Modulation of Dendritic Cell Apoptosis and CD8+ Cytotoxicity by Histamine: Role of Protein Kinase C

Abstract

Dendritic cells (DC) are able to present extracellular antigens associated with the molecules of the major histocompatibility complex class I. In a previous work, we demonstrated that the histamine (HIS), acting through H1/H4 receptors, increases the cross-presentation of soluble ovalbumin by murine DC and can enhance the recruitment of specific CD8⁺ T lymphocytes during the development of chronic inflammatory responses. Here, we studied in more depth the mechanisms underlying this enhancement. We showed that the cytotoxicity of specific CD8⁺ lymphocytes is increased in HIS-treated DC and it is lost by inhibition of vacuolar-ATPase that prevents endosome acidification. It is known that HIS acts through G protein-coupled receptors. The H1/H4 receptors are associated with a G_q subunit, which involves PKC signaling, a pathway related to the apoptotic process. Interestingly, we demonstrated for the first time that HIS prevents DC apoptosis induced by heat shock through the inhibition of caspase-3, a mechanism dependent on PKC activation, since it is reversed by its inhibition. By contrast, cytolytic activity of T lymphocytes induced by HIS-stimulated DC was independent of PKC pathway.

Figure 1

Histamine increases OVA-degradation timing. In (a), we show the ratio of OVA/β-actin at different times after HIS (0.1 μM, 37°C) stimulation. Bars represent the mean ± SE, N = 4 experiments (^∗p < 0.05). In (b), a representative Western blot is shown.

Figure 2

Histamine modulates presentation via the vacuolar pathway. (a), (i) A representative dot plot of DC region used for the analysis is shown. (a), (ii) The mean percentage of OVA257-264-H2K^b+ DC is significantly higher for HIS-stimulated DC. (b) We show the colocalization at the membrane of DC of class I molecules and the generated OVA epitope. (c) DC were incubated with Epo or Baf for 90 min at 37°C and then treated or not with HIS (0.1 μM). After 20 min, cells were incubated in the presence of OVA-FITC (100 μg/ml). Finally, after 2 h of culture, DC were washed and stained with anti-mouse SIINFEKL-H2K^b for 20 min at 4°C. Bars represent the % mean of OVA_257–264-H2K^b+ cells ± SE of 8 independent experiments for DC inhibited with Epo (i) and Baf (ii) (^∗p < 0.05, ANOVA and Tukey's test).

Figure 3

Histamine increases the ability of DC to activate CD8⁺ cytotoxicity. Histograms show the cytotoxicity after 24 h (a) and 48 h (b) of in vitro coculture of OVA-immunized splenic mononuclear cells (5 × 10⁶) with OVA loaded-DC stimulated or not with HIS (1.25 × 10⁵). Bars represent the mean Cx percentage ± SE of 6 independent experiments. In (c), we show a representative in vitro Cx histogram plot. The cytotoxicity activity in vivo was evaluated in splenocytes obtained after 4 h of OVA loaded DC transfer (1 × 10⁷ cells, injected i.v. to OVA-immunized mice). In (d), the bars represent the mean percentage ± SE of 5 independent in vivo experiments. p < 0.05, Student's t-test. (e) A representative histogram is shown. (f) 10⁷ loaded DC were transferred to OTI-mice after its treatment with Baf (0.1 μM) for 90 min at 37°C. Then, DC were treated or not with HIS (0.1 μM). Bars represent the mean cytotoxicity percentage ± SE of 5 independent experiments (^∗p < 0.05, ANOVA Tukey's test).

Figure 4

CD8⁺ T lymphocyte degranulation is higher in cocultures with HIS-treated DC. Degranulation was assessed by LAMP-1 fluorescent staining and flow cytometry. (a) Bars represent the mean LAMP⁺CD8⁺ percentage ± SE of 4 experiments. ^∗p < 0.05, Student's t-test. (b) A representative dot plot is shown.

Figure 5

Histamine does not have an effect in Akt or P38 phosphorylation. DC were stimulated or not with HIS (0.1 μM) and protein expression was analyzed by Western blot in lysates obtained after 10 min of incubation at 37°C. In (a), we show a representative gel. The histograms represent the quantification of Akt (b) and pP38 (c) expressed as the protein/β-actin ratio. Bars represent the means ± SE, n = 4.

Figure 6

Histamine modulates PKC and caspase activation. (a), (i) Bars represent the mean ± SE of 8 experiments (^∗p < 0.05, Student's t-test). (a), (ii) A representative Western blot of PKC is shown. In (b), (i) and (ii), we show the quantification of procaspase-3 and caspase-3, respectively. Bars represent the mean ratio ± SE of 5 experiments, ^∗p < 0.05, Student's t-test. (b), (iii) We show a representative gel for procaspase-3 and cleaved caspase-3. (c) Analysis of cleaved caspase-3 at different time points was performed. We show one representative blot.

Figure 7

Histamine prevents DC apoptosis. We performed an assay with Annexin-V and propidium iodide. (a) Bars represent mean percentage Annexin⁺ cells ± SE, n = 5. ^∗∗p < 0.05, Student's t-test. (b) A representative dot plot is shown.

Figure 8

PKC modulates the apoptosis of DC stimulated by HIS. DC were treated with or without Baf (0.1 μM). After 90 min, DC were stimulated or not with HIS (0.1 μM). In (a), bars represent the quantification of PKC as the mean ± SE of 5 obtained from Western blot experiments, p < 0.05, ANOVA and Tukey's test. In (b), a representative gel is shown. (c) Histograms show the cytotoxicity after 48 h of in vitro coculture of OVA-immunized splenic mononuclear cells (5 × 10⁵) with OVA loaded-DC stimulated or not with HIS (1.25 × 10⁵). Bars represent the mean of cytotoxicity percentage ± SE of 6 independent experiments. p < 0.05, ANOVA, and Tukey's test. In figure (d), after inhibition of PKC pathway, HIS-stimulated DC were exposed to 42°C. Finally, apoptosis was analyzed 24 h later by flow cytometry. In (d), bars represent the mean ± SE, n = 5 (p < 0.05, ANOVA Tukey's test). In (e), a representative dot plot is shown with Annexin-V and propidium iodide. (f) TNF-α production in DC cultures was analyzed in the supernatants of apoptotic cells at 24 h by ELISA. The bars represent the mean ± SEM of 4 experiments. The figure shows mean concentration values (pg/ml). Asterisks indicate statistical significance (^∗p < 0.05, ANOVA and Tukey's posttest).