Giulia Lanzilli

Anti-inflammatory Effect of Resveratrol and Polydatin by In Vitro IL-17 Modulation

Interleukin-17 (IL-17) is a proinflammatory cytokine produced, although not exclusively, by T helper 17 recently identified as a distinct T helper lineage mediating tissue inflammation. IL-17 is known to be involved in a number of chronic disorders although the mechanisms regulating its production in inflammatory disease are still unclear. The beneficial properties of the polyphenolic compound resveratrol including its anti-inflammatory, antioxidant, and antitumor effects, its role in the aging process and in the prevention of heart and neurodegenerative diseases are well-known. In addition, derivatives of resveratrol, including glucosylated molecules as polydatin have been linked to similar beneficial effects. We have investigated the effects of resveratrol and polydatin on the in vitro production of IL-17 in a model of inflammation in vitro. The results obtained by activated human peripheral blood mononuclear cells, stimulated with anti-CD3/anti-CD28 monoclonal antibodies and treated with these polyphenolic compounds at different concentrations show that both decrease IL-17 production in a concentration-dependent manner. This study confirms the anti-inflammatory activity of resveratrol and its derivatives and suggests a potential clinical relevance in the therapy of inflammatory diseases.

In vitro effects of an immunostimulating bacterial lysate on human lymphocyte function.

MLBL is an oral immunostimulating vaccine consisting of bacterial standardized lysates obtained by mechanical lysis of different strains of Gram-positive and Gram-negative bacteria that can cause acute and chronic infections of the respiratory tract. Previous studies suggested a stimulating effect of MLBL both on humoral and cellular immune responses. In the present study, the in vitro effects of MLBL on human lymphocyte effector functions and its mechanisms of action were evaluated. The results show that the most remarkable effects of MLBL on the immune system are: i) activation of the IL-2 receptor (IL-2Rα) on different lymphocyte subsets (B, CD4+ T and CD8+ T cells) involved both in humoral and cellular immune responses; ii) induction of cytokine synthesis (IL-2, IL-10, IL-12, IFNγ) in the immune competent cells that induce and regulate immune responses; iii) generation of CD4+ and CD8+ effector T cells. Overall, these results suggest that the therapeutic effect of MLBL on acute and recurrent infections of the respiratory tract is related to its ability to activate the responses of different subsets of immune competent cells both for humoral and cellular immunity. Moreover, these effects can be induced either by direct immune cell activation or through the generation and activation of immune effector cells.

Antiviral activity of individual versus combined treatments with interferon α, β and γ on early infection with HTLV-I in vitro

We have shown previously that infection of mononuclear cells derived from neonatal cord (CBMC) or adult peripheral (PBMC) blood with HTLV-1 can be controlled in vitro by treatment with interferon (IFN) alpha, beta or gamma. The activity of IFNs was mainly related to the induction of an active antiviral competence in hosts immune effector cells. The antiviral activity of IFN-boosted CBMC could be ascribed both to a positive regulation of cell-mediated immunity and to inhibition of viral infection. Data described herein provide further information on the mechanisms of the antiviral activity of IFNs and compare the activity of each type of IFN with the association of alpha + beta, alpha + gamma and beta + gamma IFNs, at a concentration of 100 or 1000 IU/ml. When added at the onset of the co-culture of CBMC with lethally irradiated, virus-donor MT-2 cells, IFNs could protect host CBMC by inhibiting HTLV-1 infection in terms of reduced proviral integration and a lower percentage of virus-positive cells, until 4 weeks of culture. Infection of CBMC was inhibited at a comparable extent by either individual or combined IFN treatments. However, a clearcut inhibition of HTLV-I transcription was found only when alpha 100 + beta 1000 IU/ml and especially alpha 1000 + gamma 100 IU/ml combined treatments were tested. When the chronically infected, virus-producing MT-2 cells were treated with IFNs, a remarkable inhibition of HTLV-I transcription was found only after multiple treatments. However, MT-2 cells became resistant to the antiviral activity of IFN gamma, but not to that of IFN alpha or beta. These data provide further information on the control of HTLV-I replication mediated by IFNs at different steps of the viral life cycle, being therefore relevant to the clinical use of combined IFNs in the treatment of acute infection. Moreover, IFNs could be used to prevent the establishment of a persistent infection, which is a prerequisite for developing adult T-cell leukemia (ATL) and/or virus-associated myelopathy.

Effects of cocaine administration to influenza virus-immunized mice on cytokine profiles of individual splenic CD4+ and CD8+ T cells.

We have analysed the effects of cocaine, administered to mice during the in vivo differentiation of effector T cells stimulated by antigen (influenza virus) recognition, on the frequency of IL‐2‐, IL‐4‐ and interferon‐gamma (IFN‐γ)‐expressing CD4+ and CD8+ T cells. Each animal was injected intraperitoneally with 10 mg/kg of cocaine 6, 24, 48 and 72 h after immunization with A/PR8 influenza virus (PR8). This enabled the determination of the pharmacological effects of cocaine on T cells during the initial step of the immune response, which is characterized by the production of large amounts of immunoregulatory cytokines. The distribution of IL‐2‐, IL‐4‐ and IFN‐γ‐producing CD4+ and CD8+ T cells was assayed on unseparated PR8‐immune spleen cells, obtained from mice treated with cocaine or vehicle, and restimulated in vitro with UV‐inactivated PR8 virus. The frequency of T cells singly or co‐expressing the above three cytokines was determined at single‐cell level by simultaneous flow cytometric analysis of intracellular cytokines and surface antigen expression. In parallel, the levels of IL‐2, IL‐4 and IFN‐γ in the culture supernatants were quantified by ELISA. The results showed that cocaine, administered during the in vivo virus‐induced differentiation of T cells, caused an increase of both the frequencies of CD8+ T cells singly and co‐expressing IL‐2 and IFN‐γ and the levels of these cytokines in virus‐restimulated spleen cell culture supernatants, compared with those of untreated controls. In contrast, no effect was found on IL‐4‐positive CD8+ T cells and on IL‐2‐, IFN‐γ‐ and IL‐4‐positive CD4+ T cells. Our findings suggest that the immunomodulatory effects of cocaine may be due to the up‐regulation of the production of IL‐2 and IFN‐γ by CD8+ T cells with a type 0 cytokine profile.

Determination of cytokine co-expression in individual splenic CD4+ and CD8+ T cells from influenza virus-immune mice.

We have studied the patterns of interleukin‐2 (IL‐2), IL‐4 and interferon‐γ (IFN‐γ) co‐expression displayed by individual splenic CD4+ and CD8+ T cells in response to influenza virus immunization. Unseparated spleen cells obtained from mice intraperitoneally (i.p.) injected with A/PR8 (H1N1) influenza virus (PR8) were cultured for 24 hr in the presence of ultraviolet‐inactivated PR8. As controls, cultures of both naive spleen cells stimulated with PR8 or of immune cells lacking the inactivated virus were used. The frequencies of CD4+ and CD8+ T cells expressing IL‐2, IL‐4 and IFN‐γ were determined by three‐colour flow cytometric analysis of fixed and saponin‐permeabilized cells fluorescent‐stained for either CD4 or CD8 surface molecules and for one of the following combinations of two intracellular cytokines: IL‐2/IL‐4, IL‐2/IFN‐γ and IL‐4/IFN‐γ. The results showed that immunization with influenza virus induces in both CD4+ and CD8+ T cells a heterogeneity of cytokine response patterns that do not follow the type 1/type 2 polarized response model, but with substantial differences between the two populations. In fact, the analysis of the phenotypes of virus‐immune CD8+ T cells revealed similar significant proportions of cells either expressing any one of the three cytokines or co‐expressing combinations of them (i.e. IL‐4/IL‐2, IL‐4/IFN‐γ and IL‐2/IFN‐γ), whereas immune CD4+ T cells were seen to express almost exclusively a single cytokine per cell. The observed patterns of cytokine production suggest that influenza virus immunization induces the expression of a type 0 cytokine pattern at both population and single cell levels in CD8+ T cells and exclusively at the population level in CD4+ T cells.

HSP70 production and inhibition of cell proliferation in Molt-4 T-cells after cell-to-cell transmission of HTLV-I: effect of PGA1.

Infection with HTLV-I is associated with leukemic transformation of mature CD4+ T lymphocytes. PGA1, a powerful inhibitor of tumour cell proliferation, can prevent the clonal expansion of HTLV-I-infected cells following acute infection of cord blood-derived mononuclear cells. Since the antiproliferative effect of PGA1 on HTLV-I transformed, chronically infected MT-2 cell line was associated with induction of HSP70, we have investigated the effect of PGA1 on cell cycle progression and HSP70 production in a leukemic T-cell line (Molt-4) shortly after exposure to HTLV-I in a cell-to-cell transmission model. Rate of cell proliferation and HSP70 expression were studied within one duplication cycle of Molt-4 cells after exposure to HTLV-I. Growth of both control and virus-exposed cultures was inhibited by treatment with PGA1 (4 micrograms/ml) and cell cycling was arrested preferentially at the G1/S interphase. Synthesis of HSP70 was induced within 3 h by PGA1 in control and virus-exposed Molt-4 cells and became undetectable from overnight onward, though the protein accumulated in the cells. The arrest of growth was observed from overnight up to 48 h so that treated cells almost missed one cycle. Interestingly, HSP70 transcript and protein persisted at remarkably high levels in Molt-4 cells exposed to HTLV-I in the absence of PGA1, showing that HSP70 expression can be directly activated during primary infection with this human retrovirus. Moreover, in these cocultures, treatment with PGA1 or heat shock was not able to increase further the elevated level of HSP70 found in untreated cocultures, suggesting that during the early period of the virus-transmission phase, HTLV-I could interfere with HSP70 induction by other inducers.

Anti-apoptotic seminal vesicle protein IV inhibits cell-mediated immunity

The in vitro effect of seminal vesicle protein IV (SV-IV) on the cytotoxic activity of human natural or acquired cellular immunity has been investigated by standard immunological procedures, a (51)Cr-release cytotoxicity assay, and labeled-ligand binding experiments. The data obtained demonstrate that: (1) fluoresceinated or [(125)I]-labeled SV-IV binds specifically to the surface of human purified non-adherent mononuclear cells (NA-MNC); (2) SV-IV suppresses the cytotoxicity of natural killer (NK) cells against K562 target cells, that of IL-2-stimulated NK (LAK) cells against DAUDI target cells, and that of VEL antigen-sensitized cytotoxic T lymphocytes (CTLs) against VEL target cells; (3) treatment of K562 target cells alone with SV-IV decreases their susceptibility to NK-induced lysis. These findings indicate that the protein SV-IV has a marked in vitro inhibitory effect on NK, LAK and CTL cytotoxicity, providing a better understanding of its immune regulatory functions.

In vitro combined effects of human interferons and interleukin-2 on natural cell-mediated cytotoxicity

In vitro modulation of natural cell-mediated cytotoxicity (NCMC), following sequential treatment of human mononuclear cells (MNC) with cytokines was investigated. Recombinant Interleukin-2 (IL2) used in combination with interferons (IFNs) induced variable effects on the cytolytic function of different MNC preparations obtained from 16 healthy donors. When MNC were treated with IFNs on day 4, after IL2 induction of LAK cells, increase or no change in cytotoxic activity was found. On the other hand, either no change or decrease in LAK activity occurred when MNC were treated with IFNs on day 0 before exposure to IL2. In this case the effect of IFNs on NCMC did not correlate with their activity on cell proliferation or on TAC antigen expression. In conclusion the present study points out that the NCMC of MNC of healthy donors, subjected to IL2 treatment in vitro, can be significantly increased by IFNs. However this effect is largely schedule-dependent (i.e. detectable with IL2-IFNs but not with IFNs-IL2 sequence), and can be obtained in a relatively limited number of cases. Moreover it is suggested that these in vitro studies could provide preclinical bases for a rational approach to in vivo treatment with cytokine cascade in a clinical setting.

In vitro end points for the assessment of cellular immune response-modulating drugs

Background: The concept of immunotoxicology and the development of a battery of immune-function assays to screen potential immunotoxic compounds have been increasingly used in the past. Immunotoxic outcome generally seems appropriate to evaluate the risk in drug development. Improving this approach is possible, by using methods now available, to study the effect of a chemical compound on the immune system. Objective: The goal of this review is to provide an overview of the current and recent methodologies for testing the immunological effect and immunotoxic risks in drug candidates. Methods: The methodological details here discussed include a synthetic description of the immunocompetent cells in cell-mediated immunity and the choice of the most appropriate assay (bioassays, immunoassays, molecular biology techniques, flow cytometry). Conclusion: This review offers an assessment of in vitro models to study the toxic impact of (bio)pharmaceuticals on cellular immune system and aid drug scientists in understanding the significance and the methods to approach immunotoxicology.