Histamine induces CD86 expression and chemokine production by human immature dendritic cells.

Mast cells and immature dendritic cells (DC) are in close contact in peripheral tissues. Upon activation, mast cells release histamine, a mediator involved in the immediate hypersensitivity reaction. We therefore tested whether histamine could affect human DC activation and maturation. Histamine induces CD86 expression on immature DC in a dose-dependent (significant at 10(-7) M) and transient manner (maximal after 24-h stimulation). Histamine also transiently up-regulates the expression of the costimulatory and accessory molecules, CD40, CD49d, CD54, CD80, and MHC class II. As a consequence, immature DC exposed for 24 h to histamine stimulate memory T cells more efficiently than untreated DC. In addition, histamine induces a potent production of IL-6, IL-8, monocyte chemoattractant protein 1, and macrophage-inflammatory protein 1alpha by immature DC and also up-regulates IL-1beta, RANTES, and macrophage-inflammatory protein 1beta but not TNF-alpha and IL-12 mRNA expression. Histamine activates immature DC through both the H1 and H2 receptors. However, histamine-treated DC do not have a phenotype of fully mature cells, as they do neither show significant changes in the expression of the chemokine receptors, CCR5, CCR7 and CXC chemokine receptor 4, nor expression of CD83 de novo. These data demonstrate that histamine activates immature DC and induces chemokine production, thereby suggesting that histamine, via stimulation of resident DC, may participate locally in T cell stimulation and in the late inflammatory reaction associated with allergic disorders.

Soluble CD86 is a costimulatory molecule for human T lymphocytes.

CD86 is an important costimulatory molecule for the priming and activation of naive and memory T cells, respectively. Here, we show that soluble CD86 is detected in human serum. Soluble CD86 is produced by resting monocytes and results from an alternatively spliced transcript (CD86deltaTM) characterized by deletion of the transmembrane domain. Recombinant CD86deltaTM binds to CD28 and CTLA-4 and induces the activation of T cells after stimulation with anti-CD3 mAb. CD86deltaTM also induces IFNgamma production by virus-specific CD8+ memory human T cells stimulated with the Flu M1 peptide. The concentrations of soluble CD86 found in human serum are sufficient to induce biological activity. Soluble CD86 molecule, therefore, appears to be a functional costimulatory molecule playing a potentially important role in immune surveillance.

OmpA targets dendritic cells, induces their maturation and delivers antigen into the MHC class I presentation pathway.

We analyzed the interaction between a bacterial cell wall protein and dendritic cells (DCs). Outer membrane protein A from Klebsiella pneumoniae (kpOmpA) specifically bound to professional antigen presenting cells and was endocytosed by immature DCs via a receptor-dependent mechanism. kpOmpA signaled through Toll-like receptor 2, induced DCs to produce interleukin 12 and induced maturation of DCs. Whole antigen that was coupled to kpOmpA and injected into mice was taken up by DCs and delivered to the conventional cytosolic MHC class I presentation pathway. kpOmpA also primed antigen-specific CD8+ CTLs in the absence of CD4+ T cell help or adjuvant and elicited therapeutic immunity to antigen-expressing tumors. Thus, OmpA belongs to a class of proteins that are able to elicit CTL responses to exogenous antigen.

Human NK cells constitutively express membrane TNF-alpha (mTNFalpha) and present mTNFalpha-dependent cytotoxic activity.

We have evaluated the expression and the involvement of membrane-associated TNF-alpha (mTNF-alpha) in human NK cell-mediated cytotoxicity. Results from FCM analysis show that peripheral blood NK cells constitutively express mTNF-alpha. In contrast, mTNF-alpha expression is undetectable on resting T cells, B cells and monocytes. Western blotting analysis confirmed that freshly purified NK cells express the 17-kDa soluble form (sTNF-alpha) and the 26-kDa transmembrane form of TNF-alpha. Stimulation with IL-2, IL-15 and IL-18 up-regulates TNF-alpha mRNA, sTNF-alpha and mTNF-alpha expression in NK cells. The role of mTNF-alpha in the cytotoxic activity of resting NK cells has been evaluated in in vitro cytotoxic assays using freshly purified NK cells fixed with paraformaldehyde as effector cells (in order to avoid the participation of cytotoxic soluble mediators such as perforin, granzymes or sTNF-alpha) and the TNF-alpha-sensitive Fas ligand- and TRAIL-resistant cell line KYM-1-D4 as target cell. Results show that fixed NK cells kill the KYM-1-D4 cells and that neutralizing anti-TNF-alpha antibodies partly prevent this effect. In contrast to the other types of peripheral blood mononuclear cells NK cells from adult blood constitutively express functional mTNF-alpha in the absence of prior contact with target cells or activation. These data demonstrate a novel mechanism of cell-mediated cytotoxicity by non-acitvated human peripheral blood NK cells.

A soluble form of CTLA-4 generated by alternative splicing is expressed by nonstimulated human T cells.

CTLA-4, expressed by activated T cells, transduces an inhibitory signal. We show here that PCR amplification of the coding sequence of CTLA-4 in nonstimulated human T lymphocytes results in the amplification of two transcripts of 650 and 550 bp. Sequencing shows that the larger form codes for membrane CTLA-4 and the 550-bp transcript is a spliced variant in which exon 2 coding for the transmembrane region is deleted. This spliced cDNA has been named CTLA-4delTM. The splicing induces a frame shift which results in the addition of 22 extra amino acids before a translational termination. Activation of T cells with phorbol 12-myristate 13-acetate plus ionomycin or anti-CD3 plus anti-CD28 monoclonal antibodies induces a suppression of CTLA-4delTM mRNA expression associated with a preferential expression of the membrane CTLA-4 mRNA, showing that CTLA-4delTM mRNA expression is restricted to nonactivated T cells. A soluble immunoreactive form of CTLA-4 was detected in the serum of 14 / 64 healthy subjects. These results suggest that nonstimulated T cells may constitutively produce a soluble form of CTLA-4 which may have an important role in the regulation of immune homeostasis.

Annexin V used for measuring apoptosis in the early events of cellular cytotoxicity.

BACKGROUND: Current cytotoxic assays, including Cr release and fluorescent assays, do not directly measure the proportion of target cells which are killed by apoptosis. Cell-mediated cytotoxicity induced by CTLs and NK cells is mainly regulated by the perforin-granzyme, the Fas ligand (Fas L), and the Tumor Necrosis Factor (TNF)-alpha pathways. Perforin generates pores in the membrane of target cells, allowing granzyme B to enter and initiate apoptosis. The other effectors, Fas L and TNF-alpha act by an apoptosis mechanism, leading to DNA fragmentation. A three color flow cytometric method to measure cell-mediated cytotoxicity induced by CTLs or NK cells is described. METHODS: The fluorochromes used are: PKH-26, a stable membrane dye for the labeling of the effector cells, annexin V-FITC which allows the direct evaluation of early apoptotic cells and propidium iodide which distinguishes membrane permeabilized and late apoptotic cells. RESULTS: By eliminating through gating PKH-26 positive effector cells, we obtain a direct estimation of the percentage of target cells in the early stages of apoptosis as well as the percentage of target cells dying after late apoptosis and membrane permeabilization. The cytotoxic activity of IL-2 stimulated PBL against K562, Jurkat and KYM-1 was evaluated. CONCLUSIONS: This rapid and novel assay permits the discrimination of the cell death mechanisms occurring during a cytotoxic response and to precisely evaluate the contribution of apoptosis in the early phases of cell-mediated cytotoxicity.

Vasoactive intestinal peptide synergizes with TNF-alpha in inducing human dendritic cell maturation.

We investigated the effects of different neuropeptides on human dendritic cells (DC) maturation. Immature DC, derived from monocytes cultured for 6 days with IL-4 plus GM-CSF, have been exposed to somatostatin, substance P, or vasoactive intestinal peptide (VIP). Among these neuropeptides, only VIP induces the production of bioactive IL-12 and the neoexpression of CD83 on a fraction of the DC population, with an effect significant at 100 and 10 nM, respectively. These effects of VIP are dose-dependent, unaffected by polymixin B, and partly prevented by a VIP receptor antagonist. Although the effects of VIP alone remain modest, it synergizes with TNF-alpha to induce DC maturation. In the presence of a suboptimal concentration of TNF-alpha, which has no detectable effect on DC by itself, VIP induces the production of high levels of bioactive IL-12, the neoexpression of CD83 on almost all the DC population (with an effect significant at 10 and 0.1 nM, respectively), and the up-regulation of various adhesion and costimulatory molecule expression. Moreover, DC exposed to VIP plus a suboptimal concentration of TNF-alpha are as potent as mature DC obtained by treatment with an optimal concentration of TNF-alpha in stimulating allogenic T cell proliferation. Our data suggest that, in inflammatory sites, VIP may cooperate with proinflammatory mediators, such as TNF-alpha, to induce DC maturation.

Human effector memory T cells express CD86: a functional role in naive T cell priming.

The glycoprotein CD86 expressed on APCs provides a costimulatory signal necessary for an efficient activation of naive T cells. In contrast, there is controversy about the condition of expression and the function of CD86 on T cells. In this study, we have analyzed the phenotype and the biological activity of CD86+ T cells generated from human PBMC. Results show that CD86 expression on T cells is induced by long term stimulation via CD3 and IL-2R and is down-regulated as the cells become quiescent. The CD86-expressing cells are memory effector T cells: 1) they express CD45RO and high levels of the activation markers CD25, CD54, and HLA-Dr; 2) they selectively express CD30, CD40-ligand, and CD70; and 3) in response to stimulation, most of them produce IFN-gamma before dying by apoptosis. We then analyzed whether CD86 expressed on T cells is functional. Results show that paraformaldehyde-fixed CD86+ T cells enhance the proliferation and production of IFN-gamma by anti-CD3 mAb-stimulated naive T cells and induce proliferation of resting allogenic T cells. All these effects are prevented by neutralizing anti-CD86 mAbs. In contrast, we report no autocrine effect of CD86 in CD86+ T cell activation. In conclusion, these data show that human memory effector T cells express a functional form of CD86 that can costimulate naive T cell responses.