Clara M. Ausiello

CD38: A multi-lineage cell activation molecule with a split personality

SummaryThis review reports the characteristics of the human surface molecule CD38, a structure not linked to a definite line and predominantly expressed in early and activated phenotypes. The CD38 molecule consists of a single chain of 46 kDa, spanning the membrane and with the carboxyl terminus located in the extracellular compartment. The CD38 molecule is also involved in the transduction of activation and proliferation signals, which are line unrestricted. The gene coding for the CD38 antigen has been cloned and used for the construction of simian and mouse transfectants expressing the human molecule. These cell models are used for the analysis of several unanswered issues, mainly concerning the in vivo function of CD38, the existence of a natural ligand and of polymorphism in the population.

Cell-mediated immunity and antibody responses to Bordetella pertussis antigens in children with a history of pertussis infection and in recipients of an acellular pertussis vaccine

Cell-mediated immunity (CMI) and antibody responses to Bordetella pertussis antigens were assessed 4-6 years after primary infant immunization with diphtheria-tetanus tricomponent acellular pertussis (DTaP) or diphtheria-tetanus (DT) vaccine in a country with high endemicity of B. pertussis infection. CMI to the B. pertussis antigens (especially to the pertussis toxin [PT]) was more frequent and/or intense in DTaP than in DT recipients. No lymphoproliferation differences were found between those with and without a history of pertussis although the DT recipients produced very little interferon-gamma after antigen (particularly PT and filamentous hemagglutinin [FHA]) stimulation. In contrast, seropositivity to PT, but not to pertactin or FHA, was more frequent in DT recipients with history of pertussis than in all other subjects. Thus, years after disease or vaccination, CMI response to PT or circulating PT antibodies appears to be the main distinctive feature of pertussis-protected DTaP recipients or pertussis-affected DT recipients.

Lipopolysaccharides from Bordetella pertussis and Bordetella parapertussis Differently Modulate Human Dendritic Cell Functions Resulting in Divergent Prevalence of Th17-Polarized Responses

Bordetella pertussis and B. parapertussis are the etiological agents of pertussis, yet the former has a higher incidence and is the cause of a more severe disease, in part due to pertussis toxin. To identify other factors contributing to the different pathogenicity of the two species, we analyzed the capacity of structurally different lipooligosaccharide (LOS) from B. pertussis and LPS from B. parapertussis to influence immune functions regulated by dendritic cells. Either B. pertussis LOS and B. parapertussis LPS triggered TLR4 signaling and induced phenotypic maturation and IL-10, IL-12p40, IL-23, IL-6, and IL-1β production in human monocyte-derived dendritic cells (MDDC). B. parapertussis LPS was a stronger inducer of all these activities as compared with B. pertussis LOS, with the notable exception of IL-1β, which was equally produced. Only B. parapertussis LPS was able to induce IL-27 expression. In addition, although MDDC activation induced by B. parapertussis LPS was greatly dependent on soluble CD14, B. pertussis LOS activity was CD14-independent. The analysis of the intracellular pathways showed that B. parapertussis LPS and B. pertussis LOS equally induced IκBα and p38 MAPK phosphorylation, but B. pertussis LOS triggered ERK1/2 phosphorylation more rapidly and at higher levels than B. parapertussis LPS. Furthermore, B. pertussis LOS was unable to induce MyD88-independent gene induction, which was instead activated by B. parapertussis LPS, witnessed by STAT1 phosphorylation and induction of the IFN-dependent genes, IFN regulatory factor-1 and IFN-inducible protein-10. These differences resulted in a divergent regulation of Th cell responses, B. pertussis LOS MDDC driving a predominant Th17 polarization. Overall, the data observed reflect the different structure of the two LPS and the higher Th17 response induced by B. pertussis LOS may contribute to the severity of pertussis in humans.

Native and Genetically Inactivated Pertussis Toxins Induce Human Dendritic Cell Maturation and Synergize with Lipopolysaccharide in Promoting T Helper Type 1 Responses

The capacity of pertussis toxin (PT) to induce maturation and functional activities of human monocyte-derived dendritic cells (DCs) was investigated. Both native PT (nPT) and genetically detoxified PT (dPT) efficiently promoted expression on DCs of CD80, CD86, human leukocyte antigen-DR, and CD83 markers, alloreactive antigen presentation, and cytokine production, primarily interferon (IFN)-gamma. Although they did not affect interleukin (IL)-10 production by lipopolysaccharide (LPS)-stimulated DCs, both nPT and dPT strongly synergized with LPS for IL-12 production. PTs plus LPS-stimulated DCs secreted soluble factors fostering IFN-gamma but not IL-4 and IL-5 production by naive T cells. T helper type 1 (Th1) polarization was, as alloreactive antigen presentation, inhibited by anti-IL-12 monoclonal antibody. These findings support the notion that nPT, in addition to inducing specific immune response, is a potent Th1 adjuvant and that dPT fully preserves this adjuvanticity. The synergic interaction between PT and LPS in IL-12 production might be relevant for the mechanisms of vaccine-induced protection.

Bordetella pertussis-Infected Human Monocyte-Derived Dendritic Cells Undergo Maturation and Induce Th1 Polarization and Interleukin-23 Expression

ABSTRACT Bordetella pertussis, the causative agent of whooping cough, is internalized by several cell types, including epithelial cells, monocytes, and neutrophils. Although its ability to survive intracellularly is still debated, it has been proven that cell-mediated immunity (CMI) plays a pivotal role in protection. In this study we aimed to clarify the interaction of B. pertussis with human monocyte-derived dendritic cells (MDDC), evaluating the ability of the bacterium to enter MDDC, to survive intracellularly, to interfere with the maturation process and functional activities, and to influence the host immune responses. The results obtained showed that B. pertussis had a low capability to be internalized by—and to survive in—MDDC. Upon contact with the bacteria, immature MDDC were induced to undergo phenotypic maturation and acquired antigen-presenting-cell functions. Despite the high levels of interleukin-10 (IL-10) and the barely detectable levels of IL-12 induced by B. pertussis, the bacterium induced maturation of MDDC and T helper 1 (Th1) polarized effector cells. Gene expression analysis of the IL-12 cytokine family clearly demonstrated that B. pertussis induced high levels of the p40 and p19 subunits of IL-23 yet failed to induce the expression of the p35 subunit of IL-12. Overall our findings show that B. pertussis, even if it survives only briefly in MDDC, promotes the synthesis of IL-23, a newly discovered Th1 polarizing cytokine. A Th1-oriented immune response is thus allowed, relevant in the induction of an adequate CMI response, and typical of protection induced by natural infection or vaccination with whole-cell vaccines.

CD38 is expressed on human mature monocyte-derived dendritic cells and is functionally involved in CD83 expression and IL-12 induction

Dendritic cell (DC) maturation is characterized by the gain or loss of immunological functions and by expression of distinctive surface receptors. CD38 is an ectoenzyme that catalyzes the synthesis of cyclic ADP ribose (a potent second messenger for Ca2+ release), as well as a receptor that initiates transmembrane signaling upon engagement with its counter‐receptor CD31 or with agonistic monoclonal antibodies. Since CD38 is expressed by resting monocytes, we aimed to monitor CD38 expression during the differentiation of human monocyte‐derived DC (MDDC) and to investigate the possibility that CD38 plays a functional role during DC maturation. CD38 is down‐modulated during differentiation into immature MDDC and expressed again upon maturation. The extent of CD38 expression is dependent on the stimulus adopted (LPS > IFN‐γ > CD40 cross‐linking). Although weak, IFN‐γ consistently induces DC maturation. De novo‐synthesized CD38 is enzymatically active, and its expression in mature (m) MDDC is dependent on NF‐κB activity. However, CD38 is not merely a maturation marker but also mediates signaling in mMDDC, where it maintains its functions as a receptor. Activation via agonistic anti‐CD38 mAb induces up‐regulation of CD83 expression and IL‐12 secretion, whereas disruption of CD38/CD31 interaction inhibits CD83 expression, IL‐12 secretion and MDDC‐induced allogeneic T cell proliferation.

Bordetella pertussis Commits Human Dendritic Cells to Promote a Th1/Th17 Response through the Activity of Adenylate Cyclase Toxin and MAPK-Pathways

The complex pathology of B. pertussis infection is due to multiple virulence factors having disparate effects on different cell types. We focused our investigation on the ability of B. pertussis to modulate host immunity, in particular on the role played by adenylate cyclase toxin (CyaA), an important virulence factor of B. pertussis. As a tool, we used human monocyte derived dendritic cells (MDDC), an ex vivo model useful for the evaluation of the regulatory potential of DC on T cell immune responses. The work compared MDDC functions after encounter with wild-type B. pertussis (BpWT) or a mutant lacking CyaA (BpCyaA−), or the BpCyaA− strain supplemented with either the fully functional CyaA or a derivative, CyaA*, lacking adenylate cyclase activity. As a first step, MDDC maturation, cytokine production, and modulation of T helper cell polarization were evaluated. As a second step, engagement of Toll-like receptors (TLR) 2 and TLR4 by B. pertussis and the signaling events connected to this were analyzed. These approaches allowed us to demonstrate that CyaA expressed by B. pertussis strongly interferes with DC functions, by reducing the expression of phenotypic markers and immunomodulatory cytokines, and blocking IL-12p70 production. B. pertussis-treated MDDC promoted a mixed Th1/Th17 polarization, and the activity of CyaA altered the Th1/Th17 balance, enhancing Th17 and limiting Th1 expansion. We also demonstrated that Th1 effectors are induced by B. pertussis-MDDC in the absence of IL-12p70 through an ERK1/2 dependent mechanism, and that p38 MAPK is essential for MDDC-driven Th17 expansion. The data suggest that CyaA mediates an escape strategy for the bacterium, since it reduces Th1 immunity and increases Th17 responses thought to be responsible, when the response is exacerbated, for enhanced lung inflammation and injury.

Bordetella pertussis Inhibition of Interleukin-12 (IL-12) p70 in Human Monocyte-Derived Dendritic Cells Blocks IL-12 p35 through Adenylate Cyclase Toxin-Dependent Cyclic AMP Induction

ABSTRACT Bordetella pertussis, the causative agent of whooping cough, possesses an array of virulence factors, including adenylate cyclase toxin (ACT), relevant in the establishment of infection. Here we better define the impact of cyclic AMP (cAMP) intoxication due to the action of ACT on dendritic cell (DC)-driven immune response, by infecting monocyte-derived DC (MDDC) with an ACT-deficient B. pertussis mutant (ACT−18HS19) or its parental strain (WT18323). Both strains induced MDDC maturation and antigen-presenting cell functions; however, only ACT−18HS19 infected MDDC-induced production of interleukin-12 (IL-12) p70. Gene expression analysis of the IL-12 cytokine family subunits revealed that both strains induced high levels of p40 (protein chain communal to IL-12 p70 and IL-23) as well as p19, a subunit of IL-23. Conversely only ACT−18HS19 infection induced consistent transcription of IL-12 p35, a subunit of IL-12 p70. Addition of the cAMP analogous d-butyril-cAMP (d-cAMP) abolished IL-12 p70 production and IL-12 p35 expression in ACT−18HS19-infected MDDC. ACT−18HS19 infection induced the expression of the transcription factors interferon regulatory factor 1 (IRF-1) and IRF-8 and of beta interferon, involved in IL-12 p35 regulation, and the expression of these genes was inhibited by d-cAMP addition and in WT18323-infected MDDC. The concomitant expression of IL-12 p70 and IL-23 allowed ACT−18HS19 to trigger a more pronounced T helper 1 polarization compared to WT18323. The present study suggests that ACT-dependent cAMP induction leads to the inhibition of pathways ultimately leading to IL-12 p35 production, thus representing a mechanism for B. pertussis to escape the host immune response.

Lymphoproliferative and cytotoxic responses of human peripheral blood mononuclear cells to mannoprotein constituents of Candida albicans

Two major proteoglycan constituents (designated F1 and F2) of the cell wall of Candida albicans were separated by ion-exchange chromatography from a crude carbohydrate-rich extract (GMP), and investigated for their chemical and molecular composition, antigenicity and immunomodulatory properties in cultures of human peripheral blood mononuclear cells (PBMC). Both fractions consisted predominantly of Periodic acid-Schiff (PAS) and concanavalin A (Con A)-reactive material consisting of greater than 90% mannose, 3-5% protein and small amounts of phosphorus; each was recognized by an anti-Candida rabbit serum as well as by a monoclonal antibody (mAb AF1) directed against an oligosaccharide epitope present on the fungal cell surface. When F1 and F2 were subjected to SDS-PAGE, transblotted and stained with enzyme-conjugated mAb AF1 or Con A, most of the antibody or lectin bound to high molecular mass (greater than 200 kDa) polydisperse material, some of which was present in F2 (as in the starting GMP extract) but absent in F1. This difference was also observed in PAS-stained gels of the two fractions. The F2, but not the F1, constituent was as active as the unfractionated GMP extract in inducing lymphoproliferation, production of the cytokines interleukin-2 and interferon-gamma, and generation of cytotoxicity against a natural-killer-sensitive target cell line (K562). These immunomodulatory properties were, like those possessed by GMP, protease-sensitive and heat-stable. Treatment of PMBC cultures with a modulatory anti-T-cell receptor antibody abolished the lymphoproliferation induced by GMP and F2 but not that induced by phytohaemagglutinin, showing that the mannoprotein materials of C. albicans acted through interaction with the antigen receptor complex.

Proliferation of human peripheral blood mononuclear cells induced by Candida albicans and its cell wall fractions

Glutaraldehyde-inactivated cells and cell-wall fractions of Candida albicans were studied for their capacity to induce or inhibit the in-vitro proliferation of human peripheral blood mononuclear cells (PBMC), as measured by 3H-thymidine incorporation. Both the intact cells (CA) and a phosphorylated gluco-mannan-protein complex of the cell wall (GMP), in microgram doses, were strong inducers of PBMC proliferation, with a peak of activity at 6-9 days of culture and varying with the PBMC donor. A significant but much lower proliferation was observed on exposure of PBMC to a low-protein (less than 3% by weight) mannan component (M) of the cell wall. Both a hot-alkali extracted mannan-protein complex (M-alk), comparable to GMP in crude chemical composition, and an alkali-insoluble cell-wall glucan (GG) were inactive. None of the Candida fractions induced a lymphoproliferation of umbilical cord blood cells and all fractions, except GG, were equally effective in binding human anti-Candida antibodies as shown by a sensitive ELISA-inhibition assay. Moreover, a monoclonal antibody against the class II determinant of the HLA complex inhibited PBMC proliferation irrespective of the Candida antigen used. Taken together, the data shows that in inducing lymphoproliferation, Candida fractions act as specific antigens rather than as non-specific mitogens. Use of intact Candida cells and chemically-defined cell-wall components appears preferable to use of undefined antigenic mixtures as stimulators of PBMC proliferation.