synthesis inhibition and the extent of apoptosis was observed. Interleukin-2 (IL-2) IL-4, and IL-10 protected (IL-2 > IL-10 > IL-4) T cells from both forms of apoptosis in a dose-dependent manner. Our data suggest that spontaneous and GC-induced apoptosis regulate the human mature T-cell repertoire by acting early after the immune response and differentially affecting T-cell subsets.