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Clin Exp Immunol. 2004 August; 137(2): 359–365. doi: 10.1111/j.1365-2249.2004.02540.x. | PMCID: PMC1809105 |
Increase in Ksp37-positive peripheral blood lymphocytes in mild extrinsic asthma M Kuepper,*‡** K Koester,†** K Bratke,*¶ D Myrtek,†‡ K Ogawa,§¶ K Nagata,¶ J C Virchow, JR,* and W Luttmann* *Department of Pneumology, Medical University Clinics, Rostock, Germany †Department of Pneumology, Medical University Clinics, Freiburg, Germany ‡Department of Zoophysiology, University Oldenburg, Germany §Human Gene Sciences Center, Tokyo Medical and Dental University, Tokyo, Japan ¶Department of R & D Center, BML, Saitama, Japan Accepted May 14, 2004. 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. Keywords: bronchial asthma, CD8+ T lymphocytes, effector cells, Ksp37 Extrinsic asthma is a chronic inflammatory disease of the airways and according to current hypothesis is linked to an increase in Th2- and a decrease in Th1-like cytokines. The hypothesis of allergen-specific Th2 cells as orchestrating cells of the asthmatic inflammation is based on observations that inflammatory processes following allergen challenge are associated with an increased production of Th2-specific cytokines such as interleukin-(IL)-4, IL-5 and IL-13 as well as an augmented recruitment of Th2 cells into lung tissue following allergen challenge [reviewed in 1, 2]. Accordingly, the aetiopathology of extrinsic asthma has been linked to Th2-type inflammatory mechanisms. However, there are some studies which also report an increased Th1/Tc1 cytokine response in bronchial asthma. In addition, as Th2-dependent inflammation fails to explain all features of asthma there are increasing doubts about a strictly Th2-dependent pathogenesis of extrinsic asthma. In particular, several different experiments which tried to counterbalance inflammatory Th2 effects by inducing or augmenting Th1 responses resulted in unexpected exacerbations of inflammatory symptoms of asthma [ 3– 5]. Therefore, several recent reports suggest that in addition to Th2-like cells, other inflammatory features such as Th1- and/or Tc1-like cells or their products might play a role in the pathogenesis of chronic extrinsic asthma [ 4– 7, 10, reviewed in 3, 8, 9]. An important component of Th1/Tc1-dependent immune response are cytotoxic T lymphocytes (CTL) and their release of cytotoxic proteins such as perforin and granzymes. Recently, a new Th1/Tc1-associated serum protein termed Ksp37 (killer-specific secretory protein of 37 kDa) has been characterized, which is expressed and released by lymphocyte subpopulations, primarily by CTL and which is mainly co-expressed with perforin, suggesting that Ksp37-producing cells indeed have cytotoxic properties [ 11]. Moreover, comparative studies of Ksp37 serum levels and simultaneous assessment of the number of NK and T cells suggested these T cell subsets as the main sources for Ksp37. It has been proposed that Ksp37-serum levels reflect changes in the Ksp37-expressing cell populations [ 11]. Indications for a link between CTL and asthma has been supplied by reports of increased numbers of CD8 + T cells in BAL primarily during the early asthmatic reaction, and an increased expression of interferon (IFN)- γ in stimulated blood CD8 + T cells from patients with asthma, compared with healthy controls [ 12– 14]. In addition, a recent study reported a change in the CD8 + T cell response in patients who died from asthma [ 15]. Based on these and several other pieces of evidence, suggesting that extrinsic asthma can no longer be understood as a pure Th2-dependent disease, we tested the hypothesis that Ksp37 may indicate a participation of CTL in the pathogenesis of extrinsic asthma. We therefore compared percentages of Ksp37 + cells in patients with mild extrinsic asthma and healthy controls. In addition, the expression and release of Ksp37 which has been characterized as a Th1/Tc1-associated protein [ 11], and which is expressed in cells present in asthma, was investigated further by using flow cytometry and enzyme-linked immunosorbent assay (ELISA). Because elevated concentrations of perforin, which is a known marker for cytotoxicity, were also reported in cells from asthmatic patients [ 16] we investigated the co-expression of Ksp37 with other markers of cytotoxicity, such as granzyme B and perforin. Patient characteristics As shown in Table 1, seven non-smoking, allergic subjects (four female and three male) with mild asthma, a mean age of 27·4 ± 1·3 year (range 24–33 years) and a mean FEV 1 (forced expiratory volume in 1 s) of 88·8 ± 4·4/ (74–107/, n = 5) of predicted were included in the study. Mean total IgE levels were 230 ± 84 kilo units per litre (kU/l) (range 35–691 kU/l), and all patients had positive skin prick test reactions to at least one common aeroallergen (Allergopharma, Reinbek, Germany). All subjects had a history of intermittent wheeze, chest tightness, cough and sputum production and reversible bronchoconstriction after inhalation of allergens. There was no evidence suggesting a respiratory tract infection or symptomatic allergen exposure before or at the time of the study. All subjects received inhaled β2-agonist therapy as needed. Inhaled corticosteroids were withdrawn at least 7 days prior to the study. Seven non-smoking, non-atopic subjects (four female and three male) with no chronic illness, a comparable mean age of 26·6 ± 0·8 years (range 24–31 years) and a mean FEV 1 of 99·7 ± 3·5/ (87–112/, n = 6) of predicted were included in the study. Mean total IgE levels were 15·6 ± 5·5 kilo units per litre (kU/l) (range 2–50 kU/l), and all patients had negative skin prick test reactions (Allergopharma, Reinbek, Germany) to a panel of common aeroallergen ( Table 1). All subjects gave informed consent. The study protocol was approved by the local Ethics Committee. Cells Venous blood was drawn into sterile plastic containers containing 0·2 ml EDTA (Sarstedt, Nümbrecht, Germany) and was separated on a gradient of Ficoll-Paque with a density of 1·077 g/l (Seromed, Berlin, Germany). The band of peripheral blood mononuclear cells (PBMCs) at the interface was collected, washed twice and resuspended in phosphate buffered saline (PBS) supplemented with 2/ heat-inactivated fetal calf serum (FCS). Cell culture Secretion profiles and different cellular expression of Ksp37 in 3 × 106 PBMC/ml were measured by ELISA or relative mean fluorescence intensity (MFI), respectively, after culture in RPMI-1640 supplemented with 10/ FCS, 100 IU/ml penicillin and 100 µg/ml streptomycin in the presence or absence of 5 × 10−10m PMA/10−6 M Ca-Ionophore (Sigma, Deisenhofen, Germany) as unspecific stimulus or without any stimulus as control. For assessment of relative MFIs the PBMCs were additionally cultured in the presence or absence of Brefeldin A (10 µg/ml) (Sigma), respectively. For Ksp37-specific ELISA the culture supernatants were harvested and assayed after the indicated time-points. Cell viability was assessed by flow cytometry and prior cell staining with propidiumiodide (0·5 µg/ml). Fluorochrome-labelled and unlabelled antibodies PE-labelled antibodies against IgG 1, human CD4 and human CD8 were obtained from Dako (DK), against human CD56 from PharMingen (San Diego, USA) and against human granzyme B from Pelicluster (Amsterdam, the Netherlands). FITC-conjugated antibodies against IgG 1 and human CD8 were purchased from Dako (DK) and against human perforin from Hölzel Diagnostika (Köln, Germany). APC-labelled antibodies against human CD3 were obtained from Caltag (Hamburg, D). PerCP-conjugated streptavidin was provided by PharMingen (San Diego, USA). Biotinylated and unlabelled monoclonal antibodies against human Ksp37 (clone TDA3; IgG 1 isotype) were provided from Ogawa and Nagata [ 11], antibodies to mouse- γ-globulin from Jackson Immuno Research (USA). Specificity of the biotinylated anti-hKsp37 clone TDA3 was tested prior to its experimental use. For this purpose cells were blocked with unlabelled anti-hKsp37 and unlabelled mouse- γ-globulin, respectively. Subsequently these cells were stained with biotinylated anti-hKsp37 and PerCP-conjugated streptavidin (). Flow cytometry To analyse intracellular Ksp37 expression freshly isolated PBMC were fixed in 4/ paraformaldehyde in PBS for 10 min on ice, permeabilized in 0·1/ saponine buffer and intracellularly stained with a biotinylated anti-hKsp37-MoAb (5 µg/ml) for 30 min at 4°C. After washing in saponine buffer, cells were incubated with PerCP-conjugated streptavidin. In addition, different surface-markers were detected with corresponding MoAb (labelled with FITC, PE, PerCP or APC) in order to identify cellular subpopulations. After two washing steps the stained cells were analysed on a four-colour FACSCalibur flow cytometer (BD Biosciences, Heidelberg, Germany) using LysisII software (BD Biosciences). Identical procedures were used to detect intracellular perforin and granzyme B with the exception that directly labelled antiperforin antibodies or anti-granzyme B-antibodies were used. The relative MFI of stained cells was assessed by subtraction of fluorescence signal received by IgG1 control from those of biotinylated Ksp37-specific antibody (clone TDA3) in association with PerCP–streptavidin conjugate. The affinity of the secondary reagent PerCP-conjugated streptavidin to endogenous biotin was tested before its use. Gating strategy All plots show fixed and permeabilized PBMCs (). First, the total lymphocyte subset was gated by FSC/SSC plot () and a negative control was performed by FITC-and PE-labelled IgG1 antibodies (). Subsequently, the expression of intracellular Ksp37 in different lymphocyte subsets () CD4 T cells () CD8 T cells and () CD56 NK cells, as well as co-expression of intracellular Ksp37 with perforin () and granzyme B (), respectively, was analysed as depicted in . ELISA A Ksp37-specific sandwich ELISA was performed using a plate-bound monoclonal mouse anti-hKsp37 MoAb (clone TDA3) (10 µg/ml) as capture antibody, polyclonal rabbit anti-hKsp37 antibody (5 µg/ml) as detector antibody and rKsp37 as the standard protein. The rabbit anti-hKsp37 antibody was visualized with a HRP-conjugated goat anti-rabbit IgG (Zymed, San Francisco, CA, USA) followed by peroxidase reaction with TMB (Sigma) as substrate. Statistical analysis Normally distributed data (as assessed by Shapiro–Wilks test) are expressed as arithmetic mean values ± standard error of the mean (s.e.m.). To calculate significance levels between asthmatic and control subjects the two-tailed, unpaired Student's t-test was used. Paired data were compared using the two-tailed, paired Student's t-test. Probability values of P < 0·05 were used as the generally accepted level of statistical significance for differences between mean values. Ksp37 positive cell populations by asthmatic and healthy subjects As shown in , comparison of Ksp37 positive lymphocytes revealed significantly (P = 0·016) elevated percentages of Ksp37+ lymphocytes in patients with mild extrinsic asthma compared with healthy controls (). A subsequent differentiation of the Ksp37+ lymphocyte population by specific surface markers showed that CD3–CD56+ NK cells, predominantly the CD56dim subset, had the highest percentage of Ksp37 positive cells which was, however, not significantly different between patients with mild extrinsic asthma and healthy control subjects ( and ). Ksp37 expression in patients with mild extrinsic asthma, however, was significantly elevated in the CD8+ cell population (P = 0·010), while the Ksp37+CD4+ subsets were equally low in both groups (). Co-expression of Ksp37, granzyme B and perforin To investigate whether Ksp37 and other cytotoxic effector molecules such as perforin and granzyme B are co-expressed, cells were stained intracellularly with appropriate fluorescence-labelled antibodies. While a considerable number of cells revealed co-expression of Ksp37 and perforin or Ksp37 and granzyme B, respectively, there were no significant differences between patients with asthma and healthy controls. Similarly, the analysis of the co-expression of Ksp37, perforin and granzyme B with cell surface expression of CD3 revealed no significant differences between asthmatic patients and healthy controls ( and ). Secretion profiles of Ksp37 in PBMC from asthmatic and healthy subjects To examine differences in the secretion profile of Ksp37 between PBMC obtained from asthmatic patients and healthy controls supernatants of PMA/Ca-ionophore-stimulated and unstimulated cell cultures were analysed for Ksp37 release by ELISA. Both unstimulated PBMCs from patients with mild extrinsic asthma and from healthy controls produced Ksp37 at a nearly constant rate within 24 h in vitro with slightly increased amounts in asthmatic patients compared to healthy controls. Similar results were obtained from stimulated PBMC in both groups, but the secretion profiles were different and compatible with a non-specific stimulation of cells in vitro. In the presence of PMA/calcium-ionophore there was a significant increase in the Ksp37 secretion within 12 h in both groups compared to unstimulated cultures (P-values in a range of 0·001–0·009). Moreover, non-specific stimulation with PMA/calcium-ionophore caused a rapid increase in Ksp37 concentrations which reached its maximum within the first 6 h after which it remained on this level. After 24 h Ksp37 concentrations in the supernatants from stimulated and unstimulated cells were approximately similar in both groups investigated (). Cell viability was tested at all time-points using flow cytometry and propidium iodide. There was no evidence for an increase in the number of non-viable cells at any time-point. Relative mean fluorescence intensities of Ksp37 in PBMC from asthmatic and healthy subjects To investigate further the intracellular production and release of Ksp37 the relative MFI of Ksp37+ cells cultured for 6 h in the presence of Brefeldin A which inhibits the secretion of newly synthezised proteins and PMA/calcium-ionophore as a non-specific stimulus was analysed. In the presence of Brefeldin A the relative MFI was increased significantly in all experiments (P-values in a range of 0·001–0·039). Stimulation with PMA/calcium-ionophore resulted in a significant decrease (P = 0·001) of the relative MFI after 6 h compared to unstimulated cells, suggesting a rapid release of Ksp37 which might have been accumulated intracellularly as a response to non-specific stimulation. In addition, the relative MFI in cells from asthmatic patients was slightly increased compared to the respective controls (). In this study, for the first time we provide evidence that the percentage of Ksp37 + lymphocytes in peripheral blood of patients with mild extrinsic asthma is increased compared with healthy control subjects (). Subsequent analysis of the Ksp37 + lymphocyte subset showed that the increase in Ksp37 + cells in patients with asthma is linked primarily to CD8 + cells, suggesting that Ksp37 expression in this subpopulation might be up-regulated in bronchial asthma (). CD3 –CD56 + NK cells exhibit the largest percentage of the total Ksp37 positive cell fraction, but there was no difference in Ksp37 expression in CD3 –CD56 + NK cells between patients with asthma and healthy controls (). Our data suggest that the elevated fraction of CD8 +Ksp37 +cells reflects a true increase of this subpopulation in the peripheral blood of patients with mild extrinsic asthma. Recently, it has been reported that Ksp37 expression within the total CD8 + T cell population is limited to a subpopulation with a high cytotoxic potential which is characterized by CD27 –CD11b +CD8 + and a cytokine profile of IFN- γhighIL-4 lowIL-2 low and in addition a high degree of perforin expression [ 11, 17]. It can therefore be speculated that this particular CD8 + subset, which has a high cytotoxic potential, is of relevance for the increase in Ksp37 + expression observed in lymphocytes from patients with mild extrinsic asthma. Based on the small sample size and the relatively mild degree of asthma in our patient population we nevertheless feel that it might be appropriate to speculate that even mild allergic asthma appears to be associated with an augmented CD8 + T lymphocyte mediated cytotoxic potential. In addition, this study shows that Ksp37 is co-expressed with other cytotoxic granules, namely granzyme B and perforin, and in addition with the surface marker CD3, although this was not significantly different between patients with asthma and healthy controls (). Our data thus support previous assumptions that Ksp37 is associated selectively with cells with a cytotoxic potential [ 11]. In addition Ksp37 secretion profiles were investigated in PMA/calcium-ionophore-stimulated and unstimulated PBMC from asthmatic and healthy controls (). Similar studies have not been performed previously in patients with asthma or atopy. Ksp37 appears to be released at a constant rate in unstimulated PBMCs of both groups in vitro and can be detected in culture supernatants, suggesting a constitutive synthesis and release of Ksp37 in vitro. This finding corresponds with a previous report of a similar secretion profile and mRNA expression in PBMC from healthy subjects [ 11]. In our study the non-specific stimulus PMA/calcium-ionophore caused a significant increase in Ksp37 concentrations which reached a plateau after 6 h in PBMC cultures, both from healthy controls and patients with asthma. However, after 24 h in culture there was no difference between Ksp37 concentration from stimulated and unstimulated cultures. These secretion profiles suggest indirectly that Ksp37 can be released by stimulation-dependent degranulation. Because PMA/calcium-ionophore induces exocytotic secretion from intracellular granules in cytotoxic T cells, our findings are in agreement with the hypothesis formulated by Ogawa et al. claiming that Ksp37 secretion is mediated by degranulation of intracellularly stored granules [ 18– 23]. In addition, the early increase of Ksp37 in supernatants after only 2 h suggests that this is indeed mediated by degranulation from preformed stores, as protein de novo synthesis would most probably require longer. The results of our MFI experiments also support this hypothesis of a granular localization of Ksp37 as the relative MFI of stimulated cells was significantly decreased compared to unstimulated cells, implying a stimulation-induced exocytotic release of Ksp37 (). In addition, however, there are indications for a constitutive synthesis and release as PBMCs cultured in the presence of Brefeldin A show a significantly higher MFI for Ksp37 than cells in which intracellular transport and exocytosis was not inhibited. These observations fit with those of previous reports using immunofluorescence confocal microscopy to show that Ksp37 is located intracellularly throughout the cytoplasma and in association with granules [ 11, 23]. Ksp37 levels in PBMC culture supernatants and the MFI values of intracellular Ksp37 expression were slightly elevated in patients with asthma compared to healthy controls, irrespective of whether or not cells were stimulated with PMA/calcium-ionophore (). These data are consistent with our finding of an elevated percentage of Ksp37+ lymphocytes in asthmatic subjects and suggest that Ksp37 is indeed released from these cells in vitro, although the natural stimulus remains to be determined. In our asthmatic patient population with mild, stable extrinsic asthma the increased Ksp37 expression appears to be limited to the Ksp37 positive CD8+ effector T cell subset. Although the relevance of CD8+ T cells in the pathogenesis of bronchial asthma is still uncertain, our findings provide another indirect indication that in asthma without noticeable acute allergen contact cytotoxic mechanisms might contribute to the asthmatic phenotype. There are few data on the role of Ksp37 in other inflammatory diseases. Ogawa et al. had investigated patients suffering from infectious mononucleosis and found an increase in Ksp37 serum levels during the acute phase of Epstein–Barr virus (EBV) infection. Similar results were obtained in parvovirus B19 and cytomegalovirus (CMV) infections [ 11]. We do not assume that Ksp37 expression is characteristic for asthma, but might be another indicator of cytotoxic mechanisms present in asthma. Because CD8 + T cells and in particular CD8 + effector T cells have a role in the defence against intracellular organisms, especially viruses, our results together with previous findings of elevated Ksp37 serum levels in patients with EBV infections suggests that the immunological mechanisms underlying chronic asthma might be linked to pathological processes present during viral infections. Hoshino et al. have recently reported the expansion and decline of virus-specific CD8 + T cells in primary EBV infections [ 24]. At present the potential role of Ksp37 as a marker for extrinsic asthma remains unclear. However, the observation that Th2-mediated inflammation alone is insufficient to explain the asthmatic phenotype in combination with our findings of an elevated cytotoxic potential in patients with asthma suggests that allergen-mediated mechanisms which are associated with Th2-type reactions could appear on top of an underlying chronic Th1/Tc1-cytotoxic background, as suggested by findings in mild, stable asthmatics. Numerous studies have reported an association of CD8 + T cells, viral infections and bronchial asthma, in particular acute asthmatic exacerbations, but the underlying inflammatory mechanisms in virus-induced asthma are still unclear [ 25– 27]. In conclusion, our study provides data for a significant difference in the percentages of CD8+Ksp37+ lymphocytes between patients suffering from mild extrinsic asthma and healthy controls. 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