Histamine-treated dendritic cells improve recruitment of type 2 CD8 T cells in the lungs of allergic mice

Abstract

Histamine controls the function of dendritic cells (DCs). It appears to be required for the normal development of DCs. It also induces the chemotaxis of immature DCs and promotes the differentiation of CD4(+) T cells into cells with a T helper type 2 (Th2) profile. Moreover, we have recently shown that histamine stimulates both the uptake and the cross-presentation of antigens by DCs, supporting the theory that histamine promotes activation of CD8(+) T cells during the development of allergic pathologies. Here, we investigated whether the course of an allergic response, in a well-defined model of ovalbumin (OVA)-induced allergic airway inflammation, could be modulated by intratracheal injection of OVA-pulsed DCs previously treated with histamine (DCHISs). Compared with control DCs, DCHISs induced: (i) greater recruitment of CD8(+) T cells in the lung, (ii) greater stimulation of the production of interleukin (IL)-5 by lung CD8(+) T cells, and (iii) increased recruitment of CD11c/CD8 double-positive DCs in the lungs of allergic mice. Moreover, mice treated with DCHISs showed increased levels of serum-specific immunoglobulin E (IgE) antibodies directed to OVA, and a higher proportion of eosinophils in bronchoalveolar lavage (BAL) compared with mice treated with OVA-pulsed control DCs. Our results support the notion that histamine, by acting on DCs, increases the severity of allergic processes.

Figure 1

High levels of serum immunoglobulin E (IgE) antibodies directed to ovalbumin (OVA) in allergic mice. (a) BALB/c mice were inoculated intraperitoneally (i.p.) with OVA on days 0 and 7. On day 14, sensitized mice were challenged intranasally with OVA for 5 days. After 7 days, serum samples were obtained from allergized (A) or control (N) mice and the levels of IgE antibodies directed to OVA were determined by enzyme-linked immunosorbent assay (ELISA). Values are expressed as the arithmetic mean of the optical density (OD) ± standard error of the mean (SEM) (n = 6–8). Asterisks indicate statistical significance (**P ≤ 0·01) versus controls. (b) Representative histograms of the phenotypes of immature DCs obtained from bone marrow precursors. The thin line represents the isotype control. (c) Carboxyfluorescein succinimidyl ester (CFSE)-labelled DCs (1 × 10⁶) were injected intratracheally (i.t.). After 6 hr, lungs were processed as described in the ‘Materials and methods’. Cells were labelled with phycoerythrin (PE)-conjugated antibodies directed to CD11c and analysed by flow cytometry. A representative experiment (n = 3) is shown.

Figure 2

Histamine-pretreated dendritic cells (DCs) stimulate the recruitment of CD8⁺ T lymphocytes in the lungs of allergic mice. BALB/c mice were inoculated intraperitoneally (i.p.) with ovalbumin (OVA) on days 0 and 7. On day 14, sensitized mice were challenged intranasally with OVA for 5 days. Then, DCs obtained from bone marrow precursors, pretreated or not with 1 μm histamine (30 min at 37°) and pulsed with 100 μg/ml OVA (3 hr at 37°), were injected intratracheally (i.t.) (5 × 10⁵ cells/mouse). Control mice were injected with phosphate-buffered saline (PBS). After 7 days, mice were killed and the lungs were processed. Lung T cells were purified using anti-CD3 antibodies coupled to magnetic beads. Purified T cells were stained with phycoerythrin (PE)-labelled anti-CD8 antibodies and fluorescein isothiocyanate (FITC)-labelled anti-CD4 antibodies. Cells were analysed by flow cytometry. (a, c) Results are expressed as the percentage of CD8⁺ and CD4⁺ T cells, respectively, and represent the mean ± standard error of the mean (SEM) for eight experiments. Asterisks indicate statistical significance (*P ≤ 0·01) versus controls (PBS). (b, d) Representative dot-plots (n = 8) are shown.

Figure 3

Histamine-pretreated dendritic cells (DCs) stimulate the production of interleukin (IL)-5 by a subset of CD8⁺ T lymphocytes recruited in the lungs of allergic mice. DCs pretreated or not with histamine (1 μm; 30 min at 37°) and pulsed with ovalbumin (OVA) (100 μg/ml; 3 hr at 37°) were intratracheally (i.t.) injected (5 × 10⁵ cells/allergized mouse), as described in the Materials and Methods. After 7 days, mice were killed, the lungs were processed, and T cells were purified using anti-CD3 antibodies coupled to magnetic beads. T cells (2 × 10⁵/200 μl) were then stimulated in vitro with OVA (10 ng/ml) in the presence of brefeldin (10 μg/ml) for 18 hr. The percentages of interferon (IFN)-γ-, IL-4-, IL-13-, and IL-5-positive CD8 T cells were determined by flow cytometry. In (a), data are expressed as the percentage of CD8⁺ T cells positive for the production of each of the cytokines analysed. Data represent the mean ± standard error of the mean (SEM) for five experiments. Asterisks indicate statistical significance (*P ≤ 0·01) versus controls (phosphate-buffered saline). In (b), a representative experiment (n = 5) is shown.

Figure 4

Histamine-pretreated dendritic cells (DCs) stimulate the recruitment of CD11c⁺ CD8 alpha⁺ DCs in the lungs of allergic mice and the production of Leukotriene B4 (LTB4). DCs pretreated or not with histamine and pulsed with ovalbumin (OVA) were intratracheally (i.t.) injected (5 × 10⁵ cells/allergized mouse), as described in the Materials and Methods. After 7 days, mice were killed and the lungs were processed. Lung cell suspensions were enriched in CD11c using antibodies directed to CD11c coupled to magnetic beads. Isolated cells were analysed for the expression of CD11c and CD8α, using specific antibodies labelled with phycoerythrin (PE) and fluorescein isothiocyanate (FITC), respectively. (a) Results are expressed as the percentage of cells double-positive for CD11c and CD8α [mean ± standard error of the mean (SEM); n = 6]. (b) A dot-plot of a representative experiment is shown. (c) CD11c⁺ cells purified from the lungs (1·5 × 10⁵ cells/200 μl) were cultured for 18 hr at 37° and the production of LTB4 was analysed in the supernatants by enzyme-linked immunosorbent assay (ELISA). Results are expressed in pg/ml, and represent the mean ± SEM of eight experiments. Asterisks indicate statistical significance (*P ≤ 0·01) versus controls (phosphate-buffered saline).

Figure 5

Transfer of histamine-pretreated dendritic cells (DCs) to allergic mice increases serum levels of specific immunoglobulin E (IgE) antibodies directed to ovalbumin (OVA) and leads to a more persistent infiltration of the lungs by eosinophils. DCs pretreated or not with histamine and pulsed with OVA were intratracheally (i.t.) injected (5 × 10⁵ cells/allergized mouse), as described in the Materials and Methods. (a) After 15 days, serum samples were obtained and the levels of serum IgE antibodies directed to OVA were determined by enzyme-linked immunosorbent assay (ELISA). Data are expressed as the arithmetic mean of the optical density (OD) ± standard error of the mean (SEM) (n = 6). Asterisks indicate statistical significance (**P ≤ 0·01) versus controls [phosphate-buffered saline (PBS)]. (b) After 15 or 30 days, the percentages of eosinophils in bronchoalveolar lavage (BAL) were determined. Data are expressed as the percentage of eosinophils in BAL (arithmetic mean ± SEM; n = 6). Asterisks indicate statistical significance (*P ≤ 0·01) versus controls (PBS).