Werner Luttmann

Dendritic cell subsets in human bronchoalveolar lavage fluid after segmental allergen challenge

Background: Dendritic cells control pulmonary immune reactions. Characteristics of dendritic cells in human bronchoalveolar lavage fluid (BALF) after allergen challenge are unknown. Methods: 7 patients with allergic asthma (median 23 years, range 19–25 years) underwent segmental challenge and were lavaged 10 min and 24 h after challenge. Dendritic cell subsets and surface markers in BALF and in peripheral blood were analysed using four-colour flow cytometry. Results: Plasmacytoid dendritic cells (pDCs, median 0.06%, range 0.01–0.08%) and myeloid dendritic cells (mDCs, median 0.47%, range 0.27–0.87%) were detectable in BALF from control segments. CD1a-positive dendritic cells in BALF were identified as a subpopulation of mDCs. Both pDCs (median 0.56%, range 0.09–1.83%) and mDCs (median 1.82%, range 0.95–2.29%) increased significantly in BALF 24 h (p = 0.018 compared with the control segments for pDCs and mDCs), but not 10 min, after allergen challenge. The percentage increase in pDCs was higher than that of mDCs after allergen challenge, as reflected by an enhanced pDC:mDC ratio after allergen challenge. In peripheral blood, there was a significant decrease in mDCs (p = 0.038) and a trend to a decrease in pDCs (p = 0.068) 24 h after allergen challenge. Analysis of dendritic cell surface molecules showed that after allergen challenge, BALF dendritic cells have a less mature phenotype compared with BALF dendritic cells from control segments. Conclusion: Using a comprehensive strategy to analyse dendritic cell subsets in human BALF, we have shown for the first time that both myeloid and plasmacytoid dendritic cells accumulate in the airway lumen after allergen challenge in patients with asthma.

Granzyme K : a novel mediator in acute airway inflammation

Background: Granzymes are a subfamily of serine proteases involved in the pathogenesis of many inflammatory disorders. In contrast with granzyme A and B, the role of granzyme K (GrK) in human lung diseases is unknown. Therefore, the release and expression of GrK in allergic asthma, chronic obstructive pulmonary disease (COPD) and bronchopneumonia were investigated. Methods: Soluble GrK was quantified using an enzyme linked immunosorbent assay in the bronchoalveolar lavage fluid of patients with allergic asthma (before and after segmental allergen challenge), and in patients with mild COPD, pneumonia and in healthy controls. The molecular form of GrK was analysed by western blot. Flow cytometry was performed to determine the cellular expression of GrK. Results: Compared with healthy controls, there were normal levels of soluble GrK in the bronchoalveolar lavage fluid of patients with COPD, and patients with allergic asthma before allergen challenge. In contrast, soluble GrK was strongly increased in the bronchoalveolar lavage fluid of patients with acute bronchopneumonia. In patients with allergic asthma, there was a significant increase in soluble GrK as well as in GrK expressing CD8+ T cells in the bronchoalveolar lavage fluid 24 h and 72 h after allergen challenge. After allergen challenge, soluble GrK correlated with the percentage of GrK expressing CD8+ T cells. Finally, it was shown that the endobronchial release of the CCR5 ligand CCL3 might be a mechanism for the recruitment of GrK+CD8+ T cells after allergen challenge. Conclusion: These data provide the first evidence that expression of GrK is upregulated in acute airway inflammation, both in infectious and non-infectious diseases.

Decrease of cytotoxic T cells in allergic asthma correlates with total serum immunglobulin E

Background:  Allergic asthma has been linked to an increase in T‐helper type 2‐like cytokines and T cells, but there is growing evidence for a role of lymphocyte‐mediated cytotoxic mechanisms in the pathogenesis of asthma. Therefore, we investigated the cytotoxic potential of different lymphocyte subpopulations in patients with allergic asthma.

Interferon‐γ secretion of peripheral blood CD8+ T lymphocytes in patients with bronchial asthma: In vitro stimulus determines cytokine production

It has been postulated that T lymphocytes orchestrate the chronic inflammation in bronchial asthma. In animal models, infiltration of CD8+ T lymphocytes into the bronchial mucosa prevented bronchial hyperresponsiveness and decreased early and late phase reaction. IFN‐γ antagonizes IL‐4‐dependent IgE production as well as IL‐5‐induced proliferation and activation of eosinophils. We therefore investigated the secretion of IFN‐γ of isolated CD8+ T lymphocytes from peripheral blood of patients with allergic asthma (n = 6) and from healthy controls (n = 7) in vitro. In this setting we compared the effect of stimulation with anti‐CD3 antibodies with that of phorbol myristate acetate (PMA) and calcium‐ionophore. As expected, CD8+ T lymphocytes from peripheral blood of healthy volunteers produced significantly more IFN‐γ in the presence of PMA and calcium‐ionophore than after stimulation with anti‐CD3 antibodies. However, in subjects with allergic asthma, IFN‐γ secretion of CD8+ T cells was significantly higher when incubated with anti‐CD3 antibodies than after activation with PMA and calcium‐ionophore. While IFN‐γ secretion of CD8+ T lymphocytes of patients with allergic asthma was lower than that of healthy controls in the presence of PMA/calcium‐ionophore, it was significantly elevated when compared with normal controls after stimulation with anti‐CD3 antibodies. Thus, potent activators of cytokine secretion, such as PMA and calcium‐ionophore, induce a cytokine profile different from that induced by weaker stimulants, such as anti‐CD3 antibodies. These findings have implications for further studies investigating cytokine production of inflammatory cells in vitro.

CD8+ T cell activation and differentiation in allergic asthma and the impact of cytomegalovirus serological status

Allergic asthma is a chronic inflammatory T helper 2 (Th2)‐associated disease. There is evidence that the atopic milieu affects the development of CD8+ T cells in patients. We therefore analysed activation and differentiation states of CD8+ T cells in asymptomatic patients regarding the cytomegalovirus serological status. Memory CD8+ T cells (CCR5highCD3+CD8+), memory/effector cells (CD27+CD28–CD3+CD8+), effector cells (CD27–CD28–CD3+CD8+) and activated CD8+ T cells (CD11b+CD3+CD8+) were identified by flow cytometry in peripheral blood of 19 (seven cytomegalovirus (CMV)+/12 CMV–) patients with allergic asthma (AA) and 21 (seven CMV+/14 CMV–) healthy controls (HC). Effector and activated CD8+ T cells were significantly elevated in CMV+ HC compared to CMV– HC. There was a non‐significant trend for reduced percentages of effector CD8+ T cells in CMV+ AA (median: 10·4%, range: 4·4–33·8%) compared to CMV+ HC (median: 23·1%, range: 10·7–54·1%; P = 0·128) and in CMV– AA (median: 4·1%, range: 0·6–13·4%) compared to CMV– HC (median: 5·7%, range: 0·2–17·0%; P = 0·085). Activated CD8+ T cells were reduced significantly in CMV+ AA (median: 17·0%, range: 6·0–29·4%) compared to CMV+ HC (median: 40·4%, range: 18·9–67·0%; P = 0·004) and showed a non‐significant trend in CMV– AA (median: 15·0%, range: 2·9–24·0%) compared to CMV– HC (median: 20·2%, range: 5·8–71·0%; P = 0·060). Activated CD8+ T cells are significantly reduced in CMV+ patients with allergic asthma. Furthermore, a trend for an impaired terminal CD8+ T cell differentiation is observed in CMV+ and CMV– patients with asthma.

Increase in Ksp37-positive peripheral blood lymphocytes in mild extrinsic asthma

Killer‐specific secretory protein of 37 kDa (Ksp37), identified as a Th1/Tc1 specific secretory protein is expressed preferentially in cytotoxic T lymphocytes (CTL) and natural killer (NK) cells and might be involved in essential processes of CTL‐mediated immunity. Although extrinsic asthma is linked currently to a Th2‐dominated pathogenesis, there is increasing evidence for Th1/Tc1‐mediated processes in the aetiopathology of asthma. CTL from patients with asthma have been shown to express cytokines and effector molecules which were different from healthy controls. We hypothesized that Ksp37 could indicate the involvement of CTL in the pathogenesis of extrinsic asthma. We therefore investigated Ksp37 expression in PBMC from patients with mild extrinsic asthma (n = 7) and healthy controls (n = 7). Flow cytometric analysis was used to quantify Ksp37+ cells and to investigate cellular Ksp37 expression as relative mean fluorescence intensities (MFI). We found a significantly (P = 0·016) higher percentage of Ksp37+ cells within the total lymphocyte population obtained from patients with mild extrinsic asthma compared with healthy controls. Subdifferentiation revealed a significant difference limited exclusively to the CD8+ subset (P = 0·010). In addition, Ksp37 secretion from cultured peripheral blood mononuclear cells (PBMC) and MFI of Ksp37+ lymphocytes were increased in patients with asthma compared with healthy controls. We conclude that mild extrinsic asthma appears to be associated with an increased expression of the Tc1 related protein Ksp37. The functional role of Ksp37 in the pathogenesis of asthma remains to be elucidated.

Interleukin-4 suppresses the cytotoxic potential of in vitro generated, adaptive regulatory CD4+ T cells by down-regulation of granzyme B

Regulatory CD4+ T cells (Tregs) control immune responses using secretion of anti‐inflammatory cytokines and/or cytotoxic mechanisms and play a central role in the outcomes of several immune pathologies. Previous studies suggest an impaired function of Tregs in allergy, especially during allergen seasons, but the underlying mechanism is not known. Therefore, we analysed the impact of the T helper type 2 cytokine interleukin (IL)‐4 on in vitro generated adaptive Tregs (aTregs), which have been reported to use the granzyme B (GrB)/perforin pathway to kill autologous immune cells. aTregs were generated by co‐ligation of CD3 and CD46 on CD4+ T lymphocytes and granzyme expression was analysed using flow cytometry. To quantify GrB and perforin expression as well as IL‐10 secretion in response to IL‐4, specific enzyme‐linked immunosorbent assays were performed in cell lysates and/or culture supernatants. Using a flow cytometry‐based cytotoxicity assay the impact of IL‐4 on the cytotoxic potential of aTregs was investigated. While IL‐4 did not affect IL‐10 secretion and perforin expression in aTregs, a significant suppression of GrB synthesis was detected in the presence of IL‐4. In addition, IL‐4‐mediated suppression of GrB led to impaired cytotoxicity of aTregs against K562 target cells. In conclusion, our data suggest that IL‐4 might play a role in impaired aTreg function in allergy.