Patrick Stordeur

Deficient IL-12(p35) Gene Expression by Dendritic Cells Derived from Neonatal Monocytes

To gain insight into the defects responsible for impaired Th1 responses in human newborns, we analyzed the production of cytokines by dendritic cells (DC) derived from cord blood monocytes. We observed that neonatal DC generated from adherent cord blood mononuclear cells cultured for 6 days in the presence of IL-4 and GM-CSF show a phenotype similar to adult DC generated from adherent PBMC, although they express lower levels of HLA-DR, CD80, and CD40. Measurement of cytokine levels produced by neonatal DC upon stimulation by LPS, CD40 ligation, or poly(I:C) indicated a selective defect in the synthesis of IL-12. Determination of IL-12(p40) and IL-12(p35) mRNA levels by real-time RT-PCR revealed that IL-12(p35) gene expression is highly repressed in stimulated neonatal DC whereas their IL-12(p40) gene expression is not altered. The addition of rIFN-γ to LPS-stimulated newborn DC restored their expression of IL-12(p35) and their synthesis of IL-12 (p70) up to adult levels. Moreover, we observed that neonatal DC are less efficient than adult DC to induce IFN-γ production by allogenic adult CD4+ T cells. This defect was corrected by the addition of rIL-12. We conclude that neonatal DC are characterized by a severe defect in IL-12(p35) gene expression which is responsible for an impaired ability to elicit IFN-γ production by T cells.

ChemR23, a putative chemoattractant receptor, is expressed in monocyte‐derived dendritic cells and macrophages and is a coreceptor for SIV and some primary HIV‐1 strains

Leukocyte chemoattractants act through a rapidly growing subfamily of G protein‐coupled receptors. We report the cloning of a novel human gene encoding an orphan receptor (ChemR23) related to the C3a, C5a and formyl Met‐Leu‐Phe receptors, and more distantly to the subfamilies of chemokine receptors. ChemR23 transcripts were found to be abundant in monocyte‐derived dendritic cells and macrophages, treated or not with LPS. Low expression could also be detected by reverse transcription‐PCR in CD4+ T lymphocytes. The gene encoding ChemR23 was assigned by radiation hybrid mapping to the q21.2 – 21.3 region of human chromosome 12, outside the gene clusters identified so far for chemoattractant receptors. Given the increasing number of chemoattractant receptors used by HIV‐1, HIV‐2 and SIV as coreceptors, ChemR23 was tested in fusion assays for potential coreceptor activity by a range of viral strains. None of the tested HIV‐2 strains made use of ChemR23 as a coreceptor, but several SIV strains (SIVmac316, SIVmac239, SIVmac17E‐Fr and SIVsm62A), as well as a primary HIV‐1 strain (92UG024‐2) used it efficiently. ChemR23 therefore appears as a coreceptor for immunodeficiency viruses that does not belong to the chemokine receptor family. It is also a putative chemoattractant receptor relatively specific for antigen‐presenting cells, and it could play an important role in the recruitment or trafficking of these cell populations. Future work will be required to identify the ligand(s) of this new G protein‐coupled receptor and to define its precise role in the physiology of dendritic cells and macrophages.

Cytokine mRNA quantification by real-time PCR

Real-time PCR represents a new methodology that accurately quantifies nucleic acids. This has been made possible by the use of fluorogenic probes, which are presented in two forms, namely hydrolysis probes (also called TaqMan probes) and hybridisation probes. We decided to apply this methodology to cytokine mRNA quantification and this led us to the development of a protocol that provides an easy way to develop and perform rapidly real-time PCR on a Lightcycler instrument. It was made possible by the use of freely available software that permits a choice of both the hydrolysis probe and the primers. We firstly demonstrated that the reproducibility of the method using hydrolysis probes compares favourably with that obtained with hybridisation probes. We then applied this technique to determine the kinetics of IL-1ra, IL-1beta, IL-5, IL-13, TNF-alpha and IFN-gamma induction upon stimulation of human peripheral blood mononuclear cells (PBMC) by phytohaemagglutinin (PHA). Finally, the method was also used successfully to demonstrate that IFN-alpha induces IL-10 mRNA accumulation in human monocytes.

Recombinant interferon-alpha selectively inhibits the production of interleukin-5 by human CD4+ T cells.

The effects of recombinant IFN-alpha on the production of IL-5 by human CD4+ T cells were first analyzed on resting CD4+ T cells purified from normal PBMC and stimulated either with a combination of PMA and anti-CD28 mAb or anti-CD3 mAb cross-linked on B7-1/CD32-transfected mouse fibroblasts. We found that IFN-alpha profoundly inhibited in a dose-dependent manner IL-5 production by resting CD4+ T cells whereas IL-10 was upregulated in both systems. The addition of a neutralizing anti-IL-10 mAb to PMA and anti-CD28 mAb upregulated IL-5 production by resting CD4+ T cells but did not prevent IFN-alpha-induced IL-5 inhibition. We then analyzed the effect of IFN-alpha on the production of cytokines by differentiated type 2 helper (Th2) CD4+CD3- cells isolated from peripheral blood of two patients with the hypereosinophilic syndrome. In both cases, IFN-alpha markedly inhibited IL-5 production while it induced mild upregulation of IL-4 and IL-10. Finally, the inhibitory effect of IFN-alpha on IL-5 production was confirmed on a panel of Th2 and Th0 clones generated in vitro. In 2 out of 6 clones, IL-5 inhibition was associated with upregulation of IL-4 and IL-10. We conclude that IFN-alpha selectively downregulates IL-5 synthesis by human CD4+ T cells.

Induction of FOXP3-expressing regulatory CD4pos T cells by human mature autologous dendritic cells

Current literature suggests that T cells recognizing antigen on mature dendritic cells (DC) differentiate into effector T cells whereas tolerance is induced when antigen is presented by immature DC. We investigated the consequences of the interactions between immature or lipopolysaccharide‐matured DC and CD4pos T lymphocytes in absence of foreign antigen. While immature DC did not induce significant CD4pos T cell activation, we observed that a significant fraction of CD4pos T cells cultured with mature autologous DC displayed phenotypic features of activation and produced IL‐2, IFN‐γ, IL‐10 and TGF‐β. Furthermore, CD4pos T lymphocytes primed by mature, but not immature, autologous DC acquired regulatory properties. Indeed, when added to an allogeneic mixed leukocyte reaction, they suppressed the response of alloreactive T lymphocytes to the priming DC while responses to third‐party stimulators were spared. The generation of CD4pos T cells with regulatory function by autologous stimulation did not require the presence of natural CD4posCD25pos regulatory T cells. In addition, the acquisition ofregulatory function by CD4posCD25neg T cells stimulated by autologous mature DC was accompanied by the induction of FOXP3 expression. Our data suggest that during inflammatory conditions, presentation of self antigens by mature DC to autologous T lymphocytes could contribute to the generation of regulatory mechanisms.

Inflammation modifies the pattern and the function of Toll-like receptors expressed by human mesenchymal stromal cells.

Mesenchymal stromal cells (MSC) are involved in tissue repair and in the regulation of immune responses. MSC express Toll-like receptors (TLR) known to link innate and adaptive immunity. We hypothesized that TLR signaling could influence human MSC (hMSC) function. Here, we show that hMSC express TLR1, TLR2, TLR3, TLR4, TLR5, and TLR6 but not TLR7, TLR8, TLR9, and TLR10. In inflammatory conditions mimicked by culturing hMSC in an inflammatory environment, TLR2, TLR3, and TLR4 are upregulated, whereas TLR6 is downregulated. Interleukin (IL)-1 beta, IL-6, IL-12p35 and transforming growth factor-beta mRNAs are constitutively expressed by hMSC. Inflammation leads to an increase in IL-1 beta, IL-6, IL-12p35, and transforming growth factor-beta transcription and is characterized by IL-23p19 and IL-27p28 transcription. In this setting, poly(I:C) further augments IL-6, IL-12p35, IL-23p19, and IL-27p28 transcription, whereas lipopolysaccharide (LPS) increases IL-23p19 and IL-27p28 transcription. By upregulating TLR3 and TLR4 transcription, inflammation increases the hMSC responsiveness to LPS and poly(I:C), leading to a proinflammatory shift in their cytokine profile. The hMSC osteogenic potential does not change after TLR triggering but stimulation with LPS and poly(I:C) results in a decrease in their immunosuppressive capabilities. In conclusion, TLR activation in hMSC may affect their function and could modify their in vivo fate, especially in an inflammatory context.

Extracellular adenine nucleotides modulate cytokine production by human monocyte‐derived dendritic cells: dual effect on IL‐12 and stimulation of IL‐10

To clarify the functional consequences of adenine nucleotides action on human monocyte‐derived dendritic cells (DC), we have systematically compared the effects of adenosine 5′‐O‐(3‐thiotriphosphate) (ATPγS), an ATP analog active on the P2Y11 receptor, on the responses to three DC stimuli, TNF‐α, LPS, sCD40L, tested at various concentrations, using two different IL‐12 assays. We observed that ATPγS potentiated the IL‐12p40 release induced by TNF‐α, but also by lipopolysaccharides (LPS) and soluble CD40 ligand (sCD40L). This potentiation was observed as long as the IL‐12p40 concentration under agonist stimulation remained below a threshold value close to 10 ng/ml; inhibition was observed above this value. The combinations ATPγS‐TNF‐α and ATPγS‐sCD40L were unable to induce detectable bioactive IL‐12p70 production and at concentrations of LPS that induced a significant stimulation of IL‐12p70, the effect of ATPγS was purely inhibitory. Our results also show that ATPγS synergized with LPS and sCD40L, but not TNF‐α, to stimulate IL‐10 production. In conclusion, we have clarified the discrepancies in the literature concerningthe action of adenine nucleotides on DC and our study supports the concept that, like prostaglandin E2 and other agents increasing cyclic AMP, they favor either a Th2 response or tolerance.

Interleukin-10 as a Regulatory Cytokine Induced by Cellular Stress: Molecular Aspects

Interleukin-10 is an ubiquitous cytokine which plays a major regulatory role in the course of inflammatory responses by downregulating the synthesis of cytokines. In this paper, we summarize the major biological properties of IL-10 and the current knowledge of the molecular mechanisms by which IL-10 inhibits the expression of genes encoding proinflammatory cytokines. We then review the factors upregulating IL-10 synthesis and we present the concept that IL-10 is a stress cytokine produced not only in response to microbial pathogens but also to cellular injuries of diverse origins.

Predominance of type 1 CD4+T cells in human abdominal aortic aneurysm

The functional repertoire of T cells in abdominal aortic aneurysm (AAA) and the exact nature of aortic wall adaptive cellular immune responses still remains a matter of debate. In this study, we sought to determine whether type 1 or type 2 responses occur predominantly in human aneurysmal aortic lesions. We first examined the phenotype and cytokine secretion profile of T lymphocytes freshly isolated from aneurysmal aortic wall for comparison with their circulating counterparts using flow cytometry. We found that both populations of infiltrating CD4+ and CD8+T cells displayed a unique activated memory phenotype. In addition, we identified the presence in human aneurysmal aortic lesion of CD4+T cells producing high levels of interferon (IFN)‐γ but not interleukin (IL)‐4, reflecting their type 1 nature. Quantitative analysis of cytokine gene expression confirmed increased IFN‐γ transcript levels in infiltrating cells compared to controls. We next analysed aortic wall responses using LightCycler‐based quantitative real‐time reverse transcription‐polymerase chain reaction. Compared to control non‐diseased aortic samples, we demonstrated that whole AAA tissues exhibited high mRNA levels of IFN‐γ but not IL‐4. Overexpression of the transcription factor T‐bet in the absence of significant GATA‐3 expression further assessed the type 1 polarization of aortic wall immune responses. These findings indicate that type 1 CD4+T cells predominate in human AAA lesions. This study has important implications for the pathogenesis of aneurysm disease. Through the production of IFN‐γ, T cells may indeed contribute to orchestrate extracellular matrix remodelling.

Immune monitoring in whole blood using real-time PCR

There is a need for simple and sensitive assays to assess innate and adaptive immune responses to microbial agents and vaccines. Herein, we describe a whole blood method allowing to measure the induction of cytokine synthesis at the mRNA level. The originality of this method consists in the combination of PAXgene tubes containing an mRNA stabilizer for blood collection, the MagNA Pure instrument as an automated system for mRNA extraction and RT-PCR reagent mix preparation, and the real-time PCR methodology on the Lightcycler for accurate and reproducible quantification of transcript levels. We first demonstrated that this method is adequate to measure the induction of interleukin (IL)-1beta and IL-1 receptor antagonist (IL-1 RA) mRNA upon the addition of bacterial lipopolysaccharide (LPS) to whole blood. We then showed that this approach is also suitable to detect the production of mRNA encoding T cell-derived cytokines in whole blood incubated with tetanus toxoid as a model of in vitro immune response to a recall antigen. Finally, we demonstrated that this methodology can be used successfully to assess inflammatory as well as T cell responses in vivo, as it allowed to detect the induction of IL-1beta and IL-1 RA after injection of LPS in healthy volunteers, and also the induction of IL-2 upon recall immunisation with tetanus vaccine.