Response of human natural killer (NK) cells to NK cell stimulatory factor (NKSF): cytolytic activity and proliferation of NK cells are differentially regulated by NKSF.

Natural killer cell stimulatory factor (NKSF) is a 70-kD heterodimeric cytokine that was initially isolated from conditioned medium of human B lymphoblastoid cell lines. The effects of recombinant NKSF on the function of human peripheral blood NK cells were examined. NKSF directly augmented the cytolytic activity of freshly isolated NK cells. Both CD56dim and CD56bright NK cells demonstrated enhanced cytotoxicity after brief exposure to NKSF. In contrast, highly purified T lymphocytes did not exhibit major histocompatibility complex-unrestricted cytotoxicity after short-term culture with NKSF. Like interleukin 2 (IL-2), NKSF augmented the lysis of NK-sensitive, NK-resistant, and antibody-coated targets. Both NKSF and IL-2 induced marked upregulation of several NK cell adhesion molecules known to participate in cytolysis, including CD2, CD11a, and CD54. However, NKSF activates NK cells through a pathway distinct from that of IL-2, since the presence of anti-IL-2 receptor (anti-IL-2R) antibodies or IL-4 did not inhibit the effects of NKSF. NKSF by itself induced very little proliferation of resting NK cells. NK cells preactivated in vitro with IL-2 demonstrated enhanced proliferation to NKSF, but the degree of proliferation was always inferior to that induced by IL-2 alone. Moreover, NKSF strongly inhibited IL-2-induced proliferation of either resting or preactivated NK cells. This inhibition was not the result of decreased IL-2R expression, because NKSF-activated NK cells expressed higher levels of both IL-2Rs p75 and p55. Furthermore, NKSF did not inhibit the proliferation of mitogen-activated T cells, indicating a selective effect on NK cell proliferation. Human NK cells expanded in vivo by prolonged continuous infusions of IL-2 remained fully responsive to NKSF. Picomolar concentrations of NKSF were as effective as nanomolar concentrations of IL-2 in augmenting the cytolytic activity of NK cells expanded in vivo by IL-2. NKSF may play an important role in the regulation of human NK cell function, and its possible use as a therapeutic cytokine deserves further investigation.

The interleukin 12 p40 gene promoter is primed by interferon gamma in monocytic cells.

Interleukin (IL) 12 is a proinflammatory cytokine produced by phagocytic cells, B cells, and other antigen-presenting cells that modulates adaptive immune responses by favoring the generation of T helper type 1 cells. IL-12 mediates some of its physiological activities by acting as a potent inducer of interferon (IFN) gamma production by T and natural killer cells. IFN-gamma enhances the ability of the phagocytic cells to produce IL-12 and other proinflammatory cytokines. Thus, IL-12-induced IFN-gamma acts in a positive feedback loop that represents an important amplifying mechanism in the inflammatory response to infections. We show here that IFN-gamma enhances IL-12 production mostly by priming phagocytic cells for lipopolysaccharide (LPS)-induced transcription of the IL-12 p40 gene, which encodes the heavy chain of the IL-12 heterodimer; furthermore, IFN-gamma directly induces transcription of the IL-12 p35 gene, which encodes the light chain of IL-12, and has at least an additive effect with LPS stimulation in inducing its transcription. The priming effect of IFN-gamma on the LPS-induced p40 gene transcription requires preincubation of the cells with IFN-gamma for at least 8 h to obtain a maximal effect. The priming effect of IFN-gamma for IL-12 production is predominantly at the transcriptional level for both the p40 and the p35 gene, and no evidence for a major role of posttranscriptional or translational mechanisms was found. A 3.3-kb human IL-12 p40 promoter construct transfected into cell lines recapitulated the tissue specificity of the endogenous gene, being silent in two human T cell lines, constitutively active in two human Epstein-Barr virus-positive B lymphoblastoid cell lines, and LPS inducible in the human THP-1 and mouse RAW264.7 monocytic cell lines. Because the RAW264.7 cell line is easily transfectable and regulates the endogenous IL-12 p40 gene in response to IFN-gamma or LPS similarly to human monocytes, it was used for analysis of the regulation of the cloned human IL-12 p40 promoter. A requirement for the region between -222 and -204 in both LPS responsiveness and IFN-gamma priming was established. This region contains an ets consensus sequence that was shown to mediate activation of the promoter by IFN-gamma and LPS, as well as by a cotransfected ets-2. The -222 construct was also regulated in a tissue-specific manner. Two other elements, IRF-1 located at -730 to -719, and NF-IL6 at -520 to -512, were also studied by deletion analysis, which did not result in decreased response to IFN-gamma and LPS stimulation.

Induction of interferon gamma production by natural killer cell stimulatory factor: characterization of the responder cells and synergy with other inducers.

We previously reported that natural killer cell stimulatory factor (NKSF), a heterodimeric lymphokine purified from the conditioned medium of human B lymphoblastoid cell lines, induces interferon gamma (IFN-gamma) production from resting peripheral blood lymphocytes (PBL) and synergizes with interleukin 2 in this activity. In this study, we show that human NKSF induces IFN-gamma production from both resting and activated human PBL and from freshly isolated murine splenocytes. Human T and NK cells produce IFN-gamma in response to NKSF, but resting PBL require the presence of nonadherent human histocompatibility leukocyte antigens DR+ (HLA-DR+) accessory cells to respond to NKSF. The mechanism(s) by which NKSF induces IFN-gamma production results in accumulation of IFN-gamma mRNA, is insensitive to cyclosporin A, and synergizes with those mediated by phytohemagglutinin, phorbol diesters, anti-CD3 antibodies, and allogeneic antigens, but not by Ca2+ ionophores. The ability of NKSF to directly induce IFN-gamma production and to synergize with other physiological IFN-gamma inducers, joined with the previously described ability to enhance lymphocyte cytotoxicity and proliferation, indicates that this lymphokine is a powerful immunopotentiating agent.

Antitumor and antimetastatic activity of interleukin 12 against murine tumors.

It has recently been demonstrated that in vivo administration of murine interleukin 12 (IL-12) to mice results in augmentation of cytotoxic natural killer (NK)/lymphocyte-activated killer cell activity, enhancement of cytolytic T cell generation, and induction of interferon gamma secretion. In this study, the in vivo activity of murine IL-12 against a number of murine tumors has been evaluated. Experimental pulmonary metastases or subcutaneous growth of the B16F10 melanoma were markedly reduced in mice treated intraperitoneally with IL-12, resulting in an increase in survival time. The therapeutic effectiveness of IL-12 was dose dependent and treatment of subcutaneous tumors could be initiated up to 14 d after injection of tumor cells. Likewise, established experimental hepatic metastases and established subcutaneous M5076 reticulum cell sarcoma and Renca renal cell adenocarcinoma tumors were effectively treated by IL-12 at doses which resulted in no gross toxicity. Local peritumoral injection of IL-12 into established subcutaneous Renca tumors resulted in regression and complete disappearance of these tumors. IL-12 was as effective in NK cell-deficient beige mice or in mice depleted of NK cell activity by treatment with antiasialo GM1, suggesting that NK cells are not the primary cell type mediating the antitumor effects of this cytokine. However, the efficacy of IL-12 was greatly reduced in nude mice suggesting the involvement of T cells. Furthermore, depletion of CD8+ but not CD4+ T cells significantly reduced the efficacy of IL-12. These results demonstrate that IL-12 has potent in vivo antitumor and antimetastatic effects against murine tumors and demonstrate as well the critical role of CD8+ T cells in mediating the antitumor effects against subcutaneous tumors.

Resolution of cutaneous leishmaniasis: interleukin 12 initiates a protective T helper type 1 immune response.

Resistance to Leishmania major in mice is associated with the appearance of distinct T helper type 1 (Th1) and Th2 subsets. T cells from lymph nodes draining cutaneous lesions of resistant mice are primarily interferon gamma (IFN-gamma)-producing Th1 cells. In contrast, T cells from susceptible mice are principally Th2 cells that generate interleukin 4 (IL-4). Although existing evidence is supportive of a role for IFN-gamma in the generation of Th1 cells, additional factors may be required for a protective response to be maintained. A potential candidate is IL-12, a heterodimeric cytokine produced by monocytes and B cells that has multiple effects on T and natural killer cell function, including inducing IFN-gamma production. Using an experimental leishmanial model we have observed that daily intraperitoneal administration at the time of parasite challenge of either 0.33 micrograms IL-12 (a consecutive 5 d/wk for 5 wk) or 1.0 micrograms IL-12 per mouse (only a consecutive 5 d) caused a > 75% reduction in parasite burden at the site of infection, in highly susceptible BALB/c mice. Delay of treatment by 1 wk had less of a protective effect. Concomitant with these protective effects was an increase in IFN-gamma and a decrease in IL-4 production, as measured by enzyme-linked immunosorbent assay of supernatants generated from popliteal lymph node cells stimulated with leishmanial antigen in vitro. The reduction in parasite numbers induced by IL-12 therapy was still apparent at 10 wk postinfection. In addition, we observed that the administration of a rabbit anti-recombinant murine IL-12 polyclonal antibody (200 micrograms i.p. every other day for 25 d) at the time of infection to resistant C57Bl/6 mice exacerbated disease. These effects were accompanied by a shift in IFN-gamma production in vitro by antigen-stimulated lymph node cells indicative of a Th2-like response. These findings suggest that IL-12 has an important role in initiating a Th1 response and protective immunity.

FIZZ1 potentiates the carbachol-induced tracheal smooth muscle contraction.

FIZZ1 is an adipokine highly expressed under inflammatory conditions, and yet, little is known of its function. In this study we examine the expression and function of FIZZ1 in an ovalbumin mouse model of asthma. Trachea from naïve or ovalbumin-sensitised and -challenged mice were compared for transcriptional, functional and proteomic differences using gene microarrays, ex vivo tracheal contraction, immunohistochemistry and Western blot analysis. FIZZ1 was expressed in ovalbumin-treated, but not naïve, trachea. Naïve trachea incubated with recombinant FIZZ1 exhibited denuded epithelium and contractile hyperresponsiveness. The FIZZ1-incubated trachea also exhibited an associated increased expression of phospho-c-Raf, phospho-extracellular signal-regulated kinase 1/2, phospho-p38, MLCK and MLC-20. These data demonstrate that FIZZ1 regulates tracheal smooth muscle contraction through impairment of the epithelium and activation of the mitogen-activated protein kinase pathway in muscle.

Development of TH1 CD4+ T cells through IL-12 produced by Listeria-induced macrophages.

Development of the appropriate CD4+ T helper (TH) subset during an immune response is important for disease resolution. With the use of naïve, ovalbumin-specific alpha beta T cell receptor transgenic T cell, it was found that heat-killed Listeria monocytogenes induced TH1 development in vitro through macrophage production of interleukin-12 (IL-12). Moreover, inhibition of macrophage production of IL-12 may explain the ability of IL-10 to suppress TH1 development. Murine immune responses to L. monocytogenes in vivo are of the appropriate TH1 phenotype. Therefore, this regulatory pathway may have evolved to enable innate immune cells, through interactions with microbial pathogens, to direct development of specific immunity toward the appropriate TH phenotype.

Restoration of HIV-specific cell-mediated immune responses by interleukin-12 in vitro.

Peripheral blood mononuclear cells (PBMCs) from many asymptomatic individuals infected with human immunodeficiency virus-type 1 (HIV) are unresponsive as measured by in vitro T cell proliferation and interleukin-2 (IL-2) production to influenza virus and synthetic peptides of HIV envelope (Env). Strong influenza virus- and Env-stimulated IL-2 responses and T cell proliferation were restored when cultures were stimulated in the presence of IL-12. Interferon-gamma production by PBMCs from HIV seropositive (HIV+) patients was also restored with IL-12. Furthermore, in vitro antigen-specific production of IL-2 and proliferation of PBMCs from HIV- donors were suppressed by antibody to IL-12, but were not enhanced by addition of exogenous IL-12. Thus, IL-12 may be limiting in PBMCs from HIV+ but not HIV- individuals. These findings demonstrate that IL-12 can restore HIV-specific cell-mediated immunity in vitro in HIV-infected individuals and suggest a potential use of IL-12 in augmenting the diminished immunologic functions associated with HIV infection.

Interleukin 12 at the site of disease in tuberculosis.

Interleukin 12 (IL-12), a heterodimeric cytokine composed of p40 and p35 chains, has potent immunologic effects in vitro. We used tuberculous pleuritis as a model to study the immunoregulatory potential of IL-12 in vivo at the site of human infectious disease. Messenger RNAs for p40 and p35 were detected in pleural fluid from six of six patients by reverse-transcription polymerase chain reaction. By using an ELISA that detected both free p40 and heterodimeric IL-12, we found that mean concentrations were 585 +/- 89 pg/ml in pleural fluid of patients with tuberculous pleuritis, which were significantly higher than those in serum of the same patients (54 +/- 36 pg/ml), or in malignant pleural effusions (123 +/- 35 pg/ml). By using an ELISA specific for heterodimeric IL-12, we found that mean concentrations in pleural fluid of patients with tuberculous pleuritis were 165 +/- 28 pg/ml and undetectable in serum of the same patients, or in malignant pleural effusions. Bioactive IL-12 was detectable in five of five supernatants of pleural fluid cells stimulated with Mycobacterium tuberculosis. Addition of anti-IL-12 antibodies suppressed proliferative responses of pleural fluid cells to M. tuberculosis by 36 +/- 7%. These data indicate that IL-12 may play a role in the human immune response to infectious agents in vivo. We hypothesize that IL-12 contributes to the antimycobacterial immune response by enhancing production of interferon-gamma, facilitating development of Th1 cells and augmenting cytotoxicity of antigen-specific T cells and natural killer cells.

Potent activity of soluble B7-IgG fusion proteins in therapy of established tumors and as vaccine adjuvant.

Fusion proteins consisting of the extracellular region of murine B7.1 or B7.2 and the Fc portion of murine IgG2a (B7-IgG) were evaluated for their ability to promote antitumor responses. Therapeutic administration of soluble B7-IgG in mice with established tumors induced complete regression of the tumor and increased the survival of mice. In three models, MethA, P815, and MB49, mice with 7-day-old established tumors were cured with two to three treatment cycles of B7-IgG, given twice a week. Even in mice with an established B16/F10 tumor (a poorly immunogenic melanoma), therapeutic treatment with B7-IgG alone slowed tumor growth and increased survival significantly. Still stronger antitumor activity was achieved when B7-IgG was used as a vaccine adjuvant mixed with irradiated tumor cells. In 80% of mice with 7-day-old B16 tumors, tumors regressed completely, and mice survived for at least 80 days. In all tumor models, B7.1-IgG and B7.2-IgG had similar antitumor activity. B7-IgG-mediated tumor rejection was dependent on T cells, specifically CD8 cells, as demonstrated by the failure of B7-IgG to induce tumor regression in severe combined immunodeficient or CD8-depleted mice. In addition, mice that were cured of an established tumor were protected against a rechallenge with the same tumor for at least 4 months, suggesting the generation of memory responses. Surprisingly, the antitumor activity of B7-IgG was independent of IFN-gamma, as demonstrated by tumor rejection in IFN-gamma knockout mice. Our findings demonstrate the potent capacity of B7-IgG to generate or enhance antitumor immune responses and suggest the clinical value of B7-IgG.

Fibroblasts genetically engineered to secrete interleukin 12 can suppress tumor growth and induce antitumor immunity to a murine melanoma in vivo.

Interleukin 12 (IL-12), a disulfide-linked heterodimeric cytokine produced primarily by macrophages, is composed of light (p35) and heavy (p40) chains. It binds to a receptor on T-cells and natural killer cells, promoting the induction of primarily a TH1 response in vitro and in vivo. To determine whether paracrine IL-12 secretion can alter tumor cell growth or promote antitumor immunity, we have developed a delivery system using genetically engineered fibroblasts in murine tumor models. NIH3T3 cells were stably transfected to express 100-240 units/10(6) cells/48 h of IL-12 using expression plasmids carrying both the murine p35 and p40 genes of murine IL-12. The effects of paracrine secretion of IL-12 on tumor establishment and vaccination models were examined using the poorly immunogenic murine melanoma cell line (BL-6) in C57BL/6 mice. To determine the effects of IL-12 on tumor formation, nonirradiated BL-6 cells were inoculated s.c. into C57BL/6 mice admixed with NIH3T3 cells transfected with both subunits of mIL-12 (3T3-IL-12) or with cells transfected with only the neomycin phosphotransferase gene (3T3-Neo). Compared to mice given injections of BL-6 alone, the day of emergence of detectable tumors was significantly delayed in mice given injections of BL-6 admixed with 3T3-IL-12, but not in mice with BL-6 admixed with 3T3-Neo. Effectiveness in this system was related to the amount of IL-12 expressed by the 3T3-IL-12. To determine the ability of locally secreted IL-12 at the tumor site to induce antitumor immunity, 10(6) irradiated tumor cells mixed with 3T3-IL-12 or 3T3-Neo were injected as a vaccine, and the response to a tumor challenge was subsequently examined. With a tumor challenge of less than 1 x 10(5) nonirradiated BL-6 cells, significant delay of establishment of tumor was noted with a relatively small amount of IL-12 secretion (1.2 units/5 x 10(5) cells/48 h). Larger amounts of secreted IL-12 provided no additional therapeutic benefit. Histological examination of tumor inoculum with 3T3-IL-12 secreting a high level of IL-12 showed peritumoral accumulation of macrophages, a characteristic capsule around the tumor composed of palisades of fibroblasts, and decreased numbers of CD4+ cells in the tumor. These results suggest that local delivery of IL-12 inhibits tumor growth in a dose dependent manner but leads to the development of an antitumor immune response when IL-12 is expressed at the tumor site at the relatively small amount indicated above.(ABSTRACT TRUNCATED AT 400 WORDS)

Construction and characterization of retroviral vectors expressing biologically active human interleukin-12.

Interleukin-12 (IL-12) is a heterodimeric cytokine originally defined by its ability to induce the maturation of cytolytic lymphocytes and by its capacity to effectively synergize with IL-2 in the induction of cytolytic activity. Recent studies in mice have demonstrated the ability of IL-12 to cause tumor regression and stimulate long-term antitumor immunity in treated animals. To examine the antitumor effect of direct gene transfer of IL-12 into tumors, we have developed retroviral vectors that coordinately express both subunits of IL-12. An MFG-based retroviral vector was used to generate a recombinant retrovirus in which a long terminal repeat (LTR)-driven polycistronic transcript encodes both subunits of human IL-12: hp35 and hp40 cDNAs are linked and coexpressed using the internal ribosome entry site (IRES) from the encephalomyocarditis virus (DFG-hIL-12). In addition, two IRES sequences were used to express both subunits of IL-12 and a neomycin resistance (neoR) selectable marker gene from the same polycistronic message (TFG-hIL-12). The amphotropic DFG-hIL-12 and TFG-hIL-12 viruses were used to infect both human and murine cell lines as well as primary tumor cultures. The production of human IL-12 by the nonselected, infected cells was measured in both a PHA blast proliferation bioassay and an ELISA and ranged from 15 to 40 ng/10(6) cells per 24 hr. Following G418 selection of TFG-hIL-12-infected cells, the level of expression of IL-12 was significantly higher (up to 120 ng/10(6) cells per 24 hr). The IL-12 protein secreted by the infected cells exhibited all of the biologic activities of recombinant hIL-12: proliferation of activated natural killer (NK) and T cells, stimulation of interferon-gamma (IFN-gamma) induction by NK and T cells, and enhancement of lymphokine-activated killer (LAK) activity. These retroviral vectors expressing human IL-12 should be useful in evaluating the biological properties of IL-12 as well as for use in clinical trials for gene therapy of patients with cancer.

Dose-dependent and schedule-dependent effects of interleukin-12 on antigen-specific CD8 responses.

Interleukin-12 (IL-12) has been shown to play a central role in the innate and acquired immune responses. Its activities include enhancement of natural killer (NK) and cytotoxic T lymphocyte (CTL) activity and promotion of CD4 Th1 cell development. It has also been shown to provide potent activity as a vaccine adjuvant in generating antibody and T cell responses. We have investigated the efficacy of IL-12 protein in promoting CD8 T cell responses when it is used as an adjuvant for immunization. Studies using, as antigen, cDNA from an autologous antigen (P1A) as well as studies of responses to vaccinia virus-delivered self (gp100) and non-self (beta-galactosidase) antigens show that the dose and schedule of IL-12 administration can significantly affect adjuvant activity, leading to enhancement or suppression of antigen-specific responses.

Immunotherapy of mice with an irradiated melanoma vaccine coupled with interleukin-12.

Interleukin (IL)-12 can activate cytotoxic lymphocytes, stimulate natural killer cell activity, induce the production of INF-gamma and inhibit the development of various experimental tumors. We previously demonstrated that immunotherapy of melanoma bearing mice with an irradiated melanoma vaccine (IMV) coupled with IL-2 or GM-CSF had beneficial effects against primary melanoma growth and against subsequent spontaneous metastasis. We also had found that treatment of melanoma bearing mice with IL-12 (300 ng/day) for 4 weeks inhibited the development of primary melanoma tumors in 40% of mice. The purpose of this study was to investigate the efficacy of combined therapy of experimental melanoma with an IMV prepared from B16F10 melanoma cells coupled with IL-12 treatment. C57BL/6 mice were challenged subcutaneously in the tail with B16F10 melanoma cells and by the 45th day, more than 50% of the mice had developed visible primary melanoma tumors at the injection site. Subsequent immunotherapy of mice with IMV, when coupled with IL-12, provided partial inhibition of primary melanoma tumor growth. Optimal results against primary tumor growth were observed when IMV therapy was coupled with IL-12 at a dose of 50 ng/day. Combination of IMV with IL-12 at a dose of 100 ng/day significantly reduced melanoma metastasis to the lungs compared with control mice, and an improvement in mean survival time was observed in mice treated with a combination of IMV with IL-12 (300 ng/day).

A CTL assay requiring only 150 microliter of mouse blood.

In this paper, we present a method for measuring antigen specific cytotoxic T lymphocyte (CTL) activity from individual mouse peripheral blood samples without animal sacrifice. Peripheral blood cells are stimulated in vitro with a cocktail of antigen, cytokines, costimulatory molecules and irradiated feeder cells resulting, 7 days later, in a readily detectable antigen specific signal from a well plated under limiting dilution conditions. This highly sensitive and antigen specific assay is more efficient than conventional CTL assays and thus increases the number of mice that can be tested in a single assay. Since blood samples can be assayed from an individual mouse at multiple times during the course of an in vivo study, the assay can facilitate and strengthen correlative studies on CTL responses and in vivo results.

A new mouse model of experimental melanoma for vaccine and lymphokine therapy.

The annual incidence of malignant melanoma is estimated at 10-12 per 100,000 inhabitants in countries of central Europe and the United States, and alarmingly there has been a dramatic upward trend in that estimate. The B16 murine melanoma is a rapidly growing metastatic tumor of spontaneous origin, as are human malignant melanomas. Melanoma cells produce specific antigens which are uniquely different from normal cellular antigens, and the expression of such antigens is the cornerstone for preparation of anti-melanoma vaccines. One major problem in evaluating the effectiveness of vaccination and other biologic therapies is the variability of experimental tumor models. A new metastatic model of experimental melanoma which was developed in our laboratory imitates the major clinical stages of malignant metastatic melanoma: stage I, primary (local) tumor growth and bone marrow invasion; stage II, regional lymph node involvement; and stage III, metastasis to distant organs, such as the lungs. This model has been used successfully for screening vaccines constructed in our laboratory. Immunization with formalinized vaccines (of extracellular antigens, intact melanoma cells, or B700 antigen) or irradiated vaccines (of intact melanoma cells) partially inhibit primary melanoma tumor growth, reduce metastasis to regional lymph nodes and lungs, and significantly increase mean survival time. These anti-tumor effects were improved when polyvalent and monovalent vaccines were combined with IL-2 therapy. We also compared the immunogenic activity of vaccines made from B16 melanoma cells transfected with genes encoding murine IL-2 or GM-CSF, and effects on tumor bearing mice were compared with or without therapy using the corresponding lymphokines. In sum, comparison of antibody production, growth of primary melanoma tumors, number of surviving mice, mean survival time, and percent of mice with lung metastases, showed that the best course of immunotherapy involves vaccination of mice with irradiated B16 melanoma cells transfected to secrete GM-CSF, coupled with GM-CSF therapy.

Virulent Toxoplasma gondii strain RH promotes T-cell-independent overproduction of proinflammatory cytokines IL12 and gamma-interferon.

The aim of this study was the analysis of the cytokine response in BALB/c mice infected with the highly virulent RH or the weakly virulent Beverley strains of Toxoplasma gondii. Analysis of cytokine messages showed increased expression of IL12, IFN-gamma and TNF-alpha, but not IL4 mRNAs in spleen cells after infection with the T. gondii strains RH and Beverley. High levels of circulating IL12 and IFN-gamma were detected in the serum of mice infected with strain RH, although TNF-alpha levels remained low. In contrast, the same cytokines were detected at only low levels in the serum of mice infected with the Beverley strain. Administration of antibody against IL12 or IFN-gamma significantly delayed time to death of mice infected with strain RH compared to controls. T-Cell-deficient as well as normal mice were equally infected by strain RH, suggesting that T lymphocytes do not contribute to the response. Depletion of natural killer cells from the splenocyte population abolished the in vitro production of IFN-gamma. Together, our data suggest that the virulent strain RH induces in BALB/c mice a type 1 cytokine pattern with T-cell-independent overproduction of IL12 and IFN-gamma that may be involved in the pathogenesis of this micro-organism.

The anti-tumor effect of interleukin-12 is enhanced by mild (fever-range) thermal therapy.

The cytokine interleukin 12 (IL-12) has resulted in notable anti-tumor activity in animal models and in patients and as a result there is considerable interest in learning how to maximize its therapeutic potential while at the same time reducing its known toxic side effects. Strategies which could maintain its effectiveness while permitting reduced dosage could be especially valuable. In this study we used BALB/c mice bearing CT26 tumors as a model for testing whether combining murine IL-12 with a mild (fever range) whole body hyperthermia protocol could result in such a strategy. Our data revealed that 100 ng of IL-12/mouse/day used in combination with FR-WBH was as effective as one in which 300 ng of IL-12/mouse/day was used alone. Importantly, the mice receiving the combination treatment exhibited fewer treatment related toxicities compared to those that received high dose IL-12 alone. Initiation of the IL-12 treatment immediately after FR-WBH induced the greatest anti-tumor effect. This effect does not appear to depend on differences in IL-12-induced IFN-gamma, but may involve production of nitric oxide (NO), since treatment of mice with a NOS inhibitor, NG-monomethyl-L-arginine (L-NMA), abolishes the additive anti-tumor effect of the combination treatment. Collectively, these data suggest that modification of physiological parameters in the host by mild fever-like thermal stimuli may be an effective and feasible adjuvant for cytokine-based immunotherapeutic strategies.

Clusters of CpG dinucleotides implicated by nuclease hypersensitivity as control elements of housekeeping genes.

DNA sequences of the X-chromosome-linked hypoxanthine phosphoribosyltransferase (HPRT) and glucose 6-phosphate dehydrogenase (G6PD) genes have revealed the presence of clusters of CpG dinucleotides, raising the possibility that such clusters are involved in the control of expression of these genes, which are expressed in all tissues. Although CpG clusters are not exclusive features of the X chromosome, the analysis of X-linked genes provides the means to determine whether CpG clusters are control elements; one of the two homologous X loci in female mammals is not expressed, so that active and inactive versions of the gene can be compared. In fact, it has been shown that these CpG clusters are undermethylated when the gene is active and extensively methylated when the gene is inactive. In addition to hypomethylation, chromatin hypersensitivity to endonuclease digestion is a known hallmark of regulatory sequences in eukaryotic genes. We report here that the CpG clusters of the active hprt and g6pd genes are not only undermethylated, but also hypersensitive to MspI, DNase I and S1 nuclease, further supporting the suggestion that they are involved in the control of expression of these genes.