Effect of CD8+ T-cell depletion on bronchial hyper-responsiveness and inflammation in sensitized and allergen-exposed Brown-Norway rats

Abstract

We examined the role of CD8+ T cells in a Brown-Norway rat model of asthma, using a monoclonal antibody to deplete CD8+ T cells. Ovalbumin (OA)-sensitized animals were given anti-CD8 antibody (0.5 mg/rat) intravenously 1 week prior to exposure to 1% OA aerosol and were studied 18-24 hr after aerosol exposure. Following administration of anti-CD8 antibody, CD8+ cells were reduced to <1% of total lymphocytes in whole blood and in spleen. In sensitized animals, OA exposure induced bronchial hyper-responsiveness (BHR), accumulation of eosinophils, lymphocytes and neutrophils in bronchoalveolar lavage (BAL) fluid, and also an increase in tissue eosinophils and CD2+, CD4+ and CD8+ T cells in airways. Anti-CD8 antibody caused a further increase in allergen-induced BHR (P<0.03, compared with sham-treated animals), together with a significant increase in eosinophil number in BAL fluid (P<0.05). While CD2+ and CD4+ T cells in airways were not affected by anti-CD8 treatment, the level of CD8+ T cells was significantly reduced in sensitized, saline-exposed animals (P<0.04, compared with sham-treated rats), and sensitized and OA-challenged rats (P<0.002, compared with sham-treated rats). Using reverse transcription-polymerase chain reaction, an increase of T helper (Th)2 cytokine [interleukin (IL)-4 and IL-5], and also of Th1 cytokine [interferon-gamma (IFN-gamma) and IL-2], mRNA in the lung of sensitized and OA-exposed animals was found; after CD8+ T-cell depletion, Th1 cytokine expression was significantly reduced (P<0.02), while Th2 cytokine expression was unchanged. CD8+ T cells have a protective role in allergen-induced BHR and eosinophilic inflammation, probably through activation of the Th1 cytokine response.

Figure 1

Flow cytometric analysis of CD4⁺ and CD8⁺ T lymphocytes in peripheral blood (a) and spleen (b) for four different groups of rats. NO: non-sensitized and OA exposed, n =7; SScd8: sensitized, anti-CD8 antibody treated and saline exposed, n =4; SOsham: sensitized, mouse immunoglobulin G (IgG) treated and 1% ovalbumin (OA) challenged, n =10; and SOcd8: sensitized, anti-CD8 antibody treated and OA exposed, n =10. Cell number is expressed as percentage of total lymphocytes measured by expression of CD3⁺ T cells. There was a significant decrease in the proportion of splenic CD4⁺ T cells in the sensitized and OA-exposed rats. CD8⁺ cells were reduced to less than 1% by anti-CD8 treatment in both peripheral blood and spleen. †P < 0·03 compared with groups SScd8 and SOcd8; **P < 0·04 compared with groups NO and SScd8. Data are shown as mean±SEM.

Figure 2

(a) Mean percentage increase in lung resistance to increasing concentrations of acetylcholine (ACh) for four different groups of rats, described in the legend to Figure 1 (NO, SScd8, Sosham and SOcd8). The concentration–response curves are significantly shifted left for groups SOsham and SOcd8 by comparison with group NO. There was no effect of anti-CD8 antibody on non-sensitized ovalbumin (OA)-exposed animals, but the antibody further increased bronchial responsiveness of sensitized, allergen-exposed rats. *P < 0·05 for groups SOsham or SOcd8 compared with groups NO and SScd8; †P < 0·03 for SOsham compared with group SOcd8. (b) Mean −logPC₂₀₀, which is the negative logarithm of the provocative concentration of ACh needed to increase baseline lung resistance by 200%, for the four groups of rats detailed in Figure 1. Anti-CD8 antibody treatment significantly enhanced allergen-induced increase in −logPC₂₀₀ (P < 0·03). φP < 0·005 compared with groups NO and SScd8. Data are shown as mean±SEM.

Figure 3

Mean numbers of total cells, macrophages (Mac), eosinophils (Eos), lymphocytes (Lym) and neutrophils (Neu) in bronchoalveolar (BAL) fluid from four different groups of rats (described in the legend to Figure 1). Anti-CD8 antibody treatment further enhanced the increase in eosinophil number induced by allergen exposure of sensitized rats in BAL fluid (P < 0·02), but had no effect on influx of lymphocytes and neutrophils. **P < 0·04 compared with groups NO and SScd8. Data are shown as mean±SEM.

Figure 4

Mean eosinophil and T-lymphocyte subset (CD₂⁺, CD₄⁺ and CD₈⁺) counts in airway submucosa (expressed per mm²) of the four groups of rats described in the legend to Figure 1. Allergen exposure of sensitized rats increased the infiltration of eosinophils, as well as of T-cell subsets (CD₂⁺, CD₄⁺ and CD₈⁺). There was a reduction in CD8⁺ cell counts after anti-CD8 antibody treatment in both saline and ovalbumin (OA)-exposed animals; however, in the sensitized OA-exposed rats, anti-CD8 antibody did not completely suppress CD8⁺ T cells in tissue. CD2⁺ and CD4⁺ T-cell counts were not significantly affected, and eosinophil counts were non-significantly increased after anti-CD8 antibody treatment of sensitized and OA-exposed rats. *P < 0·05 as SOsham or SOcd8 compared with NO and SScd8 groups; ††P < 0·02 compared with the NO group. Data are shown as mean±SEM.

Figure 5

Mean interleukin (IL)-2, IL-4, IL-5, IL-10 and interferon-γ (IFN-γ) mRNA expression in rat lung, expressed as a ratio to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (a), as determined by reverse transcription–polymerase chain reaction (RT–PCR), followed by Southern blot analysis. Representative bands from Southern blot analysis are shown in (b). The expression was obtained on a radioactive probe-hybridized dot-blot of the PCR products. There was a significant increase in the expression of IL-2, IL-4, IL-5 and IFN-γ following allergen exposure of sensitized rats (group SOsham). Anti-CD8 antibody significantly reduced the increased expression of IL-2 and IFN-γ mRNA, but was without effect on IL-4 and IL-5 mRNA expression. The IL-10 mRNA expression was increased following both saline and OA exposure of sensitized rats after anti-CD8 antibody treatment. †P < 0·03 compared with groups NO and SScd8; ††P < 0·02 compared with group NO. Data are shown as mean±SEM.