Secretion of interferon-gamma by human macrophages demonstrated at the single-cell level after costimulation with interleukin (IL)-12 plus IL-18.

The interferon (IFN)-gamma component of the immune response plays an essential role in combating infectious and non-infectious diseases. Induction of IFN-gamma secretion by human T and natural killer (NK) cells through synergistic costimulation with interleukin (IL)-12 and IL-18 in the adaptive immune responses against pathogens is well established, but induction of similar activity in macrophages is still controversial, with doubts largely focusing on contamination of macrophages with NK or T cells in the relevant experiments. The possible contribution of macrophages to the IFN response is, however, an important factor relevant to the pathogenesis of many diseases. To resolve this issue, we analysed the production of IFN-gamma at the single-cell level by immunohistochemistry and by enzyme-linked immunosorbent spot (ELISPOT) analysis and unequivocally demonstrated that human macrophages derived from monocytes in vitro through stimulation with a combination of IL-12 and IL-18 or with macrophage colony-stimulating factor (M-CSF) were able to produce IFN-gamma when further stimulated with a combination of IL-12 and IL-18. In addition, naturally activated alveolar macrophages immediately secreted IFN-gamma upon treatment with IL-12 and IL-18. Therefore, human macrophages in addition to lymphoid cells contribute to the IFN-gamma response, providing another link between the innate and acquired immune responses.

Skewed expression and up-regulation of the IL-12 and IL-18 receptors in resting and activated CD4 T cells from HIV-1-infected patients.

IL-12 and IL-18 synergistically induce the production of IFN-gamma by resting and activated T cells. To evaluate whether this induction was affected in HIV-1-infected patients, PBMC or isolated CD4 T cells were cultured with IL-12 plus IL-18, anti-CD3 plus anti-CD28, or PHA for 72 h. Cell samples were labeled daily to assess the levels of IL-12 receptor beta1 (IL-12Rbeta1), IL-12Rbeta2, and IL-18Ralpha. Culture supernatants were analyzed for the presence of Th1- and Th2-related cytokines by ELISA or cytometric bead array and analyzed by flow cytometry. A twofold increase in the percentage of CD4-resting T cells expressing IL-12Rbeta1 and IL-18Ralpha from HIV-1-infected patients was observed when compared with cells from HIV-1-negative donors. Higher IL-12Rbeta1 and IL-18Ralpha expression correlated (r=0.87; P<0.007) to increased production of IFN-gamma by isolated CD4 T cells in the presence of IL-12 and IL-18. Moreover, exogenous IL-12 and IL-18 induced the up-regulation of IL-12Rbeta2 to twice higher in CD4 T cells from HIV-1-positive individuals compared with controls. Conversely, upon activation with anti-CD3 and anti-CD28 antibodies, only 25% of the CD4+ T cells from HIV-1 patients showed an increase in the IL-12beta2 when compared with 50% in healthy controls. Furthermore, the percentage of IL-12Rbeta1-positive cells correlated inversely with the CD4 nadir of patients, suggesting that deregulation of the IL-12 and IL-18 pathways may play a role in the immunopathogenesis of HIV-1 infection.

Inhibition of human immunodeficiency virus type 1 infection in macrophages by an alpha-v integrin blocking antibody.

Macrophages are key cells for HIV infection and HIV spreading inside the organism. Macrophages cultured in vitro can be successfully infected after differentiation with cytokines such as macrophage colony stimulating factor (M-CSF). In the monocyte to macrophage differentiation process with M-CSF, alphav-integrins are upregulated concomitantly with the capacity of HIV to generate a productive virus infection. In the present study we show that an anti-alphav antibody, 17E6, inhibited HIV-1 infection of primary macrophages. The effect of 17E6 on HIV-1 BaL replication in acutely infected macrophages was dose-dependent, with a 50% effective concentration (EC50) of 17+/-2 microg/ml in the absence of cytotoxicity. Similarly, a monoclonal antibody targeting the alphavbeta6 integrin (14D9.F8) also inhibited HIV-1 BaL infection in this cell type. 17E6 reduced the detection of HIV-1 BaL proviral DNA in acutely infected macrophages, but was completely ineffective against HIV-1 BaL production in chronically infected macrophages, suggesting that 17E6 inhibited HIV infection at an early stage of the virus cycle. Finally, a small molecular weight antagonist of the alphavbeta6 integrin, EMD 409849, reduced HIV replication at subtoxic concentrations. Therefore, our results suggest that alphav-containing integrins could play a role in HIV replication in macrophages and suggest that small-molecular-weight compounds might interfere with HIV replication in macrophages through the interaction with alphav integrins.