Showing papers by "Silvano Sozzani published in 2001"

Cutting Edge: Selective Usage of Chemokine Receptors by Plasmacytoid Dendritic Cells

Abstract: The existence of dendritic cell (DC) subsets is firmly established, but their trafficking properties are virtually unknown. In this study, we show that myeloid (M-DCs) and plasmacytoid (P-DCs) DCs isolated from human blood differ widely in the capacity to migrate to chemotactic stimuli. The pattern of chemokine receptors expressed by blood M-DCs and P-DCs, with the exception of CCR7, is similar. However, most chemokine receptors of P-DCs, in particular those specific for inflammatory chemokines and classical chemotactic agonists, are not functional in circulating cells. Following maturation induced by CD40 ligation, the receptors for inflammatory chemokines are down-regulated, and CCR7 on P-DCs becomes coupled to migration. The drastically impaired capacity of blood P-DCs to migrate in response to inflammatory chemotactic signals contrasts with the response to lymph node-homing chemokines, indicating a propensity to migrate to secondary lymphoid organs rather than to sites of inflammation.

Dendritic cells as a major source of macrophage-derived chemokine/CCL22 in vitro and in vivo.

Abstract: Macrophage-derived chemokine (MDC)/CCL22 is a CC chemokine active on dendritic cells (DC), NK cells and Th2 lymphocytes. The present study was aimed at comprehensively investigating MDC production in vitro and in vivo. DC were the most potent producers of MDC among leukocytes tested. Endothelial cells did not produce MDC under a variety of conditions. Signals that induce maturation (lipopolysaccharide, IL-1, TNF, CD40 ligand, recognition of bacteria and yeast) dramatically augmented MDC production, and dexamethasone and vitamin D3 blocked it. Prostaglandin E(2), which blocked the acquisition of IL-12 production and the capacity to promote Th1 generation, did not affect MDC production. Using mass spectrometry-based techniques, DC supernatants were found to contain N-terminally truncated forms of MDC [MDC(3-69), MDC(5-69) and MD(C7-69)] as well as the full-length molecule. In vivo, CD1a(+), CD83(+), MDC(+) DC were found in reactive lymph nodes, and in Langerhans' cell histiocytosis. Skin lesions of atopic dermatitis patients showed that CD1a(+) or CD1b(+) DC, and DC with a CD83(+) phenotype were responsible for MDC production in this Th2-oriented disorder. Thus, DC are the predominant source of MDC in vitro and in vivo under a variety of experimental and clinical conditions. Processing of MDC to MDC(3-69) and shorter forms which do not recognize CCR4 is likely to represent a feedback mechanism of negative regulation.

Chemokine Receptor Expression and Function in CD4+ T Lymphocytes with Regulatory Activity

Abstract: We have investigated the chemokine receptor expression and migratory behavior of a new subset of nickel-specific skin-homing regulatory CD4(+) T cells (Th(IL-10)) releasing high levels of IL-10, low IFN-gamma, and undetectable IL-4. These cells inhibit in a IL-10-dependent manner the capacity of dendritic cells to activate nickel-specific Tc1 and Th1 lymphocytes. RNase protection assay and FACS analysis revealed the expression of a vast repertoire of chemokine receptors on resting Th(IL-10), including the Th1-associated CXCR3 and CCR5, and the Th2-associated CCR3, CCR4, and CCR8, the latter at higher levels compared with Th2 cells. The most active chemokines for resting Th(IL-10), in terms of calcium mobilization and in vitro migration, were in order of potency: CCL2 (monocyte chemoattractant protein-1, CCR2 ligand), CCL4 (macrophage-inflammatory protein-1beta, CCR5 ligand), CCL3 (macrophage-inflammatory protein-1alpha, CCR1/5 ligand), CCL17 (thymus and activation-regulated chemokine, CCR4 ligand), CCL1 (I-309, CCR8 ligand), CXCL12 (stromal-derived factor-1, CXCR4), and CCL11 (eotaxin, CCR3 ligand). Consistent with receptor expression down-regulation, activated Th(IL-10) exhibited a reduced or absent response to most chemokines, but retained a significant migratory capacity to I-309, monocyte chemoattractant protein-1, and thymus and activation-regulated chemokine. I-309, which was ineffective on Th1 lymphocytes, attracted more efficiently Th(IL-10) than Th2 cells. I-309 and CCR8 mRNAs were not detected in unaffected skin and were up-regulated at the skin site of nickel-allergic reaction, with an earlier expression kinetics compared with IL-10 and IL-4. Results indicate that skin-homing regulatory Th(IL-10) lymphocytes coexpress functional Th1- and Th2-associated chemokine receptors, and that CCR8/I-309-driven recruitment of both resting and activated Th(IL-10) cells may be critically involved in the regulation of Th1-mediated skin allergic disorders.

Neutrophils produce biologically active macrophage inflammatory protein‐3α (MIP‐3α) / CCL20 and MIP‐3β / CCL19

Abstract: Macrophage inflammatory protein-3alpha (MIP-3alpha)/CCL20 and MIP-3beta/CCL19 are members of the CC chemokine subfamily which exert their effects through specific receptors, CCR6 and CCR7, respectively. Previously, we have reported that human neutrophils have the capacity to produce a number of chemokines, including IL-8/CXCL8, GROalpha/CXCL1, IP-10/CXCL10, and MIG/CXCL9. Herein, we show that neutrophils also have the ability to express and release MIP-3alpha/CCL20 and MIP-3beta/CCL19 when cultured with either LPS or TNF-alpha. We also report that MIP-3alpha/CCL20 and MIP-3beta/CCL19 production by LPS-stimulated neutrophils is negatively modulated by IL-10. Remarkably, we found that supernatants harvested from stimulated neutrophils not only induced chemotaxis of both immature and mature dendritic cells (DC), but also triggered rapid integrin-dependent adhesion of CCR6- and CCR7-expressing lymphocytes to purified VCAM-1 and ICAM-1, respectively. Importantly, both chemotaxis and rapid integrin-dependent adhesion were dramatically suppressed by anti-MIP-3alpha/CCL20 and anti-MIP-3beta/CCL19 neutralizing antibodies, indicating that MIP-3alpha/CCL20 and MIP-3beta/CCL19 present in the supernatants were both biologically active. As these chemokines are primarily chemotactic for DC and specific lymphocyte subsets, the ability of neutrophils to produce MIP-3alpha/CCL20 and MIP-3beta/CCL19 might be significant in orchestrating the recruitment of these cell types to the inflamed sites and therefore in contributing to the regulation of the immune response.

A cytokine-to-chemokine axis between T lymphocytes and keratinocytes can favor Th1 cell accumulation in chronic inflammatory skin diseases.

Abstract: The recruitment of T cells into the skin is regulated by chemokines released by resident cells. In this study, we found that normal human keratinocytes activated with Th1-derived supernatant (sup) expressed early (6-12 h) IP-10/CXCL10, MCP-1/CCL2, IL-8/CXCL8, and I-309/CCL1 mRNAs and with slower kinetics (24-96 h), RANTES/CCL5 and MDC/CCL22 mRNAs. Upon stimulation with the Th1 sup, keratinocytes secreted high levels of RANTES, IP-10, MCP-1, and IL-8 and moderate levels of I-309 and MDC. Although much less efficiently, Th2 sup could also induce keratinocyte expression of IL-8, IP-10, RANTES, and MCP-1 but not of I-309 and MDC. TARC/CCL17 was not significantly induced by any stimuli. Sup from keratinocytes activated with Th1-derived cytokines elicited a strong migratory response of Th1 cells and a limited migration of Th2 cells, whereas sup from Th2-activated keratinocytes promoted a moderate migration of Th1 and Th2 lymphocytes. Thus, keratinocytes appear considerably more sensitive to Th1- than to Th2-derived lymphokines in terms of chemokine release and can support the preferential accumulation of Th1 lymphocytes in the skin.

Transduction of human MCP-3 by a parvoviral vector induces leukocyte infiltration and reduces growth of human cervical carcinoma cell xenografts

Abstract: Background The oncosuppressive properties of some autonomous parvoviruses such as H-1 virus, together with their low pathogenicity, make them attractive vectors for tumor-directed gene therapy. Indeed, it was recently shown that these viruses became endowed with an enhanced oncosuppressive activity after they had been engineered to deliver a recognized therapeutic transgene. This prompted us to use a parvoviral vector to analyse the antineoplastic capacity of MCP-3 (monocyte chemotactic protein-3), a CC chemokine which has a broad spectrum of target cells, and can thus be considered to be a promising candidate for cancer treatment. Methods We explored the use of a parvovirus H-1-based vector encoding human MCP-3 for its antitumor potential on human cervical carcinoma cells. HeLa cells were infected in vitro with the recombinant virus hH1/MCP-3 at a low multiplicity [1 replication unit (RU)/cell] and we investigated the effect of parvovirus-mediated MCP-3 transduction on tumor formation and growth upon implantation of HeLa cells in nude mice. Results Infection of HeLa cells with hH1/MCP-3 led to secretion of high levels of MCP-3 and to significant retardation of tumor growth in recipient mice, as compared with HeLa cells that were either buffer-treated or infected with a MCP-3-free vector. Tumors from hH1/MCP-3–infected HeLa cells were heavily infiltrated with activated macrophages and showed increased numbers of dendritic cells. In addition, activated natural killer (NK) cells were also recruited into MCP-3-transduced tumors. Conclusion These observations indicate that parvovirus H-1-transduced MCP-3 is able to exert a significant antitumor activity which is mediated, at least in part, through macrophages and NK cells, under conditions in which activated T cells are lacking. Copyright © 2001 John Wiley & Sons, Ltd.

Macrophage Control of Inflammation: Negative Pathways of Regulation of Inflammatory Cytokines

Abstract: The recruitment of leukocytes from the blood compartment constitutes a multistep process which involves primary and secondary inflammatory cytokines, as well as adhesion molecules expressed on leukocytes and endothelial cells. The properties of the interleukin (IL)-1 system and of chemokines, as well as their interplay, are analysed. These mediators offer new paradigms to understand diverse pathologies, and provide tools and targets for the development of novel therapeutic strategies.

Monocytes from Wiskott-Aldrich patients differentiate in functional mature dendritic cells with a defect in CD83 expression.

Abstract: Wiskott-Aldrich syndrome (WAS) is an X-linked disorder characterized by congenital thrombocytopenia and progressive deterioration of the immune function. Dendritic cells (DC) are key effectors in the induction of specific immunity and are highly specialized in antigen uptake and subsequent migration to draining lymph nodes. DC were generated in vitro from circulating monocytes from ten WAS patients characterized by a different disease score. Immature DC showed similar morphology and membrane phenotype, as compared to normal DC. In chemotaxis assay, immature DC had a reduced migration in response to MIP-1alpha/CCL3, but efficiently endocytosed the macromolecules FITC-dextran and FITC-albumin. Upon terminal differentiation with LPS or CD40 ligand, the acquisition of a mature surface phenotype was variably achieved among WAS patients, with increased expression of CD80, CD86 and DC-LAMP. In contrast, the expression of CD83 was usually low. A defective up-regulation of CD83 was also observed in the lymph node from one WAS patient, whose DC stained positively for DC-LAMP. Mature DC from all the patients tested, but one, significantly migrated in vitro in response to MIP-3beta, a finding confirmed in vivo by the detection of HLA-DR/DC LAMP-positive cells in secondary lymphoid organs. When tested in MLR assays, both immature and mature WAS DC induced allogenic T cell proliferation in a manner comparable to control DC. Collectively these results suggest that, although many functional activities of WAS DC are essentially similar to normal DC, subtle and selective alterations of DC differentiation were also observed, with reduced migratory activity of immature DC and defective CD83 expression upon maturation.

Selective induction of phospholipase D1 in pathogen-activated human monocytes

Abstract: Phospholipase D (PLD) activation is part of the complex signalling cascade induced during phagocyte activation. Two PLD isoforms have been cloned, but their role in phagocyte functions is still poorly defined. We report that resting fresh circulating human monocytes expressed PLD1. PLD1 protein expression was rapidly down-regulated during cell culture. Lipopolysaccharide and pathogen-derived agonists (Candida albicans, arabinoside-terminated lipoarabinomannan and Gram-positive bacteria, but not mannose-capped lipoarabinomannan or double-stranded RNA) strongly induced PLD1 expression at both the mRNA and protein levels. Pro-inflammatory cytokines [interleukin (IL)-1beta and tumour necrosis factor alpha] had only a weak effect, whereas immune cytokines (IL-6 and interferon gamma), anti-inflammatory cytokines (IL-13 and IL-10) and chemoattractants (fMet-Leu-Phe and macrophage chemoattractant protein 1) were inactive. None of the agonists tested induced significant changes in the basal expression of PLD2 mRNA. Consistent with PLD1 up-regulation was the observation that PLD enzymic activity was higher in monocytes treated with active-pathogen-derived agonists than in control cells, when stimulated with PMA or with chemotactic agonists (fMet-Leu-Phe and C5a). Thus PLD2 seems to be a constitutive enzyme in circulating monocytes. Conversely, PLD1 is an inducible protein, rapidly regulated during culture conditions and selectively induced during cell activation. Therefore PLD1 might have a relevant role in immune responses against pathogens and in chronic inflammation.

CHAPTER 15 – Dendritic cells and chemokines

Abstract: Dendritic cells (DCs) respond to chemoattractants representative of different classes of chemotactic agonists, including lipid attractants, formyl peptides, and C5a generated by the complement cascade. Immature DCs, generated in vitro from monocytes, express a unique repertoire of inflammatory chemokine receptors. These receptors bind a pattern of “inflammatory” chemokines, including RANTES, MCP-3, MIP-1α, MIP-1β, and MIP-5. Langerhans cells (LCs) purified from skin or generated in vitro from CD34 precursors are characterized by the expression of CCR6, the receptor for MIP-3α, also known as LARC or exodus, in addition to the receptors expressed by monocyte DCs. The ability of precursor cells and immature DCs to migrate in response to a pattern of chemotactic signals is relevant for the accumulation of immature DCs into nonlymphoid tissues in normal conditions and during the early phase of inflammation, when local production of chemokines is strongly induced.

Transendothelial migration and reverse transmigration of in vitro cultured human dendritic cells.

Abstract: Dendritic cells (DC) are professional antigen-presenting cells (APC) that are believed to be indispensable to initiate a primary immune response (1,2). DC are migratory cells that exhibit complex trafficking properties in vivo, involving interaction with vascular and lymphatic endothelium and extracellular matrix (ECM). DC progenitors from the bone marrow enter the blood and seed nonlymphoid tissues. DC are spread all over the body, at epithelia (skin epidermis, airways, gastrointestinal, and genitourinary tracts) and at interstitial spaces of many solid organs (heart, liver, kidney), where they act as sentinels, waiting for an encounter with an antigen (1-3).

Chemotaxis of in vitro cultured human dendritic cells.

Abstract: Chemotaxis is defined as the directional locomotion of cells sensing a gradient of the stimulus. Some cell types, such as monocytes and neutrophils, can be considered as "professional migrants" and for many years the study of chemotaxis has been applied to these cells. However, other cell types including fibroblasts, melanoma cells, keratinocytes, and vascular endothelial cells exhibit directional locomotion in vitro.