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Departamento de Inmunología, Instituto de Investigaciones Hematológicas, Academia Nacional de Medicina
División de Tisioneumonología, Hospital F. J. Muiz, Buenos Aires, Argentina
Tuberculous pleuritis usually shows lymphocytic preponderance, but neutrophils are also present. Therefore, pleuritis is a good model for the study of neutrophil fate at sites of active Mycobacterium tuberculosis infection. We have previously demonstrated in vitro that M. tuberculosisinduced neutrophil apoptosis involves p38 mitogen protein kinase activation through Toll-like receptor 2. Herein, we demonstrate that, in tuberculous pleuritis, neutrophil apoptosis increases together with the expression of Toll-like receptor 2 and phosphorylated p38 (p-p38) kinase. In addition, receptors associated with activation/apoptotis (CD11b, CD64, tumor necrosis factor receptor, and Fas ligand) are up-regulated, together with a loss of CD16 expression. However, neutrophils express CD86, CD83, and major histocompatibility complex class II antigens, acquiring dendritic cell (DC) characteristics. Therefore, the cytokine milieu in the pleural space may influence signaling pathways on activated neutrophils, thereby inducing apoptosis and inhibiting their proinflammatory capacity, as well as allowing them acquire DC characteristics that influence the immune response.
Among the many clinical manifestations of tuberculosis (TB), pleuritis is of particular interest, since it resolves without therapy and patients are known to undergo a relatively effective immune response against Mycobacterium tuberculosis [1]. Pleuritis occurs in 10% of untreated individuals who have positive tuberculin test results [2]. However, tuberculous pleuritis may also develop as a complication of primary pulmonary TB [3]. Tuberculous pleuritis is caused by a severe delayed-type hypersensitivity reaction in response to the rupture of a subpleural focus of M. tuberculosis infection and subsequent escape of the bacteria or the antigens into the pleural space [2].
The inflammatory process results in increased pleural vascular permeability, leading to the accumulation of fluid enriched in proteins and to the recruitment of cells with the development of exudative pleural effusion (PE) [4]. PE is characterized by the presence of specific subsets of leukocytes [5], which, together with pleural mesothelial cells, contribute to the local production of cytokines [6]. Analysis of PEs from patients with TB usually shows a lymphocytic preponderance; however, polymorphonuclear neutrophils (PMNs) are also present, and their role in TB is not well understood.
PMNs are the first-line defense against bacterial infection, but, when appropriately activated, they also participate in chronic inflammation disease and regulation of the immune response. Once activated, PMNs show several changes in their life span and in surface-receptor expression (loss of CD16 and up-regulation of CD64, CD11b, and CD66b). We have demonstrated that circulating PMNs from patients with TB are activated [7], leading to the acceleration of spontaneous apoptosis [8]. Moreover, PMN apoptosis is induced by M. tuberculosis, in a process that involves Toll-like receptor 2 (TLR2)mediated induction of p38 mitogen-activated protein kinase (p38 MAPK) [9]. Cytokines and cytokine-producing cells are present in PEs from patients with malignant and infectious diseases [10, 11]. Patients with tuberculous pleuritis have elevated levels of tumor necrosis factor (TNF) [12, 13, 14], transforming growth factor [14, 15], interferon (IFN) [12, 16, 17], interleukin (IL)8 [18], IL-6 [19], IL-18 [17], and IL-10 [14], some of which have opposite effects on PMN survival. In addition, it has been demonstrated that, in certain bacterial infections, as well as when they are incubated with IFN- and granulocyte-macrophage colony-stimulating factor (GM-CSF) or TNF- [20], PMNs acquire characteristics of dendritic cells (DCs), thus enhancing adaptive immune responses.
The pleural space is the site of naturally occurring tuberculous inflammatory exudates; therefore, it appears to be a good model for the study of PMN fate in active M. tuberculosis infection. Given that the preactivation of peripheral PMNs from patients with TB makes them more susceptible to M. tuberculosisinduced apoptosis in vitro, we hypothesized that the cytokine milieu and bacteria/antigens present during tuberculous pleuritis trigger the apoptosis/activation process, leading PMNs to display effector functions before being removed by alveolar macrophages.
PATIENTS, MATERIALS, AND METHODS
Patients.
Informed, written consent was obtained from all patients and healthy individuals studied before their inclusion in this work. The study protocol was approved by the ethics committees of Hospital F. J. Muiz and IIHema, Academia Nacional de Medicina, Buenos Aires, Argentina. Patients with newly diagnosed moderate or large PEs were identified at the División de Tisioneumonología, Hospital F. J. Muiz. Patients were examined, and case histories were set up. Complete blood cell count, electrolyte analysis, chest x-ray, and HIV testing were performed. PEs and blood were obtained during diagnostic thoracocentesis before the initiation of chemotherapy. Exclusion criteria included a history of TB or TB treatment, suspected disseminated or meningeal TB, and HIV positivity. None of the studied subjects was receiving antituberculous or steroid therapy at the time of the study. The PEs were classified as exudates meeting at least 1 of the Light criteria [5]. Tuberculous PEs were defined as exudates with a positive Ziehl-Nielsen stain or Lowestein-Jensen culture from PE or pleural biopsy specimens. PEs were considered parapneumonic when there was acute febrile illness, with purulent sputum and pulmonary infiltrates, in the absence of malignancy or other diseases causing exudates in PEs. Malignant PEs were defined as exudates associated with a pathologic diagnosis of cancer whenever cytologic malignant cells were observed during histopathologic examination of pleural biopsy specimens.
Thoracocentesis and pleural biopsy.
Pleural fluid was collected by therapeutic thoracocentesis, and closed pleural biopsy (Abrams needle) was performed in hospitalized patients with TB (n = 25; mean age, 40 years [range, 2060 years]), parapneumonia (n = 3; age range, 2260 years), and cancer (n = 5; age range, 4050 years). In brief, after local anesthesia of the skin and subcutaneous tissue, 100200 mL of pleural fluid was aspirated under sterile conditions by use of an 18-gauge needle, which minimizes contamination of the pleural fluid with peripheral blood. Specimens were subjected to routine biochemical analysis, including tests for total protein, glucose, lactate dehydrogenase, and differential cell counts. Bacterial cultures and cytologic examinations were performed on all PEs in the central laboratory of Hospital F. J. Muiz. A second sample of the pleural fluid was dispensed into 50-mL polystyrene tubes containing heparin, to obtain PMNs from PEs (PE-PMNs). In addition, blood samples were collected from patients on the same day as thoracocentesis and from healthy control subjects (n = 10; age range, 2055 years). All control subjects had received bacille Calmette-Guérin vaccination in childhood, and their tuberculin-test status was unknown.
Sample processing.
Blood and pleural fluid samples were collected at the same time and prepared for subsequent analysis. Peripheral blood PMNs (P-PMNs) were obtained from whole heparinized blood. To obtain PE-PMNs, heparinized PEs were washed with PBS and centrifuged for 10 min at 300 g at 4°C, to sediment the cellular constituents; thereafter, cell pellets were suspended in RPMI 1640 (Gibco). Peripheral and PE cells were purified by Ficoll-Hypaque gradient centrifugation [21], followed by sedimentation in 6% dextran (Sigma). The PMN-rich supernatant was then collected, and residual red blood cells were removed by hypotonic lysis. The cells were washed and suspended at 3 × 106 cells/mL in RPMI 1640. The viability was consistently >95%, as determined by trypan blue dye exclusion. The purity of P-PMNs was >95%, as assessed by morphological examination by staining with Wright-Giemsa (Merck) and by fluorescence-activated cell sorter light-scatter patterns. The purity of PE-PMNs was variable (range, 10%80%) and depended on the number of days of disease evolution.
Flow-cytometric assessment of surface markers on PMNs.
PMNs were stained with the following monoclonal antibodies: antiCD11b-phycoerythrin, antiFas ligand (FasL) (Ancell), antiCD16fluorescein isothiocyanate (FITC) and antiCD64-FITC (Immunotech), antiTNF-R55FITC (Caltag), antiCD83-FITC, antiCD86-PE-Cy5, and antiTLR2-FITC (IgG2a,k, clone TL2.1) and MHC class IIphycoerythrin (eBioscience). The phosphorylated form of cytoplasmic protein p38 (p-p38) was measured by using a Fix and Perm kit (Caltag) and the mouse anti-human p-p38 IgM antip-p38FITCconjugated antibody (Santa Cruz Biotechnology), as described elsewhere [9]. Isotype-matched controls were used to determine autofluorescence and nonspecific staining. Samples were resuspended in Isoflow and analyzed in a FACScan (Becton Dickinson Immunocytometry Systems). Ten thousand events were collected and analyzed using CellQuest software (version 3.1f; Becton Dickinson). Results were expressed as percentages of positive cells and as mean fluorescence intensity (MFI).
PMN apoptosis.
As described elsewhere [8], we evaluated apoptosis of PMNs by staining with DNA fluorochrome propidium iodide (PI) (Sigma); apoptotic cells can be recognized by flow cytometry as cells that have less DNA than G1 cells ("sub-G1" peak). Briefly, 2.4 × 105 cells were washed in PBS and resuspended in 0.5 mL of PBS; this was added drop by drop to 4.5 mL of ice-cold 70% ethanol while vortexing. After washing, the pellet was resuspended in DNA extraction buffer and incubated for 5 min. Thereafter, cells were washed and suspended in 140 L of RNase A (500 g/mL) and 140 L of PI (100 g/mL) and incubated for 30 min at room temperature in the dark. Samples were washed in PBS before being analyzed by flow cytometry. Ten thousand events were collected in each sample, and a 488-nm laser line for excitation was used.
For microscopic assessment of PMN apoptosis, cytospin preparations were fixed in methanol, stained with May-Grünwald-Giemsa (Merck), and examined by oil-immersion light microscopy at a final magnification of 1000×. The percentage of apoptotic PMNs was determined by counting the number of cells showing features associated with apoptosis (chromatin condensation, fragmented nuclei, cytoplasmic vacuolation, and decrease in cell size). For all samples analyzed, 200400 cells/slide were counted by 2 different researchers without prior knowledge of the samples' identities.
Statistical analysis.
All values are presented as the mean ± SE of the results of a number of independent experiments. The data were evaluated statistically by use of the Mann-Whitney or Wilcoxon tests. Correlation was evaluated by use of Spearman's rank correlation test. P < .05 was considered to be significant.
RESULTS
Cellular composition of blood and PE.
A total of 33 PEs were evaluated: 25 from patients with TB, 3 from patients with bacterial parapneumonic pneumonia, and 5 from patients with cancer. None of the patients was coinfected with HIV. Causal microorganisms in the cases of parapneumonia were Staphylococcus aureus (2/3) and Streptococcus pneumoniae (1/3). Table 1 summarizes the blood and PE characteristics of the 3 groups studied.
Down-regulation of CD16 expression on tuberculous PE-PMNs.
CD16 is a low-affinity receptor for IgG (FcRIIIB) expressed on the surface of PMNs; furthermore, higher levels of CD16 on PMNs from patients with TB correlate with the activation of these cells in the periphery [7]. In addition, loss of this receptor correlates with apoptosis in PMNs cultured overnight [7, 22], allowing a differentiation between nonapoptotic (high CD16 expression) and apoptotic (low CD16 expression) PMNs. As shown in table 2, the CD16 receptor, which is overexpressed (P < .04) on P-PMNs from patients with TB, is down-regulated on PE-PMNs (P < .01) but not in PMNs from patients with parapneumonia or cancer. This confirms that the preactivation state in PMNs from patients with TB leads these cells to accelerate apoptosis as soon as they reach the site of infection.
Up-regulation of CD11b and CD64 expression on tuberculous PE-PMNs.
Another marker of PMN activation is the expression of CD64, which is also induced by IFN- [12]. In previous studies, we have observed CD64 expression on P-PMNs from patients with TB [7] as well as M. tuberculosisinduced expression of CD11b on P-PMNs from patients with TB in vitro [8]. Therefore, to evaluate whether cytokines and/or bacteria present in the PE could activate PMNs, we analyzed the expression of these receptors on PE-PMNs. Table 2 shows that expression of CD11b and CD64 in patients with TB was increased on PE-PMNs (CD11b, P < .002; CD64, P < .03), whereas, on PMNs from patients with parapneumonia or cancer, the differences were not significant, suggesting that the site of infection/inflammation determines the different patterns of PMN activation.
Increased expression of TNF-R55 and FasL on tuberculous PE-PMNs.
Both Fas/FasL and TNF- interactions have been implicated in PMN apoptosis. The effect of TNF- as an inflammatory mediator is attributable to its ability to influence PMN functions through the signaling of the 2 TNF- receptor (TNF-R) chains. Both TNF-R55 and TNF-R75 are involved in TNF-induced activation of PMN respiratory burst [23]. The presence of TNF- in the PE [12] and high soluble TNF-R serum concentrations have also been documented in patients with active TB [24]. In addition, Fas and FasL, which may be involved in in vivo apoptosis, have been found to be elevated in tuberculous PE [16, 25]. Therefore, we evaluated the expression of FasL and TNF-R55 on P-PMNs and PE-PMNs. As is shown in table 2, and in accordance with our previous results [7], the expression of TNF-R55 on P-PMNs from patients with TB was higher than that on PMNs from control individuals. In addition, its expression was increased on PE-PMNs compared with P-PMNs (P < .02) from patients with TB, whereas no differences were found in patients with parapneumonia or cancer. FasL was also found to be expressed on P-PMNs and to be enhanced on PE-PMNs from patients with TB (P < .04), whereas it was not expressed on either P-PMNs or PE-PMNs from patients with parapneumonia. These results could explain the higher response to apoptotic stimuli observed in PE-PMNs from patients with TB.
Expression of CD83, CD86, and MHC class II on tuberculous PE-PMNs.
It has been demonstrated that, under appropriate conditions, PMNs can undergo a differentiation process characterized by the acquisition of new phenotypes and functions [31] and play a more active role in the adaptive immune response. This process, often called "transdifferentiation" of PMNs to DC-like cells, is achieved by cultivation of PMNs with IFN-, GM-CSF, or a combination thereof [32]. Under these conditions, PMNs de novo synthesize MHC class II antigens and CD86. Moreover, CD83, thought to be specific for DCs, has recently been shown to be expressed by PMNs, and its synthesis has been shown to be up-regulated by TNF- [33]. Since the concentration of IFN- is higher in tuberculous PE than in other types of PE [34], and since TNF- that is also present at the site of infection is a known "priming" factor of PMNs [35], we examined the expression of MHC class II, CD83, and the costimulatory receptor CD86, on P-PMNs and PE-PMNs.
As is shown in table 3, expression of CD86 (P < .03), MHC class II (P < .01), and CD83 (P < .02) was enhanced on PE-PMNs from patients with TB but not on PE-PMNs from patients with parapneumonia or cancer, suggesting that PMNs showing characteristics of DCs at the site of M. tuberculosis infection would connect innate with specific T-cell response.
DISCUSSION
PMNs are the first cells that arrive at the site of infection to exert their microbicidal effect and produce tissue injury [36]. The PMN-mediated inflammatory response is regulated by activation of a cell death program, apoptosis [37]. Host-derived cytokines, M. tuberculosis, and bacterial products such as lipopolysaccharide (LPS) and cytokines present at the site of TB infection [1219, 38], some of them with opposite effects, may modulate the life span of PMNs. Moreover, in vivo, apoptosis of PMNs, by preventing the release of their harmful contents, allows phagocytosis by professional phagocytes, limiting the injury process [39] and contributing to the resolution of pulmonary inflammation.
Increased levels of Fas and soluble FasL, the latter a potent chemoattractant for human PMNs [40], are found in pleural fluid from patients with TB [41]. In addition, we have demonstrated an increased expression of FasL on PE-PMNs from patients with TB, which may contribute either to the generation of proteolytically cleaved soluble FasL [42] or to the induction of apoptosis. Moreover, high levels of TNF-, together with enhanced expression of TNF-R55 on PE-PMNs from patients with tuberculous pleuritis, suggest that the apoptosis of PE-PMNs could be accelerated by the interaction of TNF/TNF-R on PMNs already activated by mycobacterial antigens.
In this study, we have shown that the CD16 receptor, which is overexpressed on P-PMNs from patients with TB, is down-regulated on PE-PMNs and that only PMNs from patients with TB express CD64, the expression of which is further increased on PE-PMNs. On the contrary, no significant differences in CD16 or CD64 expression were observed on PE-PMNs from patients with parapneumonia or cancer (table 2). In this context, IFN-, a cytokine that induces CD64 expression on PMNs, was highest in tuberculous PE [43]. In contrast, the up-regulation of CD11b expression, found only on PE-PMNs from patients with TB, could be the result of the interaction with mycobacterial antigens such as peptidoglycan, lipoproteins, or M. tuberculosis itself through TLR2.
It is well known that TLR activation initiates proinflammatory gene transcription through pathways, including NF-B and MAPK cascades, with selective roles in the regulation of PMN activation and life span [44]. We have previously demonstrated that in vitro M. tuberculosisinduced PMN apoptosis involves TLR2 signaling [9]. In the present study, we have demonstrated that PE-PMNs from patients with TB show enhanced apoptosis compared with P-PMNs; this could be due to the preactivation state of these cells involving p38, which is already activated on P-PMNs from patients with TB. The fact that PMN apoptosis was induced in vitro by M. tuberculosis, whereas PE was not, suggests that the encounter between M. tuberculosis and PMNs activates TLR2 signaling, leading to the activation of p38 MAPK, which, in turn, induces PMN activation and apoptosis. Indeed, p-p38 expression was significantly higher on PE-PMNs from patients with TB, whereas no differences were observed in patients with other diseases. This dichotomy between M. tuberculosis and the 2 other gram-positive bacteria studied here could be ascribed to differences in the lipid moiety and acylation patterns, which may account for the discrimination of TLRs binding and signaling pathways to activate host immune cells. Taking into account that LPS and TNF- delay apoptosis in PMNs [43] and that patients with parapneumonia did not show features of apoptosis in PE-PMNs, the accelerated apoptosis observed in patients with TB suggests a mechanism to control PMN-mediated tissue injury characteristic of M. tuberculosis infection.
Although PMNs are not considered to be typical antigen-presenting cells, several genes encoding proteins involved in antigen presentation are up-regulated during the initial stages of PMN apoptosis [45]. It has been shown that cytokines that delay apoptosis of PMN, such as IFN- and GM-CSF, induce the synthesis of MHC class II, CD80, CD86, CD40, and CD83 and produce monocyte chemoattractant protein1 and functional CCR6 chemokine receptor [46].
In this study, we have found that PE-PMNs from patients with TB express CD86, CD83, and MHC class II antigens, suggesting that the pattern of cytokines found in the pleural space may induce the synthesis of all these molecules on PMNs, which then acquire characteristics of DCs. It has been shown that PMNs from patients with acute bacterial infections acquire CD83 but remain negative for MHC class II and, conversely, that PMNs from patients with chronic inflammatory diseases express MHC class II and CD86 but not CD83 [20]. Moreover, the addition of IFN- and GM-CSF to PMN cultures from healthy individuals induces expression of both MHC class II and CD83, whereas TNF- selectively induces the synthesis of CD83 [33]. In this context, our finding that CD83 is expressed on PE-PMNs from patients with TB does not contradict results for chronic inflammatory diseases, because both IFN- and TNF- are found on PE-PMNs of patients with TB. Accordingly, PMNs from healthy control individuals that were incubated with PEs from patients with TB acquired CD83 but did not induce apoptosis, whereas M. tuberculosis by itself induced CD83 expression at 3 hours after infection, the time at which early apoptosis was observed (data not shown), suggesting that PMNs differentiate into DC-like cells as they start to undergo apoptosis. Therefore, it is not surprising that, in this chronic disease, PMNs acquire both CD83 and MHC class II, suggesting that, in situ, they are able to participate as a bridge between innate and adaptive immune responses.
In summary, in the present study, we have demonstrated that, when PMNs extravasate at the site of M. tuberculosis infection, the expression of receptors associated with activation/apoptotis (CD11b, CD64, TNF-R55, and FasL) is up-regulated, together with a loss of CD16 expression. Therefore, PMNs in the lung could detect M. tuberculosis via TLR2, thus activating the p38 MAPK pathway, which, in turn, induces PMN activation and apoptosis, confirming the results of our previous in vitro studies [9]. Therefore, the cytokine milieu in the pleural space appears to influence the signaling pathways on activated PMNs, leading to apoptosis and inhibiting proinflammatory capacity, while these PMNs acquire DC characteristics, thus participating in the immune response or pathogenesis of TB.
Acknowledgments
We thank the medical staff of División de Tisioneumonología of Hospital F. J. Muiz for their great help in providing clinical samples from patients. We are also grateful to Christiane Dosne de Pasqualini for critical manuscript review.
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