human monocyte-derived immature DC (middle panel). Staining is performed by using FITC labeled anti-bovine IgG monoclonal antibodies. Pre-incubation of DC with IgG could be completely blocked by the competitive inhibition with anti-CD64 (FcgRI) antibodies (right panel). Data from one representative donor of two is shown. Functional activity of mature dendritic cells
An example of dietary immunomodulation is by consumption of bovine milk, which contains IgM, IgG and IgA that can bind to pathogens and allergens that are also encountered by humans [12-14]. Milk-derived and purified bovine IgG, IgA, and IgM did not bind significantly to freshly isolated human monocytes (data not shown). In contrast, bovine IgG, but not IgM, from milk as well as purified immunoglobulins did clearly bind to immature monocyte-derived DC (Figure 2, left and middle panel). Incubation of DC with IgG could be completely blocked by the competitive inhibition with anti- CD64 (FcgRI) antibodies (Figure 2, right panel), indicating that the high affinity FcgRI is responsible for binding of the monomeric IgG to DC. IgG that formed complexes with LPS, resulted in increased binding of bovine IgG to human moDC compared to monomeric bovine IgG, suggesting binding of immune complexes (Figure 3, upper panel). There is an optimal ratio between antigen and IgG for the formation of immune complexes. This binding of bovine IgG could partially be inhibited by blocking monoclonal antibodies (mAb) directed at the low affinity FcγRII (CD32), but also by mAb to the high affinity FcγRI (CD64) (Figure 3, lower panel). These data suggest that monomeric bovine IgG can bind to the high affinity human FcγRI, and that bovine IgG immune complexes could bind to the human low affinity FcγRII. Depending on the expression levels of activating and inhibitory FcγR, inflammatory or non-inflammatory downstream immune responses may be initiated leading to the production of protective IgG antibodies. This may be influenced by the type of DC the FcγR is expressed.
Figure 3. Binding of bovine IgG-LPS immune complexes to human moDCs. IgG was incubated with different doses of LPS and subsequently added to moDCs (upper panel). IgG –LPS immune complexes in three different ratios were added to moDC in the presence or absence of IgG receptor blocking antibodies (lower
panel). Unstained and FITC control show background staining, IgG mono is monomeric bovine IgG and IgG
complex shows bovine IgG pre-incubated with LPS (in three different concentrations) prior to addition to the DCs. Blocking of LPS-IgG complexes by anti (a) CD32 and aCD64 was performed with specific monoclonal antibodies.
Polarization of T-cells in allergy
Dendritic cells are professional antigen-presenting cells and are the key players in the initial stage of the interaction of e.g. allergens and the immune system that shapes the differentiation of naïve T cells. DCs display inherent plasticity and depending on their functional state, induce or inhibit T cell responses. The differentiation of naïve T cells into committed effector and regulator cells depends on complex interactions between the antigen, the antigen-presenting cell, and the milieu of cytokines in the immediate environment and expression of cell surface receptors. As described, allergy is widely considered a Th2 mediated immune maturation disease with a disturbed Th1-Th2 balance resulting in excessive amounts of IgE antibodies [7, 8].
Typically, systemic T cells are isolated from peripheral blood mononuclear cells (human) or spleen and peripheral lymph nodes (mouse) by preparing single cell suspensions from these lymphoid organs. Naïve (CD62Lhigh) T cells are purified by magnetic beads coupled with anti-CD4 antibodies and using magnetic activated cell sorting (MACS) or, alternatively, labeling of the cells by anti-CD4 antibodies coupled to a fluorochrome
A1
(e.g. FITC or PE) and using flow cytometric cell sorting (FACS) [4]. Isolated T cells can be obtained with relatively high purity (>95%) using both methodologies.
T cell polarization is obtained by culturing purified CD4+ T-cells in vitro and stimulating these cells by anti-CD3 and anti-CD28 monoclonal antibodies. All cells are cultured in the presence of IL-2 for maximal proliferation (neutral condition). For maximal Th1 development cells are cultured in the presence of IL-12, IFN-γ, and anti-IL-4 antibodies, while maximal Th2 development is obtained by stimulation in the presence of IL-4 and anti-IL-12 and anti-IFN-γ antibodies. Cells are stimulated under Th0-like conditions in the presence of monensin for 5 hours if intracellular cytokine staining (ICS) analysis will be required, or alternatively, cell supernatants are harvested 48 or 96 hours after secondary stimulation in the absence of monensin treatment [15].
Polarized T cells are pelleted and stained for surface molecules in a fluorescent activated cell-sorting (FACS) buffer. Following this, cells are fixed in paraformaldehyde and permeabilized with saponin. Cells are stained for intracellular cytokines with anti- IL-4-PE and anti-IFN-γ-FITC. Unlabeled antibody of the same clones is utilized at 10 times excess to set negative gates to control for non-specific staining. Cells are suspended in FACS buffer and then 10 000 events in the live cell gate (dead cells excluded by propidium iodine staining) will be analyzed on a flow cytometer (Figure 4). The use of propidium iodine (PI) staining of fixed and permeabilized cells provides a useful tool for setting the life cell inclusion gate for subsequent analysis of intracellular cytokine staining. This method of setting of stringent live cell gates is essential, as the use of un-stimulated cells alone does not adequately control for changes in auto-fluorescence following activation due to the inevitable increase in forward/sideward scatter in the flow cytometric analysis. Due to cell fixation and permeabilization, there is an inherent amount of non-specific staining which must be controlled for. Typically, the gates are set so that only 1% of cells are positive for the cytokine of interest in the labeled/unlabeled negative control. In order to detect intracellular cytokines, a Golgi trans- port inhibitor must be used on in vitro stimulated cells. This inhibitor affects the release or display of rapidly produced cytokines as well as other surface molecules. The concentration range in which the Golgi transport inhibitors can be used is relatively small, due to their inherent cytotoxicity. Therefore, cells must be activated to rapidly produce cytokines (hence the use of PMA and calcium ionophore) within a narrow 2-hour window for brefeldin A and a 5-hour window for monensin.
Figure 4. Maturation of the Immune Response. Absolute cytokine production of interferon-γ (upper panel) and interleukin-4 (middle panel) by T helper cells isolated from healthy control (HC) and allergic individuals, as detected by ELISA (detection limit 7.5 pg/ml) under different in vitro polarizing conditions, including Th0, Th1 and Th2 conditions. Relative cytokine production of T helper type 1 versus T helper type 2 cells (lower
panel). Absolute production is converted into a relative percentage by dividing interferon-γ or interleukin IL-4 production values by the mean IFN-γ or IL-4 concentration following Th1 or Th2 polarization, respectively, of CD4+ T cells from healthy control (HC) or allergic individuals.