Transient control of interleukin-4-producing natural killer T cells in the livers of Listeria monocytogenes-infected mice by interleukin-12.

Unconstrained development of gamma interferon (IFN-gamma)-secreting natural killer (NK) cells and T helper (Th) 1 cells is central to protection against Listeria monocytogenes. In contrast, interleukin 4 (IL-4) is considered harmful. IL-12 produced by infected macrophages promotes, and IL-4 interferes with, protective antilisterial immunity. The liver NK T lymphocytes, which are a potent source of IL-4, are downregulated at an intermediate stage of listeriosis. Here we demonstrate that endogenous IL-12 participates in the control of IL-4-producing liver NK T lymphocytes during listeriosis. The effects of L. monocytogenes infection on IL-4-producing liver NK T lymphocytes were reversed by antibody neutralization of IL-12 but not of IFN-gamma or tumor necrosis factor alpha (TNF-alpha). IL-4 production by liver NK T lymphocytes was virtually unaffected by heat-killed L. monocytogenes (HKL). Viable L. monocytogenes markedly increased the numbers of IL-12 producers in livers in parallel with an increase in macrophage numbers, whereas HKL failed to do so with similar efficiency. These results indicate that in the liver endogenous IL-12 improves protective immunity against listeriosis by downregulating IL-4-producing NK T lymphocytes. Moreover, our findings that HKL have a low level of IL-12-inducing activity and fail to control IL-4-producing NK T lymphocytes in the liver are consistent with the lesser protective capacity of HKL compared to that of live listeriae.     

IL-12/IFN-gamma/NO axis plays critical role in development of Th1-mediated experimental autoimmune encephalomyelitis

The importance of the IL-12/IFN-gamma/nitric oxide (NO) axis in the pathogenesis of autoimmune diseases remains controversial. In parallel experiments, we explored the role of the IL-12/IFN-gamma/NO axis in the development of MOG 35-55-induced experimental autoimmune encephalomyelitis (EAE) in mice lacking IL-12, IFN-gamma receptor (IFN-gammaR) and inducible nitric oxide synthase (NOS2), respectively. In comparison with wide-type control mice, IL-12-/-, IFN-gammaR-/- and NOS2-/- mice displayed more severe clinical signs of EAE both in remission and at subsequent relapse. Given the relatively low IFN-gamma production in IL-12-/- mice and the lack of IFN-gamma/IFN-gammaR signaling pathway in IFN-gammaR-/- mice, IL-12-/-, IFN-gammaR-/- and NOS2-/- mice with EAE exhibited low NO production. This correlated negatively with MOG 35-55-induced T cell proliferation. Both ED1-positive macrophages and CD4-positive T cells were increased in spinal cords from IL-12-/-, IFN-gammaR-/- and NOS2-/- compared to control mice. In vitro experiments demonstrate that spleen mononuclear cells from IL-12-/-, IFN-gammaR-/- and NOS2-/-mice with EAE present stronger migration capacity when compared to control mice. These results reveal that the IL-12/IFN-gamma/NO axis plays a critical role in the development of MOG 35-55-induced EAE, possibly over failing NO production.     

Impaired interferon-gamma production in response to live bacteria and Toll-like receptor agonists in patients with ataxia telangiectasia

Ataxia telangiectasia (AT) is a pleiotropic autosomal recessive neurodegenerative disorder with associated immunodeficiency and cancer predisposition, caused by mutational inactivation of the ATM gene. Early death usually results from lymphoreticular malignancy or recurrent, chronic respiratory infections. Immune deficiency of AT patients is heterogeneous and involves both humoral and cellular responses. Reports on the number and integrity of immunocompetent cells in AT are conflicting. In the early phase of infection, the interleukin (IL)-12/interferon (IFN)-gamma axis plays a crucial role in first-line defence against pathogens. In a whole blood assay we studied the IL-12/IFN-gamma axis in the immune response of AT cells to the Toll-like receptor agonists lipopolysaccharide and heat-killed Staphylococcus aureus, as well as whole live M. bovis bacille Calmette-Guérin (BCG). The function of AT antigen-presenting cells was normal in terms of IL-12 production, while IFN-gamma production by T and natural killer (NK) cells was severely impaired, even in the presence of adequate co-stimulation by exogenous IL-12.     

Hapten-induced colitis associated with maintained Th1 and inflammatory responses in IFN-gamma receptor-deficient mice

IFN-gamma is a potent pro-inflammatory cytokine thought to be involved in the pathogenesis of Crohn's disease. To further define the role of IFN-gamma in intestinal inflammation, we studied the effects of intra-colonic 2,4,6-trinitrobenzene sulfonic acid (TNBS) instillation in mice with a functionally inactivated IFN-gamma receptor 1 (IFN-gammaR1(- / -)). Our results indicate that IFN-gamma is not necessary for the induction of hapten-induced colitis: after TNBS administration both wild-type and IFN-gammaR1(- / -) mice lost body weight, and the histological features of TNBS-induced colitis were comparable. Colons of IFN-gammaR1(- / -) mice contained a greater number of cells, represented by macrophages and CD4(+) T cells; caudal lymph node cells produced more IFN-gamma and TNF-alpha upon stimulation in vitro. Moreover, IL-18 and IL-12 p40 RNA levels were comparably up-regulated after TNBS treatment in IFN-gammaR1(- / -) wild-type mice. These findings demonstrate that IFN-gamma is dispensable for the development of TNBS-induced colitis. Importantly, the production of Th1 cytokines (e. g. IFN-gamma and TNF-alpha) by caudal lymph node T lymphocytes was enhanced rather than decreased in IFNgammaR1(- / -) mice with no evidence for default Th2 development.     

Dendritic cells stimulated with Actinobacillus actinomycetemcomitans elicit rapid gamma interferon responses by natural killer cells

Human immunoglobulin G2 (IgG2) responses are gamma interferon (IFN-gamma) dependent, and monocyte-derived dendritic cells (mDCs) promote IgG2 production. DCs spontaneously emerge from monocytes in cultures prepared from localized aggressive periodontitis (LagP) patients, and these patients have high levels of IgG2 that is reactive with Actinobacillus actinomycetemcomitans. These results prompted the hypothesis that an interaction between mDCs and A. actinomycetemcomitans promotes IFN-gamma production, and IFN-gamma is known to promote both immunopathology and protective IgG2. A. actinomycetemcomitans induced mDCs to produce interleukin-12 (IL-12), and the addition of A. actinomycetemcomitans and DCs to cultured peripheral blood lymphocytes elicited high levels of IFN-gamma within just 24 h. In contrast, IL-4 was not detectable although DC-derived IL-10 production was apparent. A. actinomycetemcomitans-stimulated macrophages prepared from the same monocytes lacked the ability to induce IL-12 or IFN-gamma responses. NK cells of the innate immune system were the primary source of this early IFN-gamma, although CD8 T cells also contributed some. The NK cell-derived IFN-gamma was IL-12 dependent, and A. actinomycetemcomitans-DC interactions were Toll-like receptor 4 dependent. A. actinomycetemcomitans and A. actinomycetemcomitans lipopolysaccharide (LPS) were more potent than Escherichia coli and E. coli LPS in the ability to induce DC IL-12 and IFN-gamma. The ability of A. actinomycetemcomitans-stimulated DCs to induce NK cells to rapidly produce IFN-gamma in the absence of detectable IL-4 suggests their potential for skewing responses toward Th1. This may help explain the presence of Th1-associated cytokines in gingival crevicular fluid (GCF) from LagP patients and the high levels of IgG2 in their serum and GCF that is reactive with A. actinomycetemcomitans.     

Interleukin-12 regulates the production of Bacille Calmette-Guérin-induced interferon-gamma from human cells in a CD40-dependent manner.

Interferon (IFN)-gamma is a cytokine which plays a critical role in the host defence against human tuberculosis infection. There is evidence that interleukin (IL)-12 can exert a potent effect in stimulating the production of IFN-gamma, and it is well known that a costimulatory signal provided by CD40 may enhance IL-12 production by monocytes/macrophages. However, it is unclear whether CD40-CD40L stimulation is able to regulate the production of mycobacterial-induced IFN-gamma through an IL-12-dependent pathway. In this study, we investigated the capacity of the Bacille Calmette-Guérin (BCG) strain of Mycobacterium bovis to induce the production of interferon-gamma through IL-12 and/or CD40 costimulation from human cells. Our data demonstrate that anti-IL-12 antibodies markedly reduced the levels of IFN-gamma produced by the BCG-stimulated human cells, while exogenous recombinant (r)IL-12 up-regulated the production of IFN-gamma. In addition, the stimulatory effect of IL-12 on BCG-induced IFN-gamma secretion was specific, as it was significantly abolished in the presence of anti-IL-12 antibodies. We also observed that the presence of an anti-CD40L monoclonal antibody significantly inhibited the production of IL-12 and IFN-gamma by human cells activated with BCG. In contrast, an isotype control antibody showed no effect on cytokine production. Furthermore, the presence of a trimeric soluble CD40L agonist (CD40T) in cultures increased the production of IL-12 and IFN-gamma. Importantly, the stimulatory capacity of CD40T on BCG-induced IFN-gamma secretion was blocked by a monoclonal antibody against IL-12, indicating that the effect of CD40T on T cells was mediated through IL-12. Together, these studies are the first to demonstrate that BCG-induced IFN-gamma production by human cells appears to be mediated by IL-12 in a CD40-dependent manner and suggest that CD40-CD40L activation may be a critical mediator in regulating the immune response to stimulation with BCG.     

Spontaneous cytokine production and its effect on induced production

Cytokines regulate cellular immune activity and are produced by a variety of cells, especially lymphocytes, monocytes, and macrophages. Multiparameter flow cytometry is often used to examine cell-specific cytokine production after in vitro phorbol 12-myristate 13-acetate and ionomycin induction, with brefeldin A or other agents added to inhibit protein secretion. Spontaneous ex vivo production reportedly rarely occurs. We examined the spontaneous production of interleukin 2 (IL-2), IL-4, IL-6, IL-8, IL-10, tumor necrosis factor alpha (TNF-alpha), and gamma interferon (IFN-gamma) by peripheral-blood B lymphocytes, T cells, CD8(-) T cells, CD8(+) T cells, CD3(-) CD16/56(+) lymphocytes (natural killer [NK] cells), CD3(+) CD16/56(+) lymphocytes (natural T [NT] cells), and/or monocytes of 316 acutely ill hospitalized persons and 62 healthy adults in Malawi, Africa. We also evaluated the relationship between spontaneous and induced cytokine production. In patients, spontaneous TNF-alpha production occurred most frequently, followed in descending order by IFN-gamma, IL-8, IL-4, IL-10, IL-6, and IL-2. Various cells of 60 patients spontaneously produced TNF-alpha; for 12 of these patients, TNF-alpha was the only cytokine produced spontaneously. Spontaneous cytokine production was most frequent in the immunoregulatory cells, NK and NT. For IL-2, IL-4, IL-6, IL-8, and IL-10, spontaneous cytokine production was associated with greater induced production. For TNF-alpha and IFN-gamma, the relationships varied by cell type. For healthy adults, IL-6 was the cytokine most often produced spontaneously. Spontaneous cytokine production was not unusual in these acutely ill and healthy persons living in an area where human immunodeficiency virus, mycobacterial, malaria, and assorted parasitic infections are endemic. In such populations, spontaneous, as well as induced, cell-specific cytokine production should be measured and evaluated in relation to various disease states.     

Retinoids synergize with interleukin-2 to augment IFN-gamma and interleukin-12 production by human peripheral blood mononuclear cells.

We have demonstrated previously that cells from both the skin and peripheral blood from patients with cutaneous T cell lymphoma (CTCL) have elevated levels of protein and mRNA for Th2 cytokines, interleukin-4 (IL-4) and IL-5, and depressed levels of Thl cytokines, IL-2 and interferon-gamma (IFN-gamma). Furthermore, IL-12 in vitro can restore IFN-gamma production by these patients' cells to near normal levels. Because retinoids exert therapeutic activity in CTCL and are potent modulators of growth and differentiation of hematopoietic cells, we investigated the role of retinoids in modulating Thl cytokine production. Peripheral blood mononuclear cells (PBMC) from normal donors and patients with CTCL were cultured with medium, IL-2, 13-cis-retinoic acid, all-trans-retinoic acid, acetretin or etretinate alone, or IL-2 plus the retinoids for 24 h, and levels of IFN-gamma were determined using ELISA. IL-2 or retinoids alone could induce low but significant levels of IFN-gamma. However, when IL-2 was cultured with each retinoid, a synergistic augmentation of IFN-gamma levels (4-fold to 90-fold) was observed except in the case of etretinate. All-trans-retinoic acid (ATRA) was the most potent IFN-y inducer. Similar studies performed using PBMC from CTCL patients indicated the IFN-gamma augmentation occurred but in a blunted manner. The IFN-y-inducing effect of ATRA and 13-cis-retinoic acid could be abrogated by addition of anti-IL-12 antibodies, suggesting that IL-12 plays a role in the synergistic upregulation of IFN-gamma. Using an IL-12 p40-specific radioimmunoassay (RIA), we confirmed the presence of IL-12 in IL-2 plus retinoid-treated culture supernatants. Purified monocytes cultured with IL-2 plus ATRA did not secrete IL-12. Only when monocytes were cocultured with lymphocytes was there an increase in IL-12 production, suggesting the involvement of a paracrine feedback loop requiring both monocytes and lymphocytes. These data suggest that retinoids can induce Th1 cytokines from normal and CTCL PBMC and that this induction may be mediated through IL-12 production.     

Preferential T(h)2 polarization by OCH is supported by incompetent NKT cell induction of CD40L and following production of inflammatory cytokines by bystander cells in vivo

The altered glycolipid ligand OCH is a selective inducer of T(h)2 cytokines from NKT cells and a potent therapeutic reagent for T(h)1-mediated autoimmune diseases. Although we have previously shown the intrinsic molecular mechanism of preferential IL-4 production by OCH-stimulated NKT cells, little is known about the extrinsic regulatory network for IFN-gamma production. Here we demonstrate that OCH induces lower production of IFN-gamma, not only by NKT cells but also by NK cells compared with alpha-galactosylceramide. OCH induced lower IL-12 production due to ineffective primary IFN-gamma and CD40 ligand expression by NKT cells, and resulted in lower secondary IFN-gamma induction. Co-injection of a sub-optimal dose of IFN-gamma and stimulatory anti-CD40 mAb compensates for the lower induction of IL-12 by OCH administration. IL-12 converts OCH-induced cytokine expression from IL-4 predominance to IFN-gamma predominance. Furthermore, CpG oligodeoxynucleotide augmented IL-12 production when co-administrated with OCH, resulting in increased IFN-gamma production. Taken together, the lower IL-12 production and subsequent lack of secondary IFN-gamma burst support the effective T(h)2 polarization of T cells by OCH. In addition, highlighted in this study is the characteristic property of OCH that can induce the differential production of IFN-gamma or IL-4 according to the availability of IL-12.     

Functional characterization of human natural killer cells responding to Mycobacterium bovis bacille Calmette-Guérin

The kinetics of activation and induction of several effector functions of human natural killer (NK) cells in response to Mycobacterium bovis bacille Calmette-Guérin (BCG) were investigated. Owing to the central role of monocytes/macrophages (MM) in the initiation and maintenance of the immune response to pathogens, two different experimental culture conditions were analysed. In the first, monocyte-depleted nylon wool non-adherent (NW) cells from healthy donors were stimulated with autologous MM preinfected with BCG (intracellular BCG). In the second, the NW cells were directly incubated with BCG, which was therefore extracellular. In the presence of MM, CD4+ T lymphocytes were the cell subset mainly expressing the activation marker, CD25, and proliferating with a peak after 7 days of culture. In contrast, in response to extracellular BCG, the peak of the proliferative response was observed after 6 days of stimulation, and CD56+ CD3- cells (NK cells) were the cell subset preferentially involved. Such proliferation of NK cells did not require a prior sensitization to mycobacterial antigens, and appeared to be dependent upon contact between cell populations and bacteria. Following stimulation with extracellular BCG, the majority of interferon-gamma (IFN-gamma)-producing cells were NK cells, with a peak IFN-gamma production at 24-30 hr. Interleukin (IL)-2 and IL-4 were not detectable in NK cells or in CD3+ T lymphocytes at any time tested. IL-12 was not detectable in the culture supernatant of NW cells stimulated with extracellular BCG. Compared to the non-stimulated NW cells, the NW cells incubated for 16-20 hr with BCG induced the highest levels of expression of apoptotic/death marker on the NK-sensitive K562 cell line. BCG also induced expression of the activation marker, CD25, and proliferation, IFN-gamma production and cytotoxic activity, on negatively selected CD56+ CD3- cells. Altogether, the results of this study demonstrate that extracellular mycobacteria activate several NK-cell functions and suggest a possible alternative mechanism of NK-cell activation as the first line of defence against mycobacterial infections.     

Intracellular expression of MICA in activated CD4 T lymphocytes and protection from NK cell-mediated MICA-dependent cytotoxicity

MICA is a stress-regulated molecule recognized by the NK cell-activating receptor NKG2D. Previously, we demonstrated that MICA is induced on activated T cells but regulation by mitogenic cytokines and its biological consequences remain unexplored. Here, we show that IL-2, IL-4, and IL-15 but not TNF-alpha or IFN-alpha induced MICA expression in T lymphocytes present in peripheral blood mononuclear cells (PBMCs), as assessed by Western blot. IL-2 effect involved Jak3/STAT5, p38 MAPK, p70(56) kinase, Lck/fyn kinases, and NF-kappaB. MICA expression was also observed in Th1 and Th2 cells. However, surface expression was not detected. T lymphocytes present in PBMCs and isolated CD4+ T lymphocytes stimulated with phorbol-12-myristate-13-acetate and ionomycin also induced MICA expression as assessed by Western blot, but only low levels were expressed at the cell surface. Activated but not resting CD4+ T lymphocytes were lysed by IL-15- or IL-2-stimulated NK cells, and susceptibility was increased when HLA class I molecules were blocked. Also, cytokine-stimulated NK cells produced more IFN-gamma after culture with activated CD4+ T lymphocytes. However, the participation of MICA in these responses, if any, was marginal. Confocal microscopy revealed that MICA is retained mostly inside activated CD4+ T cells. Our results suggest that low surface expression of MICA on activated CD4+ T lymphocytes might be a safeguard mechanism to protect them from NK cells in an inflammatory, virus-infected, or tumor microenvironment, where NK and activated CD4+ T cells are recruited.     

Essential roles of monocytes in stimulating human peripheral blood mononuclear cells with Lactobacillus casei to produce cytokines and augment natural killer cell activity

We examined the effect of a probiotic strain, Lactobacillus casei strain Shirota, on cytokine production and natural killer (NK) cell activity in human peripheral blood mononuclear cells (PBMNC). The cellular mechanisms of immunoregulation by L. casei strain Shirota were also investigated. L. casei strain Shirota stimulated PBMNC to secrete interleukin-12 (IL-12), gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and IL-10. However, depletion of monocytes from PBMNC eliminated the induction of these cytokines. L. casei strain Shirota was phagocytosed by monocytes and directly stimulated them to secrete IL-12, TNF-alpha, and IL-10. IFN-gamma production was diminished by the addition of anti-IL-12 antibody to the PBMNC cultures. Purified T cells, but not NK cells, produced IFN-gamma effectively when stimulated with L. casei strain Shirota in the presence of monocytes, indicating that monocytes triggered by L. casei strain Shirota help T cells to produce IFN-gamma through secreting IL-12. In addition, NK cell activity and CD69 expression on NK cells increased after cultivation of PBMNC with L. casei strain Shirota. When monocytes were depleted from PBMNC, L. casei strain Shirota did not enhance NK cell activity. These results demonstrate that monocytes play critical roles in the induction of cytokines and following the augmentation of NK cell activity during the stimulation of human PBMNC with L. casei strain Shirota.     

IFN-gamma and IL-12 differentially regulate CC-chemokine secretion and CCR5 expression in human T lymphocytes

Interleukin (IL)-12, especially in the presence of neutralizing anti-IL-4 monoclonal antibodies, primed CD45RO(-) T clones for high CCL3/macrophage-inflammatory protein-1alpha (MIP-1alpha) and CCL4/MIP-1beta levels. In CD4(+) and CD8(+) clones from two patients deficient for IL-12Rbeta1 (IL-12Rbeta1(-/-)), production of CCL3/MIP-1alpha and CCL4/MIP-1beta was defective. CD4(+) clones from two patients deficient for interferon-gamma (IFN-gamma) R1 (IFN-gammaR1(-/-)) produced somewhat decreased CCL4/MIP-1beta levels. IL-12 failed to prime CD4(+) or CD8(+) healthy clones for high CCL5/regulated on activation, normal T expressed and secreted (RANTES) production, although its secretion was impaired in CD4(+) clones from IL-12Rbeta1(-/-) and IFN-gammaR1(-/-) patients. CCR5 surface expression was up-regulated in resting peripheral blood mononuclear cells and CD4(+) clones from both kinds of patients, rendering them more susceptible to CCR5-dependent (R5) HIV-1 infection. Neutralization of IFN-gamma increased CCR5 expression and decreased CC-chemokine secretion by CD4(+) clones from healthy and IL-12Rbeta1(-/-) individuals, suggesting an IFN-gamma-dependent control of CCR5 expression. These data provide the first documented analysis of chemokine secretion and chemokine receptor expression on T cells from IL-12 and IFN-gamma receptor-deficient patients and dissect the role of IL-12 and IFN-gamma on inducing inflammatory chemokine secretion and down-regulating CCR5 expression in human T cells.     

Brucella abortus as a potential vaccine candidate: induction of interleukin-12 secretion and enhanced B7.1 and B7.2 and intercellular adhesion molecule 1 surface expression in elutriated human monocytes stimulated by heat-inactivated B. abortus.

Development of a vaccine which is capable of generating a strong cellular immune response associated with gamma interferon (IFN-gamma) production and cytotoxic T-cell development requires that the immunogen be capable of inducing the secretion of interleukin-12 (IL-12), which is a pivotal factor for the differentiation of Th1 or Tc1 cells. We have previously shown that the heat-inactivated gram-negative bacterium Brucella abortus can induce IFN-gamma secretion by T cells. In the present study, we demonstrate that B. abortus and lipopolysaccharide (LPS) from B. abortus can induce IL-12 p40 mRNA expression and protein secretion by human elutriated monocytes (99% pure). p40 mRNA was detected within 4 h, and p40 protein could be measured at 24 h. This induction was abrogated by anti-CD14 monoclonal antibody, suggesting that monocytes recognize B. abortus via their receptor for LPS. The biological activity of IL-12 secreted by B. abortus-stimulated monocytes was demonstrated by its ability to upregulate IFN-gamma mRNA expression in T cells separated from monocytes and B. abortus by a transwell membrane. The B. abortus-induced IL-12 also enhanced NK cytolytic activity against K562 target cells. B. abortus was shown to rapidly increase the expression of the costimulatory molecules B7.1 and B7.2 and intercellular adhesion molecule 1 on human monocytes. Together, these data indicate that B. abortus can directly activate human monocytes and provide the cytokine milieu which would direct the immune response towards Th1-Tc1 differentiation.     

Different natural killer (NK) receptor expression and immunoglobulin E (IgE) regulation by NK1 and NK2 cells

Many studies concerning the role of T cells and cytokines in allergy have been performed, but little is known about the role of natural killer (NK) cells. Accordingly, the expression of co-stimulatory, inhibitory and apoptosis receptors, cytokine profiles and their effect on immunoglobulin isotypes were investigated in polyallergic atopic dermatitis (AD) patients with hyper immunoglobulin E (IgE) and healthy individuals. AD patients showed significantly decreased peripheral blood NK cells compared to healthy individuals. Freshly isolated NK cells of polyallergic patients spontaneously released higher amounts of interleukin (IL)-4, IL-5, IL-13 and interferon (IFN)-gamma compared to healthy individuals. NK cells were differentiated to NK1 cells by IL-12 and neutralizing anti-IL-4 monoclonal antibodies (mAb), and to NK2 cells by IL-4 and neutralizing anti-IL-12 mAb. Following IL-12 stimulation, NK cells produced increased levels of IFN-gamma and decreased IL-4. In contrast, stimulation of NK cells with IL-4 inhibited IFN-gamma, but increased IL-13, production. The effect of NK cell subsets on IgE regulation was examined in co-cultures of in vitro differentiated NK cells with peripheral blood mononuclear cells (PBMC) or B cells. NK1 cells significantly inhibited IL-4- and soluble CD40-ligand-stimulated IgE production; however, NK2 cells did not have any effect. The inhibitory effect of NK1 cells on IgE production was blocked by neutralization of IFN-gamma. Except for CD40, NK cell subsets showed different expression of killer-inhibitory receptors and co-stimulatory molecules between the polyallergic and healthy subjects. These results indicate that human NK cells show differences in numbers, surface receptor and cytokine phenotypes and functional properties in AD.     

Induction of IFN-gamma-producing CD4+ natural killer T cells by Mycobacterium bovis bacillus Calmette Guérin

The CD4+ natural killer (NK)T cells in the liver are potent IL-4 producers and hence may promote Th2 cell development. Following Mycobacterium bovis bacillus Calmette Guérin (BCG) infection, IL-4-producing CD4+ NKT cells become undetectable in liver mononuclear cells of normal density (interface between 40 and 70% Percoll) by flow cytometry. The present study shows that M. bovis BCG infection changes the density of liver CD4+ NKT cells and shifts cytokine production from IL-4 to IFN-gamma. The number of CD4+ NK1+ TCR alpha/beta(intermediate) cells increased in the low-density fraction (<40% Percoll density gradient) in parallel to the reduction of this cell population in the fraction of normal density. The number of IL-4-producing cells, however, was small and high frequencies of IFN-gamma-secreting cells were identified in the low-density fraction after TCR/CD3 ligation. Accordingly, selected low-density CD4+ NKT cells encompassed high numbers of IFN-gamma producers and minute numbers of IL-4-secreting cells. Induction of low-density CD4+ NKT cells by M. bovis BCG was abrogated by endogenous IL-12 neutralization which also caused increased bacterial growth in the liver. We assume that M. bovis BCG infection changes cytokine secretion by the CD4+ NKT cell population from IL-4 to IFN-gamma through IL-12 induction. Thus, CD4+ NKT cells may contribute to host resistance against intracellular bacteria prior to conventional IFN-gamma-producing Th1 cells.     

FcR+ and FcR- monocytes differentially secrete monokines during pokeweed mitogen-induced T-cell-monocyte interactions.

Monocyte subpopulations which differ in the expression of Fc receptor for human IgG (FcRI) differentially regulate the T-cell-dependent, pokeweed mitogen (PWM)-induced, polyclonal B-cell response. We, thus, studied the cytokine production in human peripheral blood monocyte and T-lymphocyte cultures activated with this lectin. Monocytes or their FcR+ and FcR- subpopulations stimulated with PWM were cultured with or without T lymphocytes or their CD4+ and CD8+ subsets. Both monocyte subpopulations cultured alone produced similar amounts of tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6), but FcR- monocytes showed significantly enhanced ability to secrete interleukin-1 (IL-1). T cells, especially CD4+, added to monocyte cultures enhanced IL-1 production. This enhancement was presumably due to interferon-gamma (IFN-gamma) release by T lymphocytes, since this lymphokine enhanced IL-1 secretion when added to PWM-stimulated cultures of monocytes. Addition of monocytes, in particular the FcR+ subpopulation, greatly enhanced production of IFN-gamma by T lymphocytes. Although both T-cell subsets produced IFN-gamma, the CD4+ cells were more efficient. These results indicate that in PWM-stimulated cultures subpopulations of monocytes differ in secretion of cytokines, which might explain their differential effect on T-cell-dependent immune responses in vitro.     

Dendritic cells derived from BCG-infected precursors induce Th2-like immune response

Human monocytes can differentiate into dendritic cells (DCs) according to the nature of environmental signals. We tested here whether the infection with the live tuberculosis vaccine bacillus Calmette-Guerin (BCG), which is known to be limited in preventing pulmonary tuberculosis, modulates monocyte and DC differentiation. We found that monocytes infected with BCG differentiate into CD1a- DCs (BCG-DCs) in the presence of granulocyte macrophage-colony stimulating factor and interleukin (IL)-4 and acquired a mature phenotype in the absence of maturation stimuli. In addition, BCG-DCs produced proinflammatory cytokines (tumor necrosis factor alpha, IL-1beta, IL-6) and IL-10 but not IL-12. BCG-DCs were able to stimulate allogeneic T lymphocytes to a similar degree as DCs generated in the absence of infection. However, BCG-DCs induced IL-4 production when cocultured with human cord-blood mononuclear cells. The induction of IL-4 production by DCs generated by BCG-infected monocytes could explain the failure of the BCG vaccine to prevent pulmonary tuberculosis.