Franca Gerosa

Reciprocal activating interaction between natural killer cells and dendritic cells

We analyzed the interaction between human peripheral blood natural killer (NK) cells and monocyte-derived immature dendritic cells (DC). Fresh NK cells were activated, as indicated by the induced expression of the CD69 antigen, and their cytolytic activity was strongly augmented by contact with lipopolysaccharide (LPS)-treated mature DC, or with immature DC in the presence of the maturation stimuli LPS, Mycobacterium tuberculosis or interferon (IFN)-α. Reciprocally, fresh NK cells cultured with immature DC in the presence of the maturation stimuli strongly enhanced DC maturation and interleukin (IL)-12 production. IL-2–activated NK cells directly induced maturation of DC and enhanced their ability to stimulate allogeneic naive CD4+ T cells. The effects of NK cells were cell contact dependent, although the secretion of IFN-γ and TNF also contributed to DC maturation. Within peripheral blood lymphocytes the reciprocal activating interaction with DC was restricted to NK cells, because the other lymphocyte subsets were neither induced to express CD69, nor induced to mature in contact with DC. These data demonstrated for the first time a bidirectional cross talk between NK cells and DC, in which NK cells activated by IL-2 or by mature DC induce DC maturation.

The Reciprocal Interaction of NK Cells with Plasmacytoid or Myeloid Dendritic Cells Profoundly Affects Innate Resistance Functions

A reciprocal activating interaction between NK cells and dendritic cells (DC) has been suggested to play a role in the functional regulation of these cells in immunity, but it has been studied only using in vitro generated bone marrow- or monocyte-derived DC. We report that human peripheral blood plasmacytoid DC (pDC) and myeloid DC are necessary to induce NK cell function depending on the type of microbial stimulus. pDC and myeloid DC are required for strongly increased NK cytolytic activity and CD69 expression, in response to inactivated influenza virus or CpG-containing oligonucleotides and poly(I:C), respectively. Secreted type I IFN is required and sufficient for the augmentation of NK cell cytolytic activity in the coculture with pDC or myeloid DC, whereas CD69 expression is dependent on both type I IFN and TNF. In addition, in response to poly(I:C), myeloid DC induce NK cells to produce IFN-γ through a mechanism dependent on both IL-12 secretion and cell contact between NK cells and myeloid DC, but independent of type I IFN. IL-2-activated NK cells have little to no cytolytic activity for immature myeloid DC and pDC, but are able to induce maturation of these cells. Moreover, IL-2-activated NK cells induce, in the presence of a suboptimal concentration of CpG-containing oligonucleotides, a strong IFN-α and TNF production. These data suggest that the reciprocal functional interaction between NK cells and either pDC or myeloid DC may play an important physiological role in the regulation of both innate resistance and adaptive immunity to infections.

Differential regulation of interleukin 12 and interleukin 23 production in human dendritic cells

We analyzed interleukin (IL) 12 and IL-23 production by monocyte-derived dendritic cells (mono-DCs). Mycobacterium tuberculosis H37Rv and zymosan preferentially induced IL-23. IL-23 but not IL-12 was efficiently induced by the combination of nucleotide-binding oligodimerization domain and Toll-like receptor (TLR) 2 ligands, which mimics activation by M. tuberculosis, or by the human dectin-1 ligand β-glucan alone or in combination with TLR2 ligands, mimicking induction by zymosan. TLR2 ligands inhibited IL-12 and increased IL-23 production. DC priming with interferon (IFN) γ strongly increased IL-12 production, but was not required for IL-23 production and inhibited IL-23 production induced by β-glucan. The pattern of IL-12 and IL-23 induction was reflected in accumulation of the IL-12p35 and IL-23p19 transcripts, respectively, but not IL-12/23p40. Although IL-23, transforming growth factor β, and IL-6 contained in the supernatants of activated mono-DCs played a role in the induction of IL-17 by human CD4+ T cells, IL-1β, in combination with one or more of those factors, was required for IL-17 production, and its production determined the differential ability of the stimuli used to elicit mono-DCs to produce soluble factors directing IL-17 production. Thus, the differential ability of pathogens to induce antigen-presenting cells to produce cytokines regulates the immune response to infection.

Regulation of interleukin-12/interleukin-23 production and the T-helper 17 response in humans.

Summary: Interleukin‐12 (IL‐12) and IL‐23 share a common chain. Yet, their production in response to pathogens is differentially regulated, and their functions are distinct and often antithetic. IL‐12 is involved in the induction or amplification of the T‐helper (Th) type 1 response, whereas IL‐23 has been associated with the generation of the Th17 response and IL‐17 production. Mycobacterium tuberculosis and yeast zymosan induce IL‐23, but in the absence of other stimuli, no IL‐12 is induced in human dendritic cells (DCs). The stimulation of IL‐23 by M. tuberculosis was mostly explained by the triggering of Toll‐like receptor (TLR2) and the cytoplasmic receptor nucleotide oligomerization domain (NOD)‐containing protein 2, whereas zymosan induces IL‐23 primarily by stimulating the β‐glucan receptor dectin‐1 alone or in combination with TLR2. IL‐23, IL‐6, transforming growth factor (TGF‐β1), and IL‐1β in supernatants from activated human DCs induce human naive CD4+ T cells to produce IL‐17. These data are consistent with various recent reports that TGF‐β is an inducer of IL‐17 production both in human and in mouse cells. However, IL‐1 is necessary in combination with some or all of the other cytokines to induce IL‐17 production in human T cells. The ability of various stimuli to induce Th17 cells depends not only on their induction of IL‐23, IL‐6, and TGF‐β production in DCs but also on their ability to activate directly or indirectly the inflammasome and to induce IL‐1β.

Biosynthesis and Posttranslational Regulation of Human IL-12

IL-12 is a heterodimeric proinflammatory cytokine consisting of a light α-chain, formerly defined as p35, disulfide-linked to a heavier β-chain, formerly defined as p40. The β-chain is also produced in large excess in a free form, and disulfide-linked β-chain homodimers with anti-inflammatory effects are produced in the mouse. We analyzed the biosynthesis and glycosylation of IL-12 in human monocytes, and in a cell line stably transfected with IL-12 α and β genes (P5-0.1). The IL-12 heterodimer and free β-chain were immunoprecipitated from supernatants and cell lysates of metabolically labeled cells and resolved in SDS-PAGE. Whereas the β-chain showed similar pI pattern whether in the free form or associated in the heterodimer, either in the secreted or intracellular form, the α-chain in the secreted heterodimer was much more acidic than that present in the intracellular heterodimer. Deglycosylation experiments with neuraminidase and Endo-F combined with two-dimensional PAGE of single bands of the intracellular vs extracellular IL-12 heterodimer revealed that the α-chain was extensively modified with sialic acid adducts to N-linked oligosaccharides before secretion. N-glycosylation inhibition by tunicamycin (TM) did not alter free β-chain secretion, while preventing the IL-12 heterodimer assembling and secretion. Pulse-chase experiments indicated that IL-12 persists intracellularly for a long period as an immature heterodimer, and that glycosylation is the regulatory step that determines its secretion. β-chain disulfide-linked homodimers were observed in TM-treated P5-0.1 cells, but in neither TM-treated nor untreated monocytes.

Specific IgE antibodies in twenty-eight workers with diisocyanate-induced bronchial asthma

A specific IgE‐mediated response was evaluated in twenty‐eight workers exposed to TDI or MDI, with diagnosis of occupational asthma and positive to bronchial provocative challenge. The presence of anti‐diisocyanate IgE was observed in 27% of subjects exposed to TDI and 83% of those exposed to MDI, particularly in individuals who experienced an acute massive exposure. An immediate‐type response to bronchial provocative test was found in 66% of individuals with specific antibodies. Specific IgE are prevalent (91%) in subjects who developed symptoms before 6 years of exposure to isocyanates. The results suggest an association between the presence of specific IgE, early asthmatic symptoms and heavy episodic exposure.

Constitutive expression of CD69 in interspecies T-cell hybrids and locus assignment to human chromosome 12

In this study we describe the generation and characterization of interspecies somatic cell hybrids between human activated mature T cells and mouse BW5147 thymoma cells. A preferential segregation of human chromosomes was observed in the hybrids. Phenotypic analysis of two hybrids and their clones demonstrated coexpression of CD4 and CD69 antigens in the same cells. Segregation analysis of an informative family of hybrids followed by molecular and karyotype studies clearly demonstrated that the locus encoding CD69 antigen mapped to human chromosome 12. Although the expression of CD69 antigen is an early event after T-lymphocyte activation and rapidly declines in absence of exogenous stimuli, in the hybrids described in this study the expression was constitutive, similarly to what was previously found in early thymocyte precursors and mature thymocytes. In this respect it was important to note that the behavior of the hybrids in culture strongly suggested a dominant influence of the thymus-derived mouse tumor cell genome in controlling the constitutive expression of human CD69. These hybrids may thus provide a system to study the genetic and molecular mechanisms controlling the expression and function of this activation antigen.

Acute induction and priming for cytokine production in lymphocytes

When T-lymphocytes (CD4+, CD8+, or TCR gamma delta +) and NK cells proliferate in vivo or in vitro in response to exposure to antigen or other stimuli, they often segregate into subsets with the ability to produce either type-1 [interferon-gamma (IFN-gamma) and interleukin-2 (IL-2)] or type-2 cytokines (IL-4, IL-5, IL-6 and IL-10). IL-12 induces the differentiation of type-1 cytokine-producing T-cells primarily through its ability to prime them for high IFN-gamma production; however, paradoxically IL-12 also primes T-cells for high production of the type-2 cytokine IL-10. Priming of T-cells for IL-4 production requires the presence of IL-4, but it is maximally observed in cultures containing both IL-4 and IL-12. IL-12, in addition to priming T-cells for high IFN-gamma and IL-10 production, is also a potent acute inducer of expression of the IFN-gamma gene in T- and NK-cells, and, to a much lower extent, of the IL-10 gene. IL-4, which has a very powerful effect in priming T-cells for IL-4 production, does not appear to have a significant ability to directly activate the expression of the IL-4 gene. Thus, IL-12 and IL-4 affect the expression of type-1 and type-2 cytokine genes by two different mechanisms: an acute induction of gene expression which is rapid and reversible, and a priming of the genes to a highly responsive state to restimulation, a state that is stable and probably irreversible.

IFN-γ and R-848 Dependent Activation of Human Monocyte-Derived Dendritic Cells by Neisseria meningitidis Adhesin A

A soluble recombinant form of Neisseria meningitidis adhesin A (NadAΔ351–405), proposed as a constituent of anti-meningococcal B vaccines, is here shown to specifically interact with and immune-modulate human monocyte-derived dendritic cells (mo-DCs). After priming with IFN-γ and stimulation with NadAΔ351–405, mo-DCs strongly up-regulated maturation markers CD83, CD86, CD80, and HLA-DR, secreted moderate quantities of TNF-α, IL-6, and IL-8, and produced a slight, although significant, amount of IL-12p70. Costimulation of mo-DCs with NadAΔ351–405 and the imidoazoquinoline drug R-848, believed to mimic bacterial RNA, increased CD86 in an additive way, but strongly synergized the secretion of IL-12p70, IL-1, IL-6, TNF-α, and MIP-1α, especially after IFN-γ priming. CD86/CD80 overexpression correlated with the occupation of high-(kd ∼ 80 nM) and low-(kd ∼ 4 μM) affinity binding sites for NadAΔ351–405. Alternatively, secretion of IL-12p70 and TNF-α, IL-6, and IL-8 corresponded to the occupation of high- or low-affinity receptors, respectively. Mo-DCs matured by IFN-γ and NadAΔ351–405 supported the proliferation of naive CD4+ T lymphocytes, inducing the differentiation of both IFN-γ and IL-4 producing phenotypes. Our data show that NadA not only is a good immunogen but is as well endowed with a proimmune, self-adjuvating, activity.

IFN-γ 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‐1α (MIP‐1α) and CCL4/MIP‐1β levels. In CD4+ and CD8+ clones from two patients deficient for IL‐12Rβ1 (IL‐12Rβ1−/−), production of CCL3/MIP‐1α and CCL4/MIP‐1β was defective. CD4+ clones from two patients deficient for interferon‐γ (IFN‐γ) R1 (IFN‐γR1−/−) produced somewhat decreased CCL4/MIP‐1β 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‐12Rβ1−/− and IFN‐γR1−/− 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‐γ increased CCR5 expression and decreased CC‐chemokine secretion by CD4+ clones from healthy and IL‐12Rβ1−/− individuals, suggesting an IFN‐γ‐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‐γ receptor‐deficient patients and dissect the role of IL‐12 and IFN‐γ on inducing inflammatory chemokine secretion and down‐regulating CCR5 expression in human T cells.