Streptococcal preparation OK-432: a new maturation factor of monocyte-derived dendritic cells for clinical use

For vaccinations based on dendritic cells (DCs), maturation of DCs is critical to the induction of T-cell responses. We tested the efficacy of streptococcal preparation OK-432 as a Good Manufacturing Practice (GMP)-grade maturation agent. OK-432 is currently used in Japan as a cancer immunotherapy drug. Immature monocyte-derived dendritic cells (imMo-DCs) isolated from human peripheral blood monocytes stimulated with granulocyte-macrophage colony stimulating factor and interleukin-4 were exposed to maturation factors, i.e., lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF-alpha) plus prostaglandin E2 (PGE2), and OK-432 for 2 days. OK-432 increased expression of activation- and maturation-related molecules such as HLA-DR, CD80, CD83, and CD86 in imMo-DCs at levels similar to that of TNF-alpha plus PGE2, and higher than that of LPS. All agents examined induced allogeneic T-cell proliferation at a similar level. Only OK-432 caused significant production of interleukin-12 (IL-12) p70 and interferon gamma (IFN-gamma) at both the mRNA and protein levels in imMo-DCs. Neutralizing antibody against IL-12 p70 blocked IFN-gamma secretion from OK-432-stimulated Mo-DCs. IL-12 p70 produced by OK-432-stimulated imMo-DCs induced secretion of IFN-gamma by CD4+ T cells. OK-432 and LPS activated nuclear factor kappa B (NF-kappaB) in imMo-DCs. Both secretion of IL-12 p70 and IFN-gamma and activation of NF-kappaB induced by OK-432 were suppressed when imMo-DCs were pretreated with cytochalasin B. These results indicate that uptake of OK-432 by imMo-DCs is an early critical event for IL-12 p70 production and that NF-kappaB activation induced by OK-432 also contributes partially to IL-12 p70 production. In conclusion, OK-432 is a GMP-grade maturation agent and may be a potential tool for DC-based vaccine therapies.     

Human dendritic cells discriminate between viable and killed Toxoplasma gondii tachyzoites: dendritic cell activation after infection with viable parasites results in CD28 and CD40 ligand signaling that controls IL-12-dependent and -independent T cell production of IFN-gamma

We studied how the interaction between human dendritic cells (DC) and Toxoplasma gondii influences the generation of cell-mediated immunity against the parasite. We demonstrate that viable, but not killed, tachyzoites of T. gondii altered the phenotype of immature DC. DC infected with viable parasites up-regulated the expression of CD40, CD80, CD86, and HLA-DR and down-regulated expression of CD115. These changes are indicative of DC activation induced by T. gondii. Viable and killed tachyzoites had contrasting effects on cytokine production. DC infected with viable T. gondii rather than DC that phagocytosed killed parasites induced secretion of high amounts of IFN-gamma by T cells from T. gondii-seronegative donors. IFN-gamma production in response to DC infected with viable parasites required CD28 and CD40 ligand (CD40L) signaling. In addition, this IFN-gamma response was dependent in part on IL-12 secretion. Production of IL-12 p70 occurred after interaction between T cells and DC infected with viable T. gondii, but not after incubation of T cells with DC plus killed tachyzoites. IL-12 synthesis was inhibited by blockade of CD40L signaling. IL-12-independent IFN-gamma production required CD80/CD86-CD28 interaction and, to a lesser extent, CD40-CD40L signaling. Taken together, T. gondii-induced activation of human DC is associated with T cell production of IFN-gamma through CD40-CD40L-dependent release of IL-12 and through CD80/CD86-CD28 and CD40-CD40L signaling that mediate IFN-gamma secretion even in the absence of bioactive IL-12.     

Systemic inflammatory priming in normal pregnancy and preeclampsia: the role of circulating syncytiotrophoblast microparticles

Systemic inflammatory responsiveness was studied in normal human pregnancy and its specific inflammatory disorder, pre-eclampsia. Compared with nonpregnancy, monocytes were primed to produce more TNF-alpha throughout normal pregnancy, more IL-12p70 in the first and second trimesters, and more IL-18 in the first trimester only. Intracellular cytokine measurements (TNF-alpha and IL12p70) showed little change by comparison. IFN-gamma production was suppressed in all three trimesters. In pre-eclampsia, IL-18 secretion was increased. Secreted but not intracellular measures of TNF-alpha and IL-12p70 were also further enhanced compared with normal pregnancy. Inhibition of IFN-gamma production was lost and involved both CD56(+) NK and CD56(-) lymphocyte subsets. We determined whether circulating syncytiotrophoblast microparticles (STBM) could contribute to these inflammatory changes. Unbound STBM could be detected in normal pregnancy by the second trimester and increased significantly in the third. They were also bound in vivo to circulating monocytes. Women with pre-eclampsia had significantly more circulating free but not cell-bound STBMs. STBMs prepared by perfusion of normal placental lobules stimulated production of inflammatory cytokines (TNF-alpha, IL12p70, and IL-18 but not IFN-gamma) when cultured with PBMCs from healthy nonpregnant women. Inflammatory priming of PBMCs during pregnancy is confirmed and is established by the first trimester. It is associated with early inhibition of IFN-gamma production. The inflammatory response is enhanced in pre-eclampsia with loss of the IFN-gamma suppression. Circulating STBMs bind to monocytes and stimulate the production of inflammatory cytokines. It is concluded that they are potential contributors to altered systemic inflammatory responsiveness in pregnancy and pre-eclampsia.     

TGF-beta and IL-10 regulation of IFN-gamma produced in Th2-type schistosome granulomas requires IL-12

Interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta) regulate CD4+ T cell interferon-gamma (IFN-gamma) secretion in schistosome granulomas. The role of IL-12 was determined using C57BL/6 and CBA mice. C57BL/6 IL-4-/- granuloma cells were stimulated to produce IFN-gamma when cultured with IL-10 or TGF-beta neutralizing monoclonal antibody. In comparison, C57BL/6 wild-type (WT) control granuloma cells produced less IFN-gamma. IL-12, IL-18, and soluble egg antigen stimulated IFN-gamma release from C57BL/6 IL-4-/- and WT mice. IFN-gamma production in C57 IL-4-/- and WT granulomas was IL-12 dependent, because IL-12 blockade partly abrogated IFN-gamma secretion after stimulation. All granuloma cells released IL-12 (p70 and p40), and IL-12 production remained constant after anti-TGF-beta, anti-IL-10, recombinant IL-18, or antigen stimulation. C57 WT and IL-4-/- mouse granuloma cells expressed IL-12 receptor (IL-12R) beta1-subunit mRNA but little beta2 mRNA. TGF-beta or IL-10 blockade did not influence beta1 or beta2 mRNA expression. CBA mouse dispersed granuloma cells released no measurable IFN-gamma, produced IL-12 p70 and little p40, and expressed IL-12R beta2 and little beta1 mRNA. In T helper 2 (Th2) granulomas of C57BL/6 WT and IL-4-/- mice, cells produce IL-12 (for IFN-gamma production) and IL-10 and TGF-beta modulate IFN-gamma secretion via mechanisms independent of IL-12 and IL-12R mRNA regulation. We found substantial differences in control of granuloma IFN-gamma production and IL-12 circuitry in C57BL/6 and CBA mice.     

Cigarette smoke extract suppresses human dendritic cell function leading to preferential induction of Th-2 priming

Dendritic cells (DC) are key regulators of immune responses. In the current study, we hypothesized that cigarette smoke-induced aberrance in DC function is an important mechanism by which smokers develop cancer, infection, and allergy--diseases common in smokers. We demonstrate that cigarette smoke extract (CSE) inhibits DC-mediated priming of T cells, specifically inhibiting the secretion of IFN-gamma whereas enhancing the production of IL-4 in the MLR. Conditioning with CSE did not effect cytokine (IL-10, IL-6, or IL-12) production from immature DCs, but significantly inhibited IL-12p70 release by LPS-matured DCs. In contrast, IL-10 secretion by LPS-activated CSE-conditioned DCs was enhanced when compared with control DCs. CSE also induced cyclooxygenase-2 protein levels in maturing DCs and significantly augmented endogenous PGE2 release. Conditioning of DCs with CSE also suppressed LPS-mediated induction of CD40, CD80, and CD86, and suppressed maturation-associated CCR7 expression. Although CSE has been reported to induce apoptosis of fibroblasts and epithelial cells, the immunomodulatory effects observed with CSE were not due to diminished DC viability. The effects of CSE on DC function were not exclusively mediated by nicotine, because equivalent, or even higher concentrations of nicotine than those found in CSE, failed to suppress DC-induced T cell priming. These data provide evidence that soluble components extracted from cigarette smoke suppress key DC functions and favor the development of Th-2 immunity.     

Human dendritic cells respond to Helicobacter pylori, promoting NK cell and Th1-effector responses in vitro

Helicobacter pylori infection leads to chronic gastric inflammation. The current study determined the response of human APCs, NK cells, and T cells toward the bacteria in vitro. Human monocyte-derived dendritic cells (DC) were incubated with bacteria for 48 h. Intact H. pylori at a multitude of infection 5 stimulated the expression of MHC class II (4- to 7-fold), CD80, and CD86 B7 molecules (10- to 12-fold) and the CD83 costimulatory molecule (>30-fold) as well as IL-12 secretion (>50-fold) in DCs, and thereby, strongly induced their maturation and activation. CD56(+)/CD4(-) NK cells, as well as CD4(+)/CD45RA(+) naive T cells, were isolated and incubated with DCs pulsed with intact bacteria or different cellular fractions. Coculture of H. pylori-pulsed DCs with NK cells strongly potentiated the secretion of TNF-alpha and IFN-gamma. Coculture of naive T cells with H. pylori-pulsed DCs significantly enhanced TNF-alpha, IFN-gamma, and IL-2 secretion as well as T-bet mRNA levels, while GATA-3 mRNA was lowered. However, the effect appeared attenuated compared with coculture with Escherichia coli. A greater stimulation was seen with naive T cells and DCs pulsed with H. pylori membrane preparations. Intact H. pylori potently induced the maturation and activation of human monocyte-derived DC and thereby promote NK and Th1 effector responses. The strong activation of NK cells may be important for the innate immune response. Th1-polarized T cells were induced especially by incubation with membrane preparations of H. pylori, suggesting that membrane proteins may account for the specific adaptive immune response.     

Generation of dendrite cells and the use of immunomodulators of bacterial origin as cell differentiation inducers

The generation of ripe dendrite cells (DC) of marrow origin was obtained with the use of the vaccine Immunovac-BN-4, an immunomodulator of microbial origin, as well as Klebsiella pneumoniae LPS and TNF-alpha, as ripening inducers. These inducers equally led to the ripening of DC. The generation of ripe DC was characterized by morphological, phenotypical and functional changes. The immunophenotype of cells altered from CD34+, CD38-, CD40-, CD80-, CD86-, MHC I-, MHC II-, F4/80- to CD34-, CD38+, CD40+, CD80+, MHC I+, MHC II+, F4/ 80(low). In parallel with the ripening of DC their phagocytic activity decreased. In culture medium with ripe DC the levels of such cytokines as IL-1b, IL-6, IL-12, IFN-gamma, TNF-alpha significantly increased and the production of IL-4 decreased. The content of IL-2 and IL-10 remained unchanged.     

Flow cytometrical determination of regulatory cytokines (IL-10, IL-12) and circulating dendritic cell cytokines in allergic asthmatic children

Interleukin (IL)-10 and IL-12 have been suggested to be key regulators in the pathogenesis of allergic asthma. Several of the secretion products of dendritic cells (DC), such as IL-12, IL-10, IL-1beta and TNF-alpha, are considered to play a role in allergic asthma. This study compares the production of IL-10 and IL-12 in allergic asthmatic children (n = 17) and controls (n = 14) by measuring their extracellular secretion in whole blood samples after stimulation, using a microsphere-based immunoassay. Additionally, we assessed intracellular production of IL-1beta, TNF-alpha, IL-12 and IL-10 by circulating DC in stimulated whole blood samples of asthmatic and healthy children. The concentration of IL-10 in the supernatants of LPS-stimulated whole blood was significantly lower in allergic asthmatic children as compared to healthy children (463 (207-768) vs 881 (364-2626) pg/mL; p = 0.005). When a combined LPS and IFN-gamma stimulation was used, IL-10 production decreased significantly as compared to LPS alone, especially in healthy children. Consequently, no difference in IL-10 production after LPS/IFN-gamma stimulation was found between healthy and allergic children. In contrast to isolated LPS stimulation, stimulation with LPS/IFN-gamma induced higher IL-12 production; allergic asthmatic children showed a significantly lower IL-12 secretion after LPS/IFN-gamma stimulation as compared to healthy children (20 (5-247) vs 208 (7-775) pg/mL; p=0.03). Moreover, the number of IL-12 producing CD11c-positive DC (DC1) tended to be lower in asthmatic children compared to healthy children (0.05 (0.00-0.45) vs 0.27 (0.00-0.83) 10(6)/L) and correlated with the extracellular release of IL-12 in asthmatic children (r = 0.65; p = 0.016). The number of IL-1beta and TNF-alpha producing CD11c-positive DC (DC1) was comparable between healthy and asthmatic children. We hypothesize that the decreased production of IL-10 and IL-12 is responsible for Th2 polarized responses in allergic asthmatic children.     

Selective suppression of IL-12 production by chemoattractants

We investigated the ability of chemoattractants to affect IL-12 production by human monocytes and dendritic cells. We found that pretreatment of monocytes with macrophage chemoattractant proteins (MCP-1 to -4), or C5a, but not stromal-derived factor-1, macrophage inflammatory protein-1alpha, RANTES, or eotaxin, inhibited IL-12 p70 production in response to stimulation with Staphylococcus aureus, Cowan strain 1 (SAC), and IFN-gamma. The production of TNF-alpha and IL-10, however, was minimally affected by any of the chemoattractants. The degree of inhibition of IL-12 p70 production by MCP-1 to -4 was donor dependent and was affected by the autocrine inhibitory effects of IL-10. In contrast, C5a profoundly suppressed IL-12 production in an IL-10-independent fashion. Neither TGF-beta1 nor PGE2 was important for the suppression of IL-12 by any of the chemoattractants tested. The accumulation of mRNA for both IL-12 p35 and p40 genes was inhibited by chemokine pretreatment. Interestingly, MCP-1 to -4 and C5a did not suppress IL-12 production by monocyte-derived dendritic cells (DC) stimulated with CD40 ligand and IFN-gamma or by SAC and IFN-gamma, suggesting that these factors may act at the site of inflammation to suppress IL-12 and IFN-gamma production rather than in the lymph node to affect T cell priming. Despite the inability of C5a to inhibit IL-12 production by DCs, the receptor for C5a (CD88) was expressed by these cells, and recombinant C5a induced a Ca2+ flux. Taken together, these results define a range of chemoattractant molecules with the ability to suppress IL-12 production by human monocytes and have broad implications for the regulation of immune responses in vivo.     

Lutzomyia longipalpis salivary peptide maxadilan alters murine dendritic cell expression of CD80/86, CCR7, and cytokine secretion and reprograms dendritic cell-mediated cytokine release from cultures containing allogeneic T cells

Leishmania protozoan parasites, the etiologic agent of leishmaniasis, are transmitted exclusively by phlebotomine sand flies of the genera Phlebotomus and Lutzomyia. In addition to parasites, the infectious bite inoculum contains arthropod salivary components. One well-characterized salivary component from Lutzomyia longipalpis is maxadilan (MAX), a vasodilator acting via the type I receptor for the pituitary cyclic AMP activating peptide. MAX has been shown to elicit immunomodulatory effects potentially dictating immune responses to Leishmania parasites. When exposed to MAX, both resting and LPS-stimulated dendritic cells (DCs) show reduced CD80 and CD86 expression on most DCs in vitro. However, CD86 expression is increased significantly on a subpopulation of DCs. Furthermore, MAX treatment promoted secretion of type 2 cytokines (IL-6 and IL-10) while reducing production of type 1 cytokines (IL-12p40, TNF-alpha, and IFN-gamma) by LPS-stimulated DCs. A similar trend was observed in cultures of MAX-treated DCs containing naive allogeneic CD4(+) T cells: type 2 cytokines (IL-6 and IL-13) increased while type 1 cytokines (TNF-alpha and IFN-gamma) decreased. Additionally, the proinflammatory cytokine IL-1beta was increased in cultures containing MAX-treated mature DCs. MAX treatment of LPS-stimulated DCs also prevented optimal surface expression of CCR7 in vitro. These MAX-dependent effects were evident in DCs from both Leishmania major-susceptible (BALB/c) and -resistant (C3H/HeN) murine strains. These data suggest that modification of DC phenotype and function by MAX likely affects crucial cellular components that determine the pathological response to infection with Leishmania.     

IL-12/IL-18-dependent IFN-gamma release by murine dendritic cells.

Dendritic cells (DC) develop in GM-CSF-stimulated cultures from murine bone marrow progenitors in serum-free (or low serum) medium. CD11c(+) myeloid DC from 7-day cultures stimulated with TNF-alpha, IFN-alpha, IFN-gamma, or LPS up-regulated surface expression of CD40 and CD86 costimulator and MHC class II molecules, did not up-regulate the low "spontaneous" release of IL-18, and did not release IFN-gamma. Stimulation of in vitro-generated DC with exogenous IL-12 and IL-18 (but not with IL-4 or LPS plus IL-18) induced IFN-gamma expression and release in 15-20% of the DC (detectable by FACS analyses or ELISA). Endogenous IL-12 p70 produced by DC in response to ligation of CD40 stimulated IFN-gamma release when exogenous IL-18 was supplied. In vivo-generated, splenic CD8alpha(+) and CD8alpha(-) DC (from immunocompetent and immunodeficient H-2(d) and H-2(b) mice) cultured with IL-12 and IL-18 released IFN-gamma. The presence of LPS during the stimulation of DC with IL-18 plus endogenous (CD40 ligation) or exogenous IL-12 did not affect their IFN-gamma release. In contrast, splenic DC pretreated in vitro or in vivo by LPS strikingly down-regulated IFN-gamma release in response to stimulation by IL-18 and (endogenous or exogenous) IL-12. Hence, DC are a source of early IFN-gamma generated in response to a cascade of cytokine- and/or cell-derived signals that can be positively and negatively regulated.     

IL-12p70-dependent Th1 induction by human B cells requires combined activation with CD40 ligand and CpG DNA

The detection of microbial molecules via Toll-like receptors (TLR) in B cells is not well characterized. In this study, we found that both naive and memory B cells lack TLR4 (receptor for LPS) but express TLR9 (receptor for CpG motifs) and produce IL-6, TNF-alpha, and IL-10 upon stimulation with CpG oligonucleotides (ODN), synthetic mimics of microbial DNA. Consistent with the lack of TLR4, purified B cells failed to respond to LPS. Similar to CpG ODN, CD40 ligand (CD40L) alone induced IL-6, TNF-alpha, and IL-10. Production of these cytokines as well as IgM synthesis was synergistically increased when both CpG ODN and CD40L were combined. Unlike IL-6, TNF-alpha, and IL-10, the Th1 cytokine IL-12p70 was detected only when both CpG ODN and CD40L were present, and its induction was independent of B cell receptor cross-linking. CpG ODN did not increase the capacity of CD40L-activated B cells to induce proliferation of naive T cells. However, B cells activated with CpG ODN and CD40L strongly enhanced IFN-gamma production in developing CD4 T cells via IL-12. Together, these results demonstrate that IL-12p70 production in human B cells is under the dual control of microbial stimulation and T cell help. Our findings provide a molecular basis for the potent adjuvant activity of CpG ODN to support humoral immune responses observed in vivo, and for the limited value of LPS.     

CD86 and IL-12p70 Are Key Players for T Helper 1 Polarization and Natural Killer Cell Activation by Toll-Like Receptor-Induced Dendritic Cells

Background

Dendritic cells (DCs) determine the activation and polarization of T cells via expression of costimulatory molecules and secretion of cytokines. The function of DCs derived from monocytes ex vivo strongly depends on the composition of the maturation cocktail used.

Methodology/Principal Findings

We analyzed the effect of costimulatory molecule expression and cytokine secretion by DCs on T and natural killer (NK) cell activation by conducting a head-to-head comparison of a Toll-like receptor (TLR) agonist-based cocktail with the standard combination of proinflammatory cytokines or IL-10 alone. We could show that TLR-induced DCs are characterized by a predominance of costimulatory over coinhibitory molecules and by high secretion of IL-12p70, but not IL-10. Functionally, these signals translated into an increase in IFN-γ secreting Th1 cells and a decrease in regulatory T cells. T cell activation and polarization were dependent on IL-12p70 and CD86, but remarkably not on CD80 signaling. By means of IL-12p70 secretion, only TLR-induced DCs activated NK cells.

Conclusions/Significance

TLR-matured DCs are highly suitable for application in immunotherapeutic strategies that rely on strong type 1 polarization and NK cell activation. Their effects particularly depend on high CD86 expression and IL-12p70 secretion.     

Type I IFN-induced, NKT cell-mediated negative control of CD8 T cell priming by dendritic cells

We investigated the negative effect of type I IFN (IFN-I) on the priming of specific CD8 T cell immunity. Priming of murine CD8 T cells is down-modulated if Ag is codelivered with IFN-I-inducing polyinosinic:polycytidylic acid (pI/C) that induces (NK cell- and T/B cell-independent) acute changes in the composition and surface phenotype of dendritic cells (DC). In wild-type but not IFN-I receptor-deficient mice, pI/C reduces the plasmacytoid DC but expands the CD8(+) conventional DC (cDC) population and up-regulates surface expression of activation-associated (CD69, BST2), MHC (class I/II), costimulator (CD40, CD80/CD86), and coinhibitor (PD-L1/L2) molecules by cDC. Naive T cells are efficiently primed in vitro by IFN-I-stimulated CD8 cDC (the key APC involved in CD8 T cell priming) although these DC produced less IL-12 p40 and IL-6. pI/C (IFN-I)-mediated down modulation of CD8 T cell priming in vivo was not observed in NKT cell-deficient CD1d(-/-) mice. CD8 cDC from pI/C-treated mice inefficiently stimulated IFN-gamma, IL-4, and IL-2 responses of NKT cells. In vitro, CD8 cDC that had activated NKT cells in the presence of IFN-I primed CD8 T cells that produced less IFN-gamma but more IL-10. The described immunosuppressive effect of IFN-I thus involves an NKT cell-mediated change in the phenotype of CD8 cDC that favors priming of IL-10-producing CD8 T cells. In the presence of IFN-I, NKT cells hence impair the competence of CD8 cDC to prime proinflammatory CD8 T cell responses.     

Expression of inflammatory markers in pig amnion after intraamniotic infection with nonpathogenic or enteropathogenic<Emphasis Type="Italic">Escherichia coli</Emphasis>

The pig amnion was in vivo intraamniotically infected with E. coli for 10 h at 80-85 d of gestation either with the nonpathogenic O86 strain or enteropathogenic O55 strain. TNF-alpha, IL-10, IL-1beta and IFN-gamma were determined in amniotic fluids by ELISA, the expression of cytokines and some other inflammatory markers was determined by immunohistochemistry. Intraamniotic infection induced high levels of TNF-alpha in amniotic fluids which correlated with bacterial virulence whereas IL-10 was induced only by O86. The IL-1beta level did not increase significantly and was expressed in all infected membranes. IFN-gamma was negligible or absent. TNF-alpha, IL-12p40, calprotectin, HSP65 and gp91phox were found by immunohistochemistry only in amnion membranes infected with the enteropathogenic strain 055.     

Differential expansion, activation and effector functions of conventional and plasmacytoid dendritic cells in mouse tissues transiently infected with Listeria monocytogenes

Dendritic cells (DC) are crucial in generating immunity to infection. Here we characterize changes in DC in terms of number, activation and effector functions, focusing on conventional DC (cDC) and plasmacytoid DC (pDC), in Listeria-infected mice. Kinetic studies showed a subset- and tissue-specific expansion of cDC and upregulation of CD80 and CD86 on splenic and mesenteric lymph node (MLN) cDC after intragastric infection. Expansion of pDC was more prolonged than cDC, and pDC upregulated CD86 and MHC-II, but not CD80, in both the spleen and MLN. cDC were an important source of IL-12 but not TNF-alpha during infection, while pDC made neither of these cytokines. Instead other CD11c(int) cells produced these cytokines. Using five-colour flow cytometry and double intracellular cytokine staining, we detected phenotypically similar CD11c(int)CD11b(+)Gr1(+) cells with distinct capacities to produce TNF-alpha/IL-12 or TNF-alpha/iNOS (inducible nitric oxide synthase) in Listeria-infected tissues. IL-12p70 was also produced by sorted CD11c(hi) and CD11c(int)CD11b(+)Gr1(+) cells. Furthermore, production of TNF-alpha, iNOS and IL-12 was differentially dependent on cellular localization of the bacteria. Cytosol-restricted bacteria induced TNF-alpha and iNOS-producing cells, albeit at lower frequency than wild-type bacteria. In contrast, IL-12 was induced only with wild-type bacteria. These data provide new insight into the relative abundance and function of distinct CD11c-expressing populations during the early stage of Listeria infection.     

Lymphokine release, suppressor cell generation, cell surface markers, and cytotoxic activity in cancer patients receiving natural interleukin-2

We monitored patients treated for 5 days with continuous infusion of increasing doses (3 to 6 x 10(6) U/d) of natural interleukin-2 (IL-2). CD16+, CD25+, and CD56+ cells increased after treatment. Plasma tumor necrosis factor-alpha (TNF-alpha) levels, but not interferon-gamma (IFN-gamma) levels, increased during IL-2 treatment, but spontaneous and IL-2-stimulated TNF-alpha secretion in vitro remained abnormally low. However, mitogen-stimulated TNF-alpha release was normal. Mitogen-stimulated, but not IL-2-stimulated, IFN-gamma release was strongly depressed. Low spontaneous and IL-2-stimulated cytotoxicity on K562 or Daudi increased after treatment. Low suppressor cell generation also normalized after treatment. This appears to be the first reported study of immunologic monitoring of cancer patients treated with natural rather than recombinant IL-2.     

Inflammatory cytokine production in interferon-gamma-primed mice, challenged with lipopolysaccharide. Inhibition by SK&F 86002 and interleukin-1 beta-converting enzyme inhibitor

Mice challenged with lipopolysaccharide (LPS) produce variable serum levels of pro-inflammatory cytokines, and particularly low levels of interleukin-1 beta (IL-1 beta). Interferon-gamma (IFN-gamma) has been shown to be an important mediator of bacteria-induced hypersensitivity to LPS in mice. In the present study, we show that mice pretreated with IFN-gamma exhibit an enhanced capacity to produce serum IL-1 beta, IL-1 alpha, tumour necrosis factor (TNF-alpha) as well as IL-6 in response to LPS. Priming with intraperitoneal (i.p.) injection of 15 mg rat recombinant IFN-gamma, 18 hours prior to the i.p. LPS (300 mg) challenge resulted in a 4-fold increase in the LPS-stimulated release of IL-1 beta and a 2- to 7-fold increase in the release of IL-1 alpha, TNF-alpha, as well as IL-6 into the serum. LPS induced a concentration-dependent increase in the release of IL-1 beta in isolated peritoneal macrophages from IFN-gamma-primed mice whereas macrophages from unprimed mice released minute amounts of IL-1 beta. In addition, nigericin markedly enhanced the release of IL-1 beta in unprimed mice but not in macrophages from IFN-gamma primed mice. The cytokine synthesis inhibitor SK&F 86002, administered per os (100 mg/kg), 1 hour prior to LPS challenge, strongly inhibited the rise in serum levels of the four cytokines. Furthermore, treatment with the IL-1 beta converting enzyme (ICE) specific reversible inhibitor YVAD-CHO resulted in a sharp dose- and time-dependent inhibition of IL-1 beta secretion in the serum, whereas the other cytokines were not affected. In conclusion, IFN-gamma priming strongly potentiates the release of proinflammatory cytokines in the serum of mice as compared to LPS stimulation alone, and provides therefore a useful way to test the in vivo potency and selectivity of cytokine synthesis inhibitors.     

High mobility group box protein 1: an endogenous signal for dendritic cell maturation and Th1 polarization

High mobility group box protein 1 (HMGB1), a DNA binding nuclear and cytosolic protein, is a proinflammatory cytokine released by monocytes and macrophages. This study addressed the hypothesis that HMGB1 is an immunostimulatory signal that induces dendritic cell (DC) maturation. We show that HMGB1, via its B box domain, induced phenotypic maturation of DCs, as evidenced by increased CD83, CD54, CD80, CD40, CD58, and MHC class II expression and decreased CD206 expression. The B box caused increased secretion of the proinflammatory cytokines IL-12, IL-6, IL-1alpha, IL-8, TNF-alpha, and RANTES. B box up-regulated CD83 expression as well as IL-6 secretion via a p38 MAPK-dependent pathway. In the MLR, B box-activated DCs acted as potent stimulators of allogeneic T cells, and the magnitude of the response was equivalent to DCs activated by exposure to LPS, nonmethylated CpG oligonucleotides, or CD40L. Furthermore, B box induced secretion of IL-12 from DCs as well as IL-2 and IFN-gamma secretion from allogeneic T cells, suggesting a Th1 bias. HMGB1 released by necrotic cells may be a signal of tissue or cellular injury that, when sensed by DCs, induces and/or enhances an immune reaction.