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Biomedical subjects

F Granucci

Publications and source records attributed to F Granucci.

At least 19 recordsLinked to original sources
PubMed2001

Autoreactive isotype-specific T cells determine B cell frequency.

Suppressive activities involving T-B and T-T cell interactions are important to maintain immune system homeostasis. Negative control of IgG2ab+ B cells by anti-IgG2ab T cells derived from Igha mice has been well documented. Nevertheless the real contribution of anti-IgG2ab T cells, endogenously matured in Ighb mice, in controlling IgG2ab+ B cell function has never been investigated. We previously generated anti-IgG2ab TCR-transgenic mice and showed that transgenic T cells were not deleted in the thymus and that they were responsible for a complete and chronic IgG2ab suppression. Here we show that T cells expressing high density of anti-IgG2ab TCR were positively selected in the thymus with a higher efficiency in animals expressing IgG2ab, reached peripheral lymphoid organs and negatively controlled IgG2ab serum levels. Moreover, anti-IgG2ab T cells transgenic for the single TCR chain, thus undergoing normal rearrangements and normal processes of selection, also reached the periphery and suppressed IgG2ab. Interestingly, concentration of IgG2ab in serum inversely correlated with the peripheral frequency of Ig-specific T cells. Finally, T cells able to suppress IgG2ab were obtained from Ighb non-transgenic mice, indicating that anti-2ab T cells are naturally present in the periphery of Ighb animals. We propose that IgG2ab-specific T cells contribute to determine IgG2ab serum levels in Ighb mice.

Animals↗
PubMed2001

Transcriptional reprogramming of dendritic cells by differentiation stimuli.

Immature and mature dendritic cells (DC) have been well characterized functionally and phenotypically. Microorganisms or bacterial products such as lipopolysaccharide (LPS) and inflammatory molecules, including tumor necrosis factor (TNF-alpha), are both believed to activate the DC maturation program which allows DC to initiate and amplify innate and adaptive immune responses. However, there is increasing evidence that the functional state of DC, induced by different stimuli, may be relevant for the immune response outcome. Thus, we compared the transcriptional program of mature, transitional and immature DC, after either LPS or TNF-alpha stimulation. GeneChip oligonucleotide microarrays, representing approximately 6,500 murine genes and ESTs, were used for this analysis. A very diverse modulation of gene expression was observed with the two stimuli. Only LPS-treated cells showed a pattern of expression of genes compatible with a definitive growth arrest and with a suitable activation and control of the immune response.

Animals↗
PubMed2001

Analysis of the relationship between viral infection and autoimmune disease.

The clinical association between viral infection and onset or exacerbation of autoimmune disorders remains poorly understood. Here, we examine the relative roles of molecular mimicry and nonspecific inflammatory stimuli in progression from infection to autoimmune disease. Murine herpes virus 1 (HSV-1 KOS) infection triggers T cell-dependent autoimmune reactions to corneal tissue. We generated an HSV-1 KOS point mutant containing a single amino acid exchange within the putative mimicry epitope as well as mice expressing a TCR transgene specific for the self-peptide mimic to allow dissection of two pathogenic mechanisms in disease induction. These experiments indicate that viral mimicry is essential for disease induction after low-level viral infection of animals containing limited numbers of autoreactive T cells, while innate immune mechanisms become sufficient to provoke disease in animals containing relatively high numbers of autoreactive T cells.

Adoptive Transfer↗
PubMed2001

Differential activation of NF-kappa B subunits in dendritic cells in response to Gram-negative bacteria and to lipopolysaccharide.

Dendritic cell (DC) maturation is essential for the initiation of T-dependent immune responses. Nuclear factor kappa B/Rel (NF kappa B/Rel) transcription factors are ubiquitously expressed signalling molecules, known to regulate the transcription of a large number of genes involved in immune responses, including cytokines such as IL-1, IL-6, TNF-alpha and cell surface molecules (MHC class I and II, B7.2). In this study, we have compared the activation of five members of the NF-kappa B family, p65, c-Rel, p50, RelB and p52, during DC maturation in response to lipopolysaccharide (LPS) and to Salmonella typhimurium. We have shown that although the translocation of NF-kappa B occurred very early, 30 min after treatment with both S. typhimurium and LPS, bacteria-induced NF-kappa B activation was more pronounced. Four out of five members, i.e. p65, c-Rel, p50 and RelB, were similarly activated upon the two stimuli but with different kinetics. Indeed, we have observed that p65, c-Rel and p50 were translocated early, whereas RelB was translocated later in DC activation. This differential regulation suggests that the various members of NF-kappa B family can mediate distinct functions of DC physiology.

Animals↗
PubMed2001

Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria.

Penetration of the gut mucosa by pathogens expressing invasion genes is believed to occur mainly through specialized epithelial cells, called M cells, that are located in Peyer's patches. However, Salmonella typhimurium that are deficient in invasion genes encoded by Salmonella pathogenicity island 1 (SPI1) are still able to reach the spleen after oral administration. This suggests the existence of an alternative route for bacterial invasion, one that is independent of M cells. We report here a new mechanism for bacterial uptake in the mucosa tissues that is mediated by dendritic cells (DCs). DCs open the tight junctions between epithelial cells, send dendrites outside the epithelium and directly sample bacteria. In addition, because DCs express tight-junction proteins such as occludin, claudin 1 and zonula occludens 1, the integrity of the epithelial barrier is preserved.

Animals↗
PubMed2001

Inducible IL-2 production by dendritic cells revealed by global gene expression analysis.

Dendritic cells (DCs) are strong activators of primary T cell responses. Their priming ability is acquired upon encounter with maturation stimuli. To identify the genes that are differentially expressed upon maturation induced by exposure to Gram-negative bacteria, a kinetic study of DC gene expression was done with microarrays representing 11,000 genes and ESTs (expressed sequence tags). Approximately 3000 differentially expressed transcripts were identified. We found that functional interleukin 2 (IL-2) mRNA, which gave rise to IL-2 production, was transiently up-regulated at early time-points after bacterial encounter. In contrast, macrophages did not produce IL-2 upon bacterial stimulation. Thus, IL-2 is an additional key cytokine that confers unique T cell stimulatory capacity to DCs.

Animals↗
PubMed2001

Infection of dendritic cells by murine cytomegalovirus induces functional paralysis.

Cytomegalovirus (CMV), measles and HIV are the main human pathogens known to induce immunosuppression. Unlike measles and HIV, and despite the availability of a well studied animal model, little is known about the mechanisms that control CMV-induced immunosuppression. We hypothesized that dendritic cells (DCs), which are crucial in generating and maintaining immune responses, represent a target for CMV and that the transient, but profound, immunosuppression that accompanies CMV infection results from viral interference with DC functions. Here we show that DCs were permissive to murine CMV infection. In addition, DC infection prevented delivery of the signals required for T cell activation. Thus, CMV-mediated impairment of DC function may be crucial for virally induced immunosuppression and interleukin 2 is implicated as a key factor.

Animals↗
PubMed2001

Gene expression profiling in immune cells using microarray.

The recent development of DNA microarray, which offers the opportunity to study the expression of thousands of individual genes simultaneously in different biological systems, has provided new insights into the immune system. Examples discussed in this review include molecular descriptions of the differentiation program of T helper (Th) cells into Th1 and Th2 pathways and the genetic program underlying maturation of dendritic cells. It is anticipated that this new information can be used to understand gene function in both physiological and pathological conditions of the immune system.

Cell Differentiation↗
PubMed2000

Molecular events of bacterial-induced maturation of dendritic cells.

In order to protect the body from infectious microorganisms, mammals have developed powerful lines of defense, consisting in innate and adaptive immune responses. The innate response is phylogenetically more ancient and, for a long time, it has been considered to be broadly directed to microorganisms. However, the discovery of a new class of receptors, involved in recognition of patterns characteristic of groups of microorganisms (the toll-like receptor family) has re-evaluated the role of the innate immune system as a discriminating system. Indeed, there is increasing evidence that the induction of different types of effector adaptive responses are directed by the innate immune system after recognition of particular groups of pathogens. The central role of Dendritic cells (DC) in the induction of adaptive immune responses towards infectious agents has been extensively described, but, recently, a new role of DC as a link between the non-antigen- and the antigen-specific responses has been proposed. DC have, indeed, the capacity to recruit and activate cells of the innate immune system upon inflammation. Thus. understanding the interaction of bacteria with DC, and the early molecular events resulting from this interaction may shed some light on the mechanisms of initiation of the immune response to infectious agents and on aspects of invasiveness, pathogenicity, and the persistence of certain bacteria.

Animals↗
PubMed1999

Microglia induce myelin basic protein-specific T cell anergy or T cell activation, according to their state of activation.

Microglial cells are non-professional antigen-presenting cells (APC) the function of which is still controversial. Here, we studied the function of microglia derived from H-2(u) mice. We show that these microglia express a low level of B7.2 and CD40 and, interestingly, lack surface expression of B7.1. Resting and IFN-gamma-activated microglia were unable to activate naive and primed myelin basic protein (MBP)-specific CD4(+) T cells in the presence of MBP and encephalomyelitic MBP Ac1-11 peptide. Furthermore, in the presence of Ac1-11 peptide, CD4(+) TCR-transgenic T cells became anergized. Microglia became professional APC only after a multistep activation process involving both stimulation through cytokines [granulocyte-macrophage colony-stimulating factor (GM-CSF) and IFN-gamma] and cognate signaling (B7-CD28 and CD40-CD40 ligand interactions). As such they were able to present MBP to both unprimed and primed T cells. Co-culture of microglia with GM-CSF up-regulated co-stimulatory molecules, in particular B7.1. Additional activation with IFN-gamma induced MHC class II and CD40 up-regulation. CD40-CD40 ligand interaction significantly enhanced microglial ability to prime TCR-transgenic T cells and was essential for presentation of MBP to in vivo primed non-transgenic T cells. We propose that microglia may serve different functions under different inflammatory conditions, depending on the cytokine milieu and the type of cognate interaction they are involved in.

Animals↗
PubMed1999

Retroviral gene transfer, rapid selection, and maintenance of the immature phenotype in mouse dendritic cells.

We used the retroviral vector PINCO [which expresses the green fluorescent protein (GFP) as a selectable marker], to infect growth factor-dependent immature D1 dendritic cells (DC). The efficiency of infection in different experiments was between 5 and 30%, but subsequent cell sorting led to a virtually homogeneous population of GFP-positive cells. Retroviral infection did not modify the immature DC phenotype, as shown by the low expression of major histocompatibility complex and co-stimulatory molecules. Furthermore, the GFP-positive D1 cells underwent full maturation after lipopolysaccharide treatment, as indicated by a high expression of cell-surface MHC and co-stimulatory molecules, and also by strong stimulatory activity in allogeneic mixed lymphocyte reaction. The high efficiency of this retroviral system, the rapidity of the technique, and the possibility to overcome in vitro selection make this method very attractive for the stable introduction of heterologous genes into proliferating immature mouse D1 cells. Furthermore, this approach is suitable for functional studies of new DC-specific genes involved in DC maturation and survival.

Animals↗
PubMed1999

Dendritic cells as natural adjuvants.

Dendritic cells (DCs) are professional antigen presenting cells that hold the key to the induction of T-cell responses. Therefore, the use of DCs for immunotherapy to stimulate immune responses has recently raised a great deal of interest. Many clinical trials using DCs have been initiated to stimulate immune responses against tumors or infectious agents. Several issues need to be considered before DCs can be used successfully as natural adjuvants: DCs have to be generated in sufficient numbers; they should display morphological, phenotypical, and functional properties of DCs; and they should be able to present antigens. In the present review we focus on methods for the purification of DCs from human bone marrow and peripheral blood and for the optimization of in vitro cell culture systems. Methods to generate growth factor-dependent mouse DC lines are also described.

Adjuvants, Immunologic↗
PubMed1999

Early events in dendritic cell maturation induced by LPS.

Immature dendritic cells (Dcs) are characterised by high antigen uptake ability and poor T-cell stimulatory function. In contrast, mature DCs have a high stimulatory function and poor antigen uptake ability. Inflammatory stimuli induce DC maturation and migration from nonlymphoid tissues to lymphoid organs. We investigated the effect of lipopolysaccharide (LPS) on DC antigen uptake and migratory function at early and late stimulation time points. We observed that the transition from the immature to the mature state is not a progressive itinerary, but it is characterised by precise functional stages. At early time points after LPS stimulation DCs significantly decrease their intrinsic migratory ability and increase the antigen uptake function. Later, around 4 h after LPS activation, DCs show recovery of migratory ability and start to progressively lose their antigen uptake function until the mature stage in which they show poor antigen uptake and migratory activity.

Animals↗
PubMed1999

Dendritic cells at the end of the millennium.

We have recently proposed a dual role for dendritic cells (DC) in the amplification of innate immune responses and in the activation of adaptive immune responses. The DC are localized along the major routes of entry of micro-organisms, where they perform a sentinel function for incoming pathogens. Soon after interaction with appropriate stimuli, DC undergo a coordinated process of maturation and respond to danger signals by re- programming their functions. The DC first regulate leucocyte recruitment at the site of inflammation, through the production of chemokines, inflammatory cytokines and interferons, and then they acquire migratory properties and undergo a rapid switch in chemokine receptor expression. This allows them to leave the inflamed tissue and to reach the lymph node T cell area. During this migration, DC complete their maturation process and acquire the ability to prime T cell responses. Thus, DC bridge innate and adaptive immunity.

Animals↗
PubMed1999

Upon dendritic cell (DC) activation chemokines and chemokine receptor expression are rapidly regulated for recruitment and maintenance of DC at the inflammatory site.

Dendritic cells (DC) are highly motile antigen-presenting cells that are recruited to sites of infection and inflammation to antigen uptake and processing. Then, to initiate T cell-dependent immune responses, they migrate from non-lymphoid organs to lymph nodes and the spleen. Since chemokines have been involved in human DC recruitment, we investigated the role of chemokines on mouse DC migration using the mouse growth factor-dependent immature DC line (D1). In this study, we characterized receptor expression, responsiveness to chemoattractants and chemokine expression of D1 cells during the maturation process induced by lipopolysaccharide (LPS). MIP-1alpha and MIP-5 were found to be the most effective chemoattractants, CCR1 was the main receptor expressed and modulated during LPS treatment, and MIP-2, RANTES, IP-10 and MCP-1 were the chemokines modulated during DC maturation. Thus, murine DC respond to a unique set of CC and CXC chemokines, and the maturational stage determines the program of chemokine receptors and chemokines that are expressed. Since CCR1 is modulated during the early phases of DC maturation, our results indicate that the CCR1 receptor may participate in the recruitment and maintenance of DC at the inflammatory site.

Animals↗
PubMed1998

Rabbit monoclonal Fab derived from a phage display library.

Rabbit monoclonal antibodies (RmAb) are not routinely obtained by eukaryotic cell fusion techniques. Therefore, we have applied phage display technology to produce a recombinant rabbit Fab molecule directed against the KLH model antigen. The Fab fragments selected from the rabbit phage display library were subcloned in an expression vector to permit the production of a fusion protein comprising a dimer of bacterial alkaline phosphatase (phoA). This fusion protein was directly produced into the periplasmic space of Escherichia coli. We show that a crude extract containing these conjugates can be used in a direct enzyme immunoassay, as exemplified in the case of the KLH antigen.

Alkaline Phosphatase↗
PubMed1998

Molecular mimicry by herpes simplex virus-type 1: autoimmune disease after viral infection.

Viral infection is sometimes associated with the initiation or exacerbation of autoimmune disease, although the underlying mechanisms remain unclear. One proposed mechanism is that viral determinants that mimic host antigens trigger self-reactive T cell clones to destroy host tissue. An epitope expressed by a coat protein of herpes simplex virus-type 1 (HSV-1) KOS strain has now been shown to be recognized by autoreactive T cells that target corneal antigens in a murine model of autoimmune herpes stromal keratitis. Mutant HSV-1 viruses that lacked this epitope did not induce autoimmune disease. Thus, expression of molecular mimics can influence the development of autoimmune disease after viral infection.

Adoptive Transfer↗