Differential regulation of PD-1 and its ligands in allergic asthma.

BACKGROUND: Targeting PD-1/PD-1 ligand signalling is an established treatment option for cancer. The role of these molecules in allergic asthma has been investigated in several mouse studies yielding conflicting results. However, human studies investigating the expression and regulation of PD-1 and its ligands in allergic inflammation are lacking. OBJECTIVE: To analyse the expression and regulation of PD-1 and its ligands in human allergic asthma. METHODS: The well-established human asthma model of segmental allergen challenge (SAC) was used to analyse the regulation of PD-1 and its ligands PD-L1 and PD-L2 on T lymphocytes and dendritic cells by flow cytometry. The impact of immunoglobulin E (IgE)-mediated signalling on PD-L1 expression was analysed on isolated plasmacytoid dendritic cells (pDCs). RESULTS: PD-1 expression by blood CD4+ T cells was negatively associated with total and specific (against the allergen used for provocation) IgE serum concentrations. Twenty-four hours after SAC, a small decrease in endobronchial PD-1+ CD4+ T cells was accompanied by an increase in PD-L1 expression on endobronchial myeloid dendritic cells (mDCs) and pDCs. The PD-L1 up-regulation on pDCs was not induced by IgE-mediated mechanisms. In contrast, PD-L2 was only detected on endobronchial mDCs and was significantly down-regulated 24 hours after SAC. CONCLUSION AND CLINICAL RELEVANCE: This study shows, for the first time, an association of a low PD-1 expression by circulating CD4+ T cells with high total and specific (against the allergen used for provocation) IgE concentrations in allergic asthma. In addition, we demonstrate a differential regulation of PD-1 ligands on endobronchial DCs after allergen challenge which may favour Th2 inflammation. Therefore, modulating PD-1 ligand-mediated pathways might be a promising target in allergic asthma.

The dendritic cell high-affinity IgE receptor is overexpressed in both asthma and severe COPD.

BACKGROUND: The reduction of asthma exacerbations following omalizumab treatment has been related to the suppression of the high-affinity IgE receptor (FcεRI) on plasmacytoid dendritic cells (DCs). However, the FcεRI expression on DCs in chronic obstructive pulmonary disease (COPD) is unknown. OBJECTIVE: To compare FcεRI expression on DCs in COPD with patients with allergic asthma and healthy controls, and to relate the findings to clinical parameters, blood eosinophil concentrations and serum immunoglobin E (IgE) concentrations. METHODS: Using four-colour flow cytometry, FcεRI expression on blood myeloid DCs and plasmacytoid DCs was analyzed in 64 patients with COPD, 20 patients with allergic asthma, 41 asymptomatic never smokers and 21 asymptomatic current smokers. RESULTS: As compared with never smokers, current smokers displayed an increased expression of the FcεRI on myeloid and plasmacytoid DCs. In patients with COPD, the expression of the FcεRI on plasmacytoid DCs, but not myeloid DCs, increased from spirometric GOLD stage 2 to GOLD stage 4, and was correlated with several lung function parameters. Patients with severe COPD and patients with allergic asthma displayed a similar FcεRI overexpression on plasmacytoid DCs. In all groups, there was a positive correlation between total IgE serum concentrations and the FcεRI expression on plasmacytoid DCs. CONCLUSIONS AND CLINICAL RELEVANCE: Severe COPD and allergic asthma are characterized by a similar overexpression of the high-affinity IgE receptor on plasmacytoid DCs. In view of the effect of anti-IgE on exacerbations in asthma, trials investigating the effect of anti-IgE on exacerbations in severe COPD appear to be warranted.

Vitamin D binding protein and vitamin D in human allergen-induced endobronchial inflammation.

Allergic asthma is a chronic disease of the airways associated with airway hyperresponsiveness, a variable degree of airflow obstruction, airway remodelling and a characteristic airway inflammation. Factors of the vitamin D axis, which include vitamin D metabolites and vitamin D binding protein (VDBP), have been linked to asthma, but only few data exist about their regulation in the lung during acute allergen-induced airway inflammation. Therefore, we analysed the regulation of factors of the vitamin D axis during the early- and late-phase reaction of allergic asthma. Fifteen patients with mild allergic asthma underwent segmental allergen challenge. VDBP was analysed in bronchoalveolar lavage fluid (BALF) and serum using the enzyme-linked immunosorbent assay (ELISA) technique. 25-hydroxyvitamin D(3)[25(OH)D(3)] and 1,25-dihydroxyvitamin D(3)[1,25(OH)(2)D(3)] were analysed by a commercial laboratory using the liquid chromatography-mass spectrometry (LC/MS) technique. VDBP (median 2·3, range 0·2-7·1 µg/ml), 25(OH)D(3) (median 0·060, range < 0·002-3·210 ng/ml) and 1,25(OH)(2)D(3) (median < 0·1, range < 0·1-2·8 pg/ml) were significantly elevated in BALF 24 h but not 10 min after allergen challenge. After correction for plasma leakage using the plasma marker protein albumin, VDBP and 25(OH)D(3) were still increased significantly while 1,25(OH)(2)D(3) was not. VDBP and 25(OH)D(3) were correlated with each other and with the inflammatory response 24 h after allergen challenge. Serum concentrations of all three factors were not influenced by allergen challenge. In conclusion, we report a significant increase in VDBP and 25(OH)D(3) in human BALF 24 h after allergen challenge, suggesting a role for these factors in the asthmatic late-phase reaction.

Plasmacytoid dendritic cells in allergic asthma and the role of inhaled corticosteroid treatment.

BACKGROUND: Plasmacytoid dendritic cells (pDCs) infiltrate sites of acute Th2-dominant inflammation, but their role in allergic asthma remains unclear. OBJECTIVE: To characterize circulating pDCs from patients with allergic asthma outside their respective allergen season. METHODS: Adhesion molecules, co-stimulatory molecules, immunoglobulin receptors and chemokine receptors were quantified on blood pDCs from 20 patients with allergic asthma and 18 healthy controls using flow cytometry. In addition, IL-6-, TNF-α- and IFN-α-secretion were analysed after stimulating isolated pDCs with TLR7- and TLR9-ligands. RESULTS: Plasmacytoid dendritic cells from patients with allergic asthma showed an increased expression of chemokine receptors involved in inflamed tissue homing such as CCR2, CCR4, CCR9, CCR10, CXCR2, CXCR5 and CXCR6, while the expression of the lymph node homing receptor CXCR3 was down-regulated. In addition, these pDCs exhibited a higher expression of activation markers and Th2-associated molecules such as CD40, CD62L, CD64 and FcεRIα. In contrast, TLR7-mediated IL-6-, TNF-α- and IFN-α-secretion was significantly reduced in pDCs from patients with asthma. The TLR9-mediated cytokine response was only suppressed in those patients who were treated with inhaled corticosteroids (ICS) during previous allergen seasons. The same effect was observed for CD54 and OX40L expression. CONCLUSIONS: We report an increased expression of activation markers, and Th2-associated molecules, and an increased migratory potential of circulating pDCs in allergic asthma. These changes are accompanied by a reduced TLR7-mediated cytokine response. In addition, our results suggest a longterm impact of ICS treatment on the characteristics of circulating pDCs.

Differential development of plasmacytoid dendritic cells in Th1- and Th2-like cytokine milieus.

BACKGROUND: Plasmacytoid dendritic cells (pDCs) infiltrate sites of Th1- and Th2-dominant inflammation and many studies have been performed to analyse their role in these immune responses. In contrast, much less is known about the effects of a Th1 or Th2 cytokine milieu on pDC function. Therefore, we investigated the impact of Th1- and Th2-like conditions during the development of pDCs on their antigen expression and function. METHODS: PDCs were matured in vitro by the addition of IL-3 under Th1- or Th2-like conditions. Antigen expression and TLR7-ligand-induced cytokine secretion was analysed by flow cytometry and ELISA. Furthermore, the CD4(+) T-cell polarizing capacity of pDCs was determined as well as their potential to induce CD4(+) T-cell proliferation. RESULTS: PDCs matured under Th1-like conditions showed a higher expression of antigens involved in T-cell co-stimulation and antigen presentation like CD40, CD80, CD83 and HLA-DR as well as a higher secretion of IL-6 and IFN-α in response to TLR7-ligation compared to Th2-pDCs. Furthermore, Th1-pDCs induced a significantly higher CD4(+) T-cell proliferation and primed a higher percentage of CD4(+) T cells to express IFN-γ and IL-2 after TLR7-ligation compared to Th2-pDCs. In contrast, Th2-pDCs were characterized by a significant upregulation of BDCA-3 and IL-4 expression following TLR7-ligation. CONCLUSION: This study is the first to demonstrate the crucial impact of a surrounding cytokine environment on the development of pDC function including antigen expression. Based on these findings, it can be speculated that antiviral/bacterial pDC functions could be impaired during acute allergic conditions.

Functional expression of granzyme B in human plasmacytoid dendritic cells: a role in allergic inflammation.

BACKGROUND: Plasmacytoid dendritic cells (pDCs) are involved in a variety of immune functions. However, the expression of cytotoxic granule proteins like granzymes and perforin in human pDCs is still poorly understood. OBJECTIVE: The aim of this study was to systematically analyse the expression and regulation of cytotoxic granule proteins in human pDCs. METHODS: The expression of cytotoxic proteins was analysed by RT-PCR, flow cytometry, and fluorescence microscopy. The functional expression of these proteins was confirmed in a flow-cytometry-based cytotoxicity assay using K562 cells as targets. In order to analyse the regulation of pDC-derived cytotoxic proteins in infectious and allergic diseases, human pDCs were analysed after stimulation with toll-like receptor (TLR)7/9 ligands and in the human asthma model of segmental allergen challenge. RESULTS: Granzyme B (GrB), but not the granzymes A, H, K, M or perforin, was specifically expressed by human pDCs and this GrB expression was up-regulated by IL-3 stimulation. In addition, IL-3-stimulated pDCs were found to kill K562 cells in a GrB- and caspase-dependent manner. TLR7/9 ligands significantly suppressed GrB expression in pDCs. In contrast, there was an up-regulation of GrB in endobronchial pDCs 24 h after allergen challenge, and this was accompanied by enhanced GrB concentrations in bronchoalveolar lavage fluid. CONCLUSION: We report the selective expression of GrB in human pDCs and show for the first time pDC-mediated GrB- and caspase-dependent cytotoxicity against target cells. In addition, the regulation of GrB expression was investigated in vitro and in vivo providing an evidence for a specific role of pDC-derived GrB in allergic inflammation.

sCD14 in bronchoalveolar lavage 18, 42 and 162 hours after segmental allergen provocation.

Lipopolysaccharides (LPS) have been associated with a protective role in the development of asthma while higher levels of endotoxin have been linked with more severe asthma. LPS recruit neutrophils and eosinophils and activate macrophages via the CD14 receptor. The soluble CD 14 receptor (sCD14) has been found in bronchoalveolar lavage fluid in different diseases including allergic asthma. To elucidate the kinetics and the regulation of sCD14 concentrations in BAL in asthma, 18 patients with allergic asthma underwent segmental allergen challenge at different time points (10 min, 18, 42 and 162 h). In addition, CD14(+) peripheral blood mononuclear cell (PBMC-CD14(+)) cultures from seven allergic and seven non-allergic subjects were stimulated with LPS, leukotrien D(4) (LTD(4)), a combination of LPS and LTD(4), IL-17 and LTD(4) in presence of the leukotriene-receptor antagonist (LTRA) Montelukast for 6, 12 and 24 h. sCD14 concentrations in BAL and the supernatants were measured by ELISA. sCD14 concentrations in BAL were significantly increased 18 h after allergen challenge and peaked at 42 h. At 162 h, concentrations had returned to baseline levels. In PBMC-CD14(+) cultures, sCD14 levels increased significantly 24 h after stimulation with LTD(4) and Montelukast was able to block LTD(4)-induced stimulation. Allergen challenge leads to a significant increase in sCD14 concentrations in BAL and might modulate the allergen-induced inflammation. In addition, LTD(4) might play a role in the release of sCD14, and it could be speculated that sCD14 reduction by LTRA might contribute to the mechanisms of LTRA in the treatment of allergic asthma.

Acute effects of tobacco smoke on human airway dendritic cells in vivo.

Airway dendritic cells (DCs) play a key role in smoke-related lung diseases; however, the acute effects of tobacco smoke on human airway DCs in vivo are unknown. A total of 16 smokers underwent bronchoalveolar lavage at two time-points: directly after a 4-h period of nonsmoking (no smoke exposure); and directly after a 4-h period during which eight cigarettes were smoked (acute smoke exposure). Using flow cytometry, myeloid DCs (mDCs) and plasmacytoid DCs (pDCs), as well as function-associated surface molecules on mDCs, were analysed in bronchoalveolar lavage fluid (BALF) and in blood. The numbers of macrophages, lymphocytes, neutrophils, eosinophils and pDCs were unchanged in BALF following acute smoke exposure, as compared to no smoke exposure. In contrast, there was a strong increase in mDC number in BALF and a concomitant decrease in mDC number in blood following acute smoke exposure. In addition, acute smoke exposure led to an increase in the expression of the surface molecules blood dendritic cell antigen 1 and 4 and a decrease in the expression of the lung homing receptor, CC chemokine receptor 5, on mDCs in BALF. Acute tobacco smoke inhalation results in an immediate and selective recruitment of mDCs into human airways, which might reflect the very early reaction of the adaptive immune system to smoke exposure.

Adverse effects of salmeterol in asthma: a neuronal perspective.

BACKGROUND: Regular use of inhaled beta(2)-agonists has been associated with a paradoxical loss of asthma control and a deterioration of airway hyper-responsiveness, but the underlying mechanism is unknown. The neurotrophin brain-derived neurotrophic factor (BDNF) has recently been identified as a mediator of airway hyper-responsiveness in asthma. METHODS: Eighteen patients with mild allergic asthma who did not use any regular antiasthmatic therapy inhaled the long-acting beta(2)-agonist salmeterol for 2 weeks followed by 2 weeks of combination therapy with salmeterol and the corticosteroid fluticasone. Airway responsiveness to histamine and BDNF concentrations in blood were assessed prior to entry, after 14 days of salmeterol therapy and after 14 days of combination therapy. In a separate experiment, salmeterol effects on BDNF release by human peripheral blood mononuclear cells were assessed. RESULTS: Monotherapy with salmeterol significantly increased BDNF concentrations in serum and platelets. This increase was abolished by the addition of fluticasone to the treatment. The findings were confirmed in vitro: salmeterol increased the release of BDNF by mononuclear cells, and this was inhibited by co-incubation with fluticasone. Increased BDNF concentrations in serum and platelets correlated with the deterioration of airway hyper-responsiveness following salmeterol monotherapy. In contrast, there was no association between beta(2)-receptor polymorphisms and changes in airway responsiveness. CONCLUSION: Increased BDNF concentrations may underly the adverse effects of salmeterol monotherapy on airway responsiveness in asthma. TRIAL REGISTRATION NUMBER: NCT00736801.

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.

Airway dendritic cell phenotypes in inflammatory diseases of the human lung.

Airway dendritic cells (DCs) are key regulators of pulmonary immune responses. However, information is limited regarding the characteristics of airway DCs in human lung diseases. Plasmacytoid DCs (pDCs) and myeloid DCs (mDCs) were analysed using four-colour flow cytometry in bronchoalveolar lavage fluid (BALF) from nonsmoking controls and patients with sarcoidosis, idiopathic pulmonary fibrosis (IPF) and pneumonia (in the presence or absence of immunosuppression). Compared with controls, immunocompetent patients with pneumonia displayed strongly enhanced pDC counts in BALF. In contrast, pDC counts in BALF from immunocompromised patients with pneumonia were even lower than in controls. This discrepancy was not explained by a different chemotactic milieu in the airways; all patients with pneumonia were characterised by strongly increased concentrations of the pDC-attracting chemokine, CXC chemokine ligand 10, in BALF. Patients with IPF were characterised by normal percentages of DC subtypes. However, the mDCs of patients with IPF were not as mature (CD83-positive) as those of controls. Patients with sarcoidosis displayed a unique increase in CD1a-negative mDCs in the airways. In addition, there was altered expression of costimulatory molecules (increased CD80 and decreased CD86 expression) on mDCs in patients with sarcoidosis. These data suggest that inflammatory diseases of the human lung are associated with a differential phenotype and recruitment of airway dendritic cells.

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 (CCR5(high)CD3(+)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.

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. METHODS: Granzyme A, B, K, and perforin expression in peripheral blood lymphocytes was analyzed using flow cytometry. Soluble granzymes were measured in serum using specific enzyme-linked immunosorbent assays. RESULTS: Asthmatics had significantly decreased percentages of granzyme and perforin-positive CD4 T cells compared with non-atopic controls. In patients with asthma, the granzyme B and perforin-positive subset of CD8(+) T cells and natural killer T cells, which represent more differentiated cell populations, were significantly reduced, while this was not observed in the less differentiated granzyme K(+) subsets. In addition, the serum concentrations of granzyme B were significantly reduced in patients with asthma, while granzyme K concentrations were not different. Interestingly, there was a negative correlation between granzyme A, B and perforin expression in T cell subsets as well as serum granzyme B concentrations and total serum immunglobulin E. In CD3-negative natural killer cells, no differences in granzyme or perforin expression between patients with asthma and controls were detected. CONCLUSION: In allergic asthma, cytotoxic T lymphocyte subsets of a more differentiated phenotype are significantly decreased and this is correlated to serum immunglobulin E levels.

Increase in killer-specific secretory protein of 37 kDa in bronchoalveolar lavage fluid of allergen-challenged patients with atopic asthma.

BACKGROUND: Atopic asthma is linked to a T-helper type 2 dominated pathogenesis, but there is increasing evidence of Th1/Tc1-mediated processes in the aetiopathology of asthma. Killer-specific secretory protein of 37 kDa (Ksp37) is expressed in cytotoxic lymphocytes, selectively in the effector subsets of CD8+- and CD4+ T lymphocytes and in CD16+/CD56dim natural killer cells and gamma/delta T cells. This effector cell-specific expression of Ksp37 and its coexpression with perforin suggest that Ksp37 might be involved in processes mediated by cytotoxic cells. OBJECTIVE: We hypothesize that Ksp37 could indicate the involvement of cytotoxic lymphocytes in the pathogenesis of atopic asthma, and investigated Ksp37 concentration in bronchoalveolar lavage fluid (BALF) collected 10 min, 18, 42 or 162 h after segmental allergen provocation and in serum of patients with atopic asthma (n=25). METHODS: Ksp37 concentrations in BALF and serum were detected by ELISA. Flow cytometric analysis was used to assess numbers and cell subsets in BALF. RESULTS: Ksp37 increased significantly in BALF 10 min, 18 and 42 h, but not 162 h after allergen challenge compared with saline-challenged controls, while Ksp37 serum levels did not change significantly at all time-points. In addition, the increase in Ksp37 concentrations in BALF correlated with the corresponding numbers of lymphocytes. CONCLUSIONS: We conclude that Ksp37 level increased in BALF 10 min, 18 and 42 h after allergen challenge but not in peripheral blood. Our findings suggest that segmental allergen challenge in asthma is associated with an increase in Ksp37 concentrations in BALF and an influx of potentially cytotoxic T lymphocytes into the lungs.

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.

Increase in granzyme B+ lymphocytes and soluble granzyme B in bronchoalveolar lavage of allergen challenged patients with atopic asthma.

Asthma has been linked to a chronic, T-cell-mediated bronchial inflammation. Because other T-lymphocyte-mediated, chronic inflammatory disorders have been associated with elevated granzyme B (grB) expression we tested the hypothesis that atopic asthma might be associated with elevated grB levels in the bronchoalveolar compartment. Therefore we performed intracellular grB staining in lymphocytes from bronchoalveolar lavage (BAL) collected 42 h after segmental allergen provocation (SAP) in allergic patients with bronchial asthma. There was a significant increase in CD3(+), CD8(+), and CD16/56(+) lymphocytes expressing grB in BAL 42 h after SAP as compared to saline challenged controls. However, compared to peripheral blood the percentages of these lymphocyte subsets detected as grB(+) in BAL remained significantly lower. Measurement of extracellular grB in BAL fluids by a particle immunoassay revealed significantly elevated grB levels in the allergen challenged bronchoalveolar compartment 42 h following SAP in six of the eight patients (range, <1.0-348.1 pg/ml) as compared to saline challenged controls (range, <1.0-70.5 pg/ml). We conclude that total cell numbers of grB(+) lymphocyte subsets increase 42 h after SAP in the lower respiratory tract. In addition there is evidence to suggest that grB is released into the airways of asthmatic patients. This suggests a role for grB in the pathophysiological processes following SAP but its definitive role in allergic bronchial asthma needs to be established.