Heme oxygenase-1 prevents smoke induced B-cell infiltrates: a role for regulatory T cells?

Abstract

Background: Smoking is the most important cause for the development of COPD. Since not all smokers develop COPD, it is obvious that other factors must be involved in disease development. We hypothesize that heme oxygenase-1 (HO-1), a protective enzyme against oxidative stress and inflammation, is insufficiently upregulated in COPD. The effects of HO-1 modulation on cigarette smoke induced inflammation and emphysema were tested in a smoking mouse model.

Methods: Mice were either exposed or sham exposed to cigarette smoke exposure for 20 weeks. Cobalt protoporphyrin or tin protoporphyrin was injected during this period to induce or inhibit HO-1 activity, respectively. Afterwards, emphysema development, levels of inflammatory cells and cytokines, and the presence of B-cell infiltrates in lung tissue were analyzed.

Results: Smoke exposure induced emphysema and increased the numbers of inflammatory cells and numbers of B-cell infiltrates, as well as the levels of inflammatory cytokines in lung tissue. HO-1 modulation had no effects on smoke induced emphysema development, or the increases in neutrophils and macrophages and inflammatory cytokines. Interestingly, HO-1 induction prevented the development of smoke induced B-cell infiltrates and increased the levels of CD4+CD25+ T cells and Foxp3 positive cells in the lungs. Additionally, the CD4+CD25+ T cells correlated positively with the number of Foxp3 positive cells in lung tissue, indicating that these cells were regulatory T cells.

Conclusion: These results support the concept that HO-1 expression influences regulatory T cells and indicates that this mechanism is involved in the suppression of smoke induced B-cell infiltrates. The translation of this interaction to human COPD should now be pursued.

Figure 1

HO-1 protein expression. A: Protein bands for HO-1 (above band) and β-actin (loading control) detected by western blot analysis after long term smoke exposure and protoporphyrin treatment. Three animals per group are shown. B: A representative picture of the HO-1 expression (dark red) in lung tissue is shown for each group (25×). Particularly, alveolar macrophages (indicated with a closed arrow) show an increased HO-1 expression after CoPP treatment. The epithelium stains faintly in all groups and no differences were observed between the groups. The brown cells (indicated with an open arrow) are pigmented macrophages, a result of the smoke exposure. S: smoke, SS: Sham smoke. Mice were divided into 6 groups; 1. Phosphate buffered saline (PBS) + smoke, 2. Cobalt protoporphyrin (CoPP) + smoke, 3. Tin protoporphyrin (SnPP) + smoke, 4. PBS + sham smoke, 5. CoPP + sham smoke, 6. SnPP + sham smoke.

Figure 2

Emphysema development. Mean linear intercept (LMI) after long term smoke exposure and protoporphyrin treatment. Smoke groups are represented by closed symbols and sham smoke groups by open symbols. * indicates a significant effect of smoke exposure (p < 0.05). There were no interactions and no effects of CoPP or SnPP treatment.

Figure 3

Inflammatory cytokines in lung homogenate. TNF-α, IL-1α, IL-1β, IL-6, KC and MCP-1 levels expressed as pg/g lung homogenate after long term smoke exposure and protoporphyrin treatment. Smoke groups are represented by closed symbols and sham smoke groups by open symbols. The significant results of the regression analysis are depicted beneath the figures. ^# indicates a significant effect of SnPP treatment (SnPP vs. PBS, post-hoc analysis), and ** indicates a significant effect of CoPP treatment (CoPP vs. PBS, post-hoc analysis) (p < 0.05).

Figure 4

Inflammatory cells in lung tissue. Neutrophils and macrophages expressed in lung tissue after long term smoke exposure and protoporphyrin treatment. Neutrophils are expressed as numbers per mg lung homogenate. Macrophages are expressed as volume percentages. Smoke groups are represented by closed symbols and sham smoke groups by open symbols. The significant results of the regression analysis are depicted beneath the figures.

Figure 5

B-cell infiltrates in lung tissue. A: Volume percentage of B220 positive infiltrates after long term smoke exposure and protoporphyrin treatment. Smoke groups are represented by closed symbols and sham smoke groups by open symbols. The significant results of the regression analysis are depicted beneath the figure. B: Example of a B-cell infiltrate positive for B220 (red) present in lung tissue (50×).

Figure 6

CD4⁺ CD25⁺ T cells in lung tissue. A: CD4⁺CD25⁺ T cells expressed as numbers per mg lung tissue after long term smoke exposure and protoporphyrin treatment. Smoke groups are represented by closed symbols and sham smoke groups by open symbols. The significant results of the regression analysis are depicted beneath the figure. B: Example of the flow cytometry analysis: The percentage of CD25 positive cells within the CD4⁺ T cell population is shown for a PBS (left) and a CoPP (right) smoking mouse.

Figure 7

Foxp3 positive cells in lung tissue. A: Foxp3 positive cells (red nuclear staining) present in lung tissue (100×). B: Foxp3 positive cells expressed as total numbers per mm² lung tissue after long term smoke exposure and protoporphyrin treatment. Smoke groups are represented by closed symbols and sham smoke groups by open symbols. The significant results of the regression analysis are depicted beneath the figure, as well as a trend for an effect of CoPP treatment. C: Fluorescent double staining for CD3 (green) and Foxp3 (red) in lung (upper panel, 630×) and spleen (lower panel, 400×) showing that Foxp3 specifically stains T cells. The orange cells in the lung are pigmented macrophages, which show auto fluorescence.