In vitro release of interleukin-15 by broncho-alveolar lavage cells and peripheral blood mononuclear cells from patients with different lung diseases.

ARTICLE

INTRODUCTION

IL-15 is a cytokine which shares several biological activities with IL-2, i.e. it stimulates antigen driven T cell proliferation, it has costimulatory activity for proliferation and Ig-production by B cells and is an efficient activator of NK- and LAK-cells [1]. In addition to its activities on lymphoid cells, IL-15 also exhibits regulatory properties on macrophage proinflammatory cytokine release [2]. Interestingly, although IL-15 and IL-2 share only limited sequence homology both cytokines use the beta and gamma chain of the IL-2 receptor [1].

A variety of lung diseases are associated with lymphocytic alveolitis (e.g. sarcoidosis [PS], tuberculosis [TB], hypersensitivity pneumonitis [HSP]). In these cases a hypothetical (PS) or known antigen (TB, HSP) enters the lung and activates and/or recruits T cells from the peripheral blood. IL-2 is necessary for T cell proliferation and is autocrineously released by T cells. In contrast to IL-2, IL-15 is found to be released mostly by non-lymphoid cells, e.g. muscle cells, placenta, kidney, liver, heart, lung and pancreas. Among the cells of the immune system monocytes and macrophages are the major source of IL-15 [3]. In many lung diseases, alveolar macrophages (AM) are activated and release proinflammatory cytokines such as TNF-alpha, IL-6 and IL-8, and disclose an increased accessory function for T cell activation [4-8]. This leads to the assumption that IL-15 might also be released by AM and could contribute to the T cell activation in PS, TB, and HSP, which is necessary for granuloma induction. In contrast, in non-granulomatous diseases (cryptogenic fibrosing alveolitis [CFA], pneumonia [PN]) the IL-15 release should be limited.

We therefore analysed the release of IL-15 protein by cultured cells obtained by bronchoalveolar lavage (BAL) and mononuclear cells of the peripheral blood (PBMNC). Additionally, we searched for the presence of IL-15 mRNA in freshly isolated and cultured BAL cells and PBMNC and followed the kinetics of their IL-15 protein release.

METHODS

Patients

The diagnosis of pulmonary sarcoidosis was established in 30 individuals using defined criteria, including transbronchial biopsy [9]. For the purpose of this study, the patients were grouped as "clinically active sarcoidosis" (PSa, n = 15) or "clinically inactive sarcoidosis" (PSi, n = 15) using clinical criteria, i.e. new or progressing pulmonary symptoms such as cough and dyspnea, newly evolving or progressing radiographic abnormalities, and systemic symptoms as suggested by recent recommendations of the World Association of Sarcoidosis and Other Granulomatous Disorders [10]. Bronchoalveolar lavage was performed in the course of a clinically indicated bronchoscopy, either for diagnosis or re-evaluation of inflammatory activity.

Additionally, BAL cells and PBMNC from patients with tuberculosis (TB; culture-prooved M. tuberculosis, n = 9), hypersensitivity pneumonitis (HP; farmer's lung, n = 5; pigeon breeder, n = 2, parakeet keeper, n = 2), cryptogenic fibrosing alveolitis (CFA; n = 11) and pneumonia (PN; n = 8) were cultured and the cytokine concentrations estimated.

None of the patients were receiving therapy at the time of investigation or in the previous 2 months.

The control group consisted of 23 patients retrospectively free of any inflammatory or malignant pulmonary disease undergoing diagnostic bronchoscopy in the course of their clinical work-up. Only patients proven to be free of any disease that could possibly interfere with immunological tests were included the control group. The characteristics of the study groups are listed in Table 1.

Cell preparation

Bronchoalveolar lavage (BAL) was performed as previously reported [11]: 200 to 300 ml sterile saline (0.9% NaCl) were instilled in 25 ml aliquots into a lingula or middle lobe segment. Each aliquot was immediately aspirated. The cells were centrifuged at 500 x g and washed three times with phosphate-buffered saline at + 4° C. Cell differentials were determined by counting a minimum of 200 cells on a cytocentrifuge preparation (Cytospin II, Shandon INC. Pittsburgh, PA)
stained with HEMACOLOR™ (E. Merck, Darmstadt, FRG). Peripheral blood mononuclear cells were obtained from venous blood using density gradient centrifugation. These cells were used for cell culture or shock-frozen by liquid nitrogen (pre-culture).

Monoclonal antibodies and staining procedures

BAL cells were fixed on poly-L-lysine coated slides (Bio-Rad, Munich, FRG) for immunoperoxidase staining and developed with a peroxidase-antiperoxidase technique [12] using monoclonal antibodies directed against CD3, CD4, CD8, IL-2R (Ortho Diagnostic Systems, Neckargemünd, FRG) and HLA-DR (Becton Dickinson, San Jose, CA) at concentrations suggested by the supplier.

Cell culture

Washed BAL cells and PBMNC were cultured at a density of 1 x 10⁶ cells/ml for a period of 24 hours with or without l mug/ml lipopolysaccharide (LPS, Salmonella minnesota, kindly provided by Dr. Brandenburg, Borstel, Germany) in endotoxin-free RPMI 1640 (Biochrom, Berlin, FRG) supplemented with 2% heat inactivated human serum, 2 mM L-glutamine (Gibco, Karlsruhe, FRG), 100 U/ml penicillin (Gibco) and 100 mug streptomycin (Gibco) in 24-well tissue culture plates (Nunc, Wiesbaden, FRG) in 5% CO₂, humidified atmosphere at 37° C. At the end of the culture period the supernatants were harvested and stored at ­ 70° C until assayed for IL-15 activity. The remaining cells were disintegrated by a lysis-buffer containing thioisocyanate and used for PCR (post-culture).

To investigate the time-course of IL-15 release by BAL cells and PBMNC, the cells were stimulated for 2 hours with LPS (1 mug/ml) and then washed free of LPS; parallel cultures for 5, 10, 18, 23, 28, and 43 hours were initiated. The cells were washed, and fresh culture medium was added at 5, 8 or 15 hours, respectively, before the end of the culture period. The supernatants of these last culture periods were harvested and the levels of IL-15 and TNF-alpha were measured by enzyme-linked immunosorbent assay (ELISA) and expressed as pg/ml/h.

Cytokine determination

IL-15 was measured by an ELISA (Genzyme, Rüsselsheim, FRG). The assay was mainly performed as recommended by the manufacturer with the exception of an overnight incubation of the plate loaded with the samples and standards. The sensitivity of the test is given as 2 pg/ml. TNF-alpha concentrations were evaluated as described previously [6] with reagents kindly provided by Knoll AG (Ludwigshafen, FRG).

PCR

The IL-15 mRNA level of freshly frozen cells and of cells harvested after the culture period was determined using a specific primer pair (sense: AGA GTA ATG AGT TTC GGT GCC TTC GAA CTA G; antisense GAT CGG ATC CTG TCT AAG CAG CAG AGT GAT G). PCR was run under the following conditions: denaturation at 95° C for 1 min, annealing at 57° C for 1 min and elongation at 72° C for 1 min 30 s. PCR products were detected by gel-electrophoresis in a 3% agarose gel.

Statistics

Data are expressed as mean ± SD. Differences between the patient groups and the controls were analysed by the Mann-Whitney U test. Spearman's rank correlation was used to analyse correlations between different parameters within the groups. Values of p < 0.05 were considered significant.

RESULTS

IL-15 and TNF-alpha release by BAL cells

In BAL cell cultures from controls, an IL-15 concentration of 3.8 ± 1.9 pg/ml was observed. Apart from HSP (9.3 ± 9.5 pg/ml) and PSi (4.0 ± 2.6 pg/ml), all BAL cell cultures showed a significantly higher IL-15 concentration compared to controls (PSa: 8.7 ± 3.9 pg/ml, TB: 8.4 ± 1.9 pg/ml, PN: 7.8 ± 2.6 pg/ml, p ¾ 0.0001 for all comparisons; CFA: 5.7 ± 1.5 pg/ml; p ¾ 0.02; Figure 1). In the controls and in the CFA group, stimulation with LPS induced a significant increase in IL-15 release (CO: 3.8 ± 1.9 pg/ml versus 4.5 ± 2.2 pg/ml, CFA: 5.7 ± 1.5 pg/ml versus 9.5 pg/ml ± 3.9, p ¾ 0.03, for all comparisons, data not shown). In the other groups LPS-stimulation did not significantly alter IL-15 release by the BAL cells (data not shown). Furthermore, the addition of human IFN-gamma (15 IU/ml) did not change the IL-15 release by the BAL cells of three controls (5.7 ± 0.4 pg/ml versus 5.4 ± 0.6 pg/ml, data not shown).

In vitro TNF-alpha release was increased in PSa (2,472 ± 2,563 pg/ml, p ¾ 0.0001), CFA (5,016 ± 8,787 pg/ml, p ¾ 0.005), and HSP (2,307 ± 2,078 pg/ml, p ¾ 0.0001) compared to controls (360 ± 280 pg/ml, Figure 1). TNF-alpha levels were not significantly increased in PSi (2,274 ± 4,388 pg/ml), TB (851 ± 1,117 pg/ml), and PN (549 ± 592 pg/ml compared to control, Figure 1). Stimulation with LPS (1 mug/ml) resulted in a significant increase in TNF-alpha release (data not shown).

IL-15 and TNF-alpha release by cells from the peripheral blood

In PBMNC cultures, statistically significant differences could be detected between the controls (3.8 ± 2.4 pg/ml) and PSa (10.8 ± 8.9 pg/ml, p < 0.005) but not between the controls and the other groups (PSi: 4.9 ± 4.6 pg/ml; TB: 5.7 ± 1.4 pg/ml; CFA: 4.6 ± 1.6 pg/ml and HSP: 4.9 ± 3.8 pg/ml; Figure 2). LPS-stimulation did not modify the IL-15 release by the PBMNC in any of the groups (data not shown). Furthermore, the addition of human IFN-gamma did not modify the IL-15 release by PBMNC from five controls (5.4 ± 0.8 pg/ml versus 5.3 ± 1.2 pg/ml).

PBMNC from the controls released 169 ± 243 pg TNF-alpha per ml. This was significantly lower than the TNF-alpha release from the PSi group (1,756 ± 2,729 pg/ml, p ¾ 0.05), the PSa group (2,435 ± 3,891 pg/ml, p ¾ 0.002) and the HSP group (715 ± 91 pg/ml). The PBMNC from the TB group (632 ± 893 pg/ml, p ¾ 0.05), the PN group (102 ± 176 pg/ml) and the CFA group (486 ± 591 pg/ml, p ¾ 0.05) showed no significantly increased TNF-alpha release (Figure 2). Again, stimulation of the cells with LPS resulted in an increase of TNF-alpha release in all investigated groups (data not shown).

Correlations of BAL cell IL-15 release with other parameters

Statistically significant correlations between the IL-15 release by BAL cells and the percentage of CD4⁺ BAL cells (r_s = 0.8, p < 0.001), with the IL-15 release by the PBMNC (r_s = 0.7, p < 0.04) and with the TNF-alpha release (r_s = 0.6, p < 0.03) by the BAL cells could only be detected in the PSi group. No correlation could be observed between IL-15 release by BAL cells and PBMNC.

Kinetics of BAL-cell IL-15 release

Kinetic experiments with PBMNC (n = 2) revealed a minor peak in the first culture period (0-5 hours) and a major peak between 20 and 30 hours. A similar experiment with BAL cells from a control patient revealed a comparable pattern (first peak: 0-5 hours; main peak: 18-35 hours; Figure 3). For comparison we measured the TNF-alpha release in parallel. TNF-alpha release increased rapidly, after a short lag phase during the first culture period, to its maximum during the second culture period (5-10 hours) and decreased thereafter during the following culture period. The decrease was faster in the PBMNC compared to the AM (Figure 3).

IL-15-mRNA in BAL cells and PBMNC

In none of the groups tested IL-15 mRNA could be detected in freshly isolated AM or PBMNC (Figure 4). After 24 hours of culture IL-15 mRNA was detectable in AM from 3 out of 4 patients with sarcoidosis, 2 out of 5 patients with tuberculosis and in 1 out of 3 patients with HSP, but not in cultured AM from three controls. LPS amplified the mRNA induction in CO, PS and HSP. In PBMNC of patients with PS no IL-15 mRNA could be detected in freshly isolated cells, but again culturing the cells was enough to induce IL-15 mRNA.

DISCUSSION

IL-15 is a cytokine sharing properties with the T cell growth factor IL-2. We report here that BAL cells from all patient groups tested, except the HSP group, released significantly more IL-15 than AM from controls in vitro. We were not able to demonstrate IL-15 mRNA in freshly isolated BAL cells from any patient group, but IL-15 mRNA was detectable after an in vitro culture period of 24 hours in some of the patients with TB and PSa. Furthermore, kinetic studies revealed that IL-15 is released in a biphasic manner by BAL cells and PBMNC.

Although both BAL cells and PBMNC release IL-15 in low quantities, we found significant differences between the controls and patients with active sarcoidosis and tuberculosis. The IL-15 mRNA contains an AUG-rich leading sequence which results in restrictions in translation [13] and therefore the production and release of the protein is limited. Nevertheless, in rheumatoid arthritis high concentrations of IL-15 could be detected [14].

There are conflicting results concerning the expression of IL-15 mRNA in non-stimulated mouse peritoneal macrophages. Alleva et al. were not able to detect IL-15 mRNA in unstimulated peritoneal macrophages [2], whereas others found it constitutively expressed [3, 15]. In humans, IL-15 mRNA could be detected in several organ tissues, including lung, and adhesion-enriched peripheral blood monocytes. Doherty et al. found only faint bands of IL-15 mRNA in bone marrow-derived macrophages differentiated in the presence of GM-CSF, whereas in M-CSF-induced macrophages the IL-15 mRNA was easily visible, indicating that there are differentiation-dependent differences in the ability to express IL-15. However, in both lineages IL-15 mRNA could be increased by stimulation with different stimuli, the most effective being Mycobacteria plus IFN-gamma. Interestingly, GM-CSF-induced macrophages resemble alveolar macrophages particularly in the pattern of cytokines released [16]. In accordance with the above mentioned results, we could not detect IL-15 mRNA in freshly isolated AM or PBMNC, whereas adhesion to culture dishes was sufficient to induce IL-15 mRNA in AM from patients with PS or HSP. This might be due to a "primed" status of AM from patients with a particular disease. Such a pre-activation leads, e.g. in sarcoidosis, to higher TNF-alpha release after stimulation compared to controls [8]. A similar mechanism may be responsible for the adhesion-induced IL-15 mRNA in HSP and PSa.

IL-15 was discovered as a result of its biological similarities with IL-2 and most of the scientific in vitro work about its action was done in this field [1, 3, 14, 17-24]. However, the IL-15 protein concentrations used in these papers, with the exception of one [24], are very high, and up to now such concentrations could only be detected in synovial fluid of rheumatoid arthritis [14]. It has recently been demonstrated that, depending on the TCR activation, IL-15 induces a quiescent state or strong proliferation. In both cases however, the cells are less sensitive to apoptotic signals [25]. As little as 80 pg IL-15 per ml protect T cells from apoptosis. This amount is higher than we could detect in our BAL cell culture, however, it is reported that alveolar epithelial cells and a human alveolar epithelial-like cell line release IL-15 [26]. The release by both cell types in the lung may reach levels which are sufficient for antiapoptotic actions.

Additionally, IL-15 is a very potent cytokine in regulating macrophage proinflammatory cytokine production. In picomolar to attomolar concentrations IL-15 downregulates LPS-activated release of proinflammatory cytokines like TNF-alpha, IL-1 and IL-6 of mouse peritoneal macrophages, but exerts no activity on the anti-inflammatory cytokine IL-10. In high concentrations (> 10 ng/ml) however, IL-15 enhances TNF-alpha, IL-1, IL-6 and IL-10 release resulting in an imbalance of pro- and anti-inflammatory cytokines [2]. From these data Alleva et al. conclude that IL-15 is more an autocrine regulator of macrophage proinflammatory cytokine production rather than a T cell growth factor. Interestingly, we detected a correlation with the spontaneous TNF-alpha release by the BAL cells only in patients with PSi. We could demonstrate that increased TNF-alpha release by BAL cells from patients with PSi is associated with a higher risk of disease progression [27]. The positive correlation of the IL-15 and the TNF-alpha release in patients with inactive disease may be a hint that IL-15 plays a role in the initiating phase of the immune response at least in this group of patients. The increased level of IL-15 release in patients with PSa and TB implies that the released IL-15 is also involved in the immunopathogenesis of active sarcoidosis and TB, however, we could not find any correlation supporting this hypothesis. In a recent work Musso et al. demonstrated that human monocytes express membrane-bound biologically active IL-15 [28] and Agostini and coworkers detected increased expression of membrane-bound IL-15 on AM from sarcoid patients [17]. From this, it is possible to conclude that the main biological effects are maintained by membrane-bound IL-15. It is known from other molecules such as sICAM and sIL-2R, that membrane-bound molecules can be shed from the surface and be detected in cell supernatants. Thus, the significant increase in soluble IL-15 in patients with PSa and TB may, at least in part, reflect an increase in IL-15 membrane expression and shedding.

Recently it was decribed that alveolar macrophages and T-cells from sarcoid patients express higher levels of receptors for apoptotic signals compared to controls [29, 30]. The absolute number of these cells, however, is increased compared to controls. Because IL-15 acts as an anti-apoptotic factor [31], it may decrease apoptotic processes in BAL cells together with other factors such as TGF-beta [32].

Kinetic experiments revealed that BAL cells and PBMNC release IL-15 in a biphasic manner with a first peak during the first 10 hours, without any lag-phase, followed by a second increase after 15 to 20 hours and a decrease during the next 24 hours. This is completely different from TNF-alpha where we have a short lag-phase of about six hours followed by a rapid increase of TNF-alpha release (6-10 hours) and a decrease during the next 10-18 hours. The release of preformed IL-15 explains why we could detect IL-15 in our over-night culture system without detecting IL-15 mRNA in freshly isolated cells. The differences in the IL-15 concentrations might therefore be due to either an increased number of IL-15-containing cells, or to a higher amount of IL-15 stored in the cells or to an increased shedding of this molecule. The latter two possibilities are supported by higher intracellular fluorescence intensity of anti-IL-15 mAb-stained AM from patients with PS [17].

Doherty and coworkers found increased amounts of IL-15 mRNA in adherent lung cells of BCG-infected mice [15]. Additionally, stimulation of GM-CSF-elicited macrophages with M. tuberculosis but not with LPS revealed a substantial increase of IL-15 mRNA. These findings are in agreement with our data showing a significant increase in IL-15 release by BAL cells from patients with tuberculosis, although we could not detect IL-15 mRNA but increased levels of released IL-15 protein. This increase might rather be due
to a greater amount of preformed IL-15 than to a recent in vivo stimulation. Doherty et al. measured IL-15 mRNA after 7 days of infection, in most cases the incubation period in humans lasts from several weeks to many years. During this relatively long period, higher levels of intracellular IL-15 protein might be stored whilst the mRNA decays. IL-15 is found to be involved in the activation of gammadelta⁺ T cells, which are frequently observed in BAL of TB patients, and it may serve as a growth factor for these cells although the significance of these actions is under debate [20, 23].

Sarcoidosis is a systemic disorder, however, in most instances cell activation is compartmentalized, i.e. only BAL cells release cytokines [33, 34]. In PSa, however, IL-15 is released in equal amounts by BAL cells and PBMNC, reflecting the systemic nature of sarcoidosis. This is supported by the positive correlation of the IL-15 release by BAL cells and PBMNC. In the other groups no increase in IL-15 release could be detected.

Agostini and coworkers found cytoplasmic and membrane-bound IL-15 in AM from patients with active sarcoidosis but not in AM from controls [17]. These data are in accordance with our findings of increased spontaneous release of IL-15 by BAL cells from patients with sarcoidosis but not from controls. In contrast to Agostini et al., however, we could not detect IL-15 mRNA in freshly isolated BAL cells of either group. One reason for these conflicting results might be the fact that their patients had more severe changes in BAL cytology indicating a higher state of immune cell activation. However, we could not detect any correlation between the IL-15 release and the percentages of the cell populations or lymphocyte subpopulations in the BAL.

We could detect significantly increased levels of IL-15 in all patient groups and also in HSP, although without statistical significance. The total amount of IL-15 in the supernatants of PBMNC and BAL cells is very low, but biological activity has been demonstrated in concentrations as low as 0.01 pg/ml [2]. Interestingly, another source of IL-15 may be alveolar epithelial cells [26, 35] which may add to the overall IL-15 level in the lung.

CONCLUSION

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