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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 583-592, 2002
© 2002 American Thoracic Society
DOI: 10.1165/rcmb.2002-0038OC

Mycobacterium tuberculosis–Induced Activation Accelerates Apoptosis in Peripheral Blood Neutrophils from Patients with Active Tuberculosis

Mercedes Alemán, Ana García, María A. Saab, Silvia S. de la Barrera, Marta Finiasz, Eduardo Abbate and María C. Sasiain

Departamento de Inmunología, Instituto de Investigaciones Hematológicas, Academia Nacional de Medicina; and División de Tisioneumonología, Hospital F. J. Muñiz, Buenos Aires, Argentina

Address correspondence to: Dr. María del Carmen Sasiain, IIHema, Inmunología, Academia Nacional de Medicina, Pacheco de Melo 3081 (1425) Buenos Aires, Argentina. E-mail: maleman{at}hematologia.anm.edu.ar


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The activation of circulating polymorphonuclear neutrophils (PMN) from patients with active tuberculosis (TB-PMN) may be associated with induction of apoptosis. Spontaneous or Mycobacterium tuberculosis (MTB)-induced apoptosis of PMN were evaluated by microscopy, DNA content, and their binding to Annexin V at 0, 3, and 18 h. In addition, the expression of CD11b and of CD16 were evaluated as parameters of activation and apoptosis, respectively. Recently isolated TB-PMN showed a higher CD11b expression than normal PMN (N-PMN), but there were no features of apoptosis, even though an enhancement of Fas expression was observed. Spontaneous apoptosis was accelerated in TB-PMN at 3 h, but no differences were observed in TB- and N-PMN at 18 h of culture. When stimulated with MTB, both TB- and N-PMN steadily increased CD11b expression along the culture period. MTB induced apoptosis of N-PMN at 3 h with loss of CD16 expression. By contrast, MTB delayed the apoptotic rate of TB-PMN, preserving the CD16 receptor at 3 h, whereas it accelerated apoptosis at 18 h, increasing at the same time the expression of CD11b. Taken together, these data suggest that the acceleration of apoptosis observed in TB-PMN could be associated with the MTB-induced activation.

Abbreviations: Annexin V, AV • bronchoalveolar lavage fluid, BALF • fetal calf serum, FCS • granulocyte macrophage colony-stimulating factor, GM-CSF • interferon {gamma}, IFN-{gamma} • interleukin, IL • lipopolysaccharide, LPS • mean fluorescence intensity, MFI • Mycobacterium tuberculosis, MTB • polymorphonuclear neutrophils, PMN • propidium iodide, PI • radical oxygen intermediates, ROI • tumor necrosis factor-{alpha}, TNF-{alpha}


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In the early phases of pulmonary infection, polymorphonuclear neutrophils (PMN) migrate, via the action of chemotactic factors, through the capillaries to the interstitium and, ultimately, to the alveolar air spaces. This mobilization and subsequent activation are important for microbicidal function (1). Although tuberculosis (TB) lesions are characterized by a predominant migration of monocytes/macrophages to the site of infection, the earliest response to mycobacteria is primarily an influx of PMN (2) that play a significant role in the acute phase of TB (3). Nevertheless, as the disease evolves, an increased number of PMN is observed in the bronchoalveolar lavage fluid (BALF) of the affected lung (4). The interaction between PMN and endothelial cells is mediated by an important group of adhesion molecules, members of the ß2 integrin family, composed of four heterodimers with distinct {alpha} subunits (CD11a through CD11d) sharing a ß subunit (CD18, ß2). These receptors are dramatically upregulated on the surface of activated PMN and mediate various leukocyte adhesion-dependent events (5).

It has been demonstrated that PMN exposed to mycobacteria display bactericidal responses (such as phagocytosis), generation of intracellular reactive oxygen intermediates (ROI), exocytosis of specific granules, and killing of mycobacteria (6–8). The powerful mediators that help PMN to be effective killers, such as oxidants and elastase, can also injure endothelial cells and may produce structural damage (9). Therefore, the lack of timely removal of these cells from the inflamed sites may result in tissue damage or progression to chronic inflammation (10).

PMN undergo spontaneous apoptosis in culture and concomitantly downregulate the surface expression of the CD16 molcule exposing at the same time phosphatidylserine (PS) on their surface (11). PS exposure is evident in PMN after 3 h of culture and, at later time points, many of the apoptotic cells proceed to secondary necrosis. This fact makes PMN susceptible to engulfment by macrophages (12) avoiding the secretion of toxic contents. In vitro, PMN rapidly undergo apoptosis when cultured for 18–24 h (13), and this process is initiated by Fas–FasL interactions (14). Inflammatory mediators can modulate PMN apoptosis in vitro. Bacterial products such as lipopolysaccharide (LPS), and some host-derived cytokines such as interleukin (IL)-1, interferon (IFN)-{gamma}, granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-8 retard PMN apoptosis (15), whereas tumor necrosis factor (TNF)-{alpha} and ROI induce the process (16, 17). Besides, several activation-dependent neutrophil functions lead to changes in second messenger systems (18), which are associated with induction of apoptosis in PMN.

We have previously demonstrated that circulating PMN from patients with active TB have enhanced parameters of activation, such as superoxide anion generation and high production of TNF-{alpha} and IL-1ß, upon stimulation with MTB (19). These signals could either promote or delay the apoptosis of PMN. Apoptosis, in turn, could be responsible for the shutdown of granulocyte secretory process and the removal of the intact senescent cells by macrophages, representing a pivotal point in the control of inflammation. The aim of this study was to evaluate the in vitro spontaneous apoptosis in activated peripheral PMN from patients with TB and to determine whether MTB or TNF-{alpha} affect the apoptosis in these cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients
A total of 25 hospitalized patients with active TB were studied. Their informed consent for experimentation was obtained according to the Ethics Commission of the Hospital Francisco J. Muñiz. All the patients had advanced radiographic abnormalities on chest X-ray. According to the classification of the American Tuberculosis Society, 13 of them had bilateral disease with massive affectation and multiple cavities, and the remaining 12 had unilateral involvement of two or more lobes with cavities. All the patients studied had a sputum smear positive for acid-fast bacilli, and the presence of MTB was confirmed by a sputum-positive culture. All of them were seronegative for HIV and had not used intravenous drugs. Those patients with other infectious or underlying disease were ruled out. One patient had a previous overcome pulmonary TB, and no patients had extrapulmonary TB. Patients were treated with a standard four antituberculosis-drug regimen of isoniazid (8 mg/kg/d), rifampicin (10 mg/kg/d), pyrazinamide (25 mg/kg/d) and ethambutol (25 mg/kg/d) for 1–30 d. The mean age was 32 yr (range, 18–56 yr). A total of 20 healthy volunteer blood-donors (N) were also studied. The mean age was 30 yr (range, 20–60 yr). All N had received BCG vaccination in childhood and their tuberculin-test status was unknown.

Antigens
The MTB H37-Rv strain was kindly provided by the Mycobacteria service from Instituto Nacional de Enfermedades Infecciosas, ANLIS, Dr. C. G. Malbrán (Buenos Aires, Argentina). Stocks of these organisms were maintained in 7H11 agar (Difco Laboratories, Detroit, MI), at 37°C in 5% CO2/95% air. A midlogarithmic phase of mycobacteria was scraped from agar, suspended in phosphate-buffered saline free of pyrogen, and sonicated to break up clumps. After settling, the supernatant was transferred to a new tube and then heated for 20 min at 80°C and adjusted to an OD600 of 1, which corresponds to a bacterial suspension of ~ 1 x 108 bacteria/ml. This antigenic preparation contains particulate and soluble antigens.

PMN Purification
Human PMN were isolated from heparinized venous blood by Ficoll-Hypaque gradient centrifugation (20) followed by sedimentation in 3% dextran (Sigma Chemical Co., St. Louis, MO). The PMN-rich supernatant was then collected, and residual red blood cells were removed by hypotonic lysis. The cells were washed immediately and resuspended at 3 x 106 cells/ml in RPMI-1640 medium (Gibco, Grand Isle, NY) supplemented with 1% heat-inactivated fetal calf serum (FCS) (Gibco) and 50 µg/ml gentamycin (complete media, CM). The viability was consistently > 95% as determined by trypan blue dye exclusion. The purity of the final PMN preparation was up to 95% as assessed by morphologic examination by staining with Wright-Giemsa and by FACS light scatter patterns.

Cultures were performed by incubating 1 ml of a PMN suspension (3 x 106 cells) in Falcon 2063 tubes at 37°C in a humidified 5% CO2 incubator without stimulation (control), or stimulated with heated MTB (1 x 106 bacteria/ml) or TNF-{alpha} (25 ng/ml) for 3 and 18 h.

Measurement of PMN Apoptosis
Flow cytometry. The percentage of apoptotic neutrophils was assessed based on the Annexin V-FITC (Sigma) protein binding assay (21). Briefly, 5 µl of Annexin V-FITC (10 µg/ml) and 5 µl of propidium iodide (PI) (250 mg/ml) (Sigma) were added to 1.2 x 106 cells suspended in 500 µl of binding buffer (20 mmol/liter HEPES, 132 mmol/liter NaCl, 6 mmol/liter KCl, 2.5 mmol/liter CaCl2, 1 mmol/liter MgSO4, 1.2 mmol/liter potassium phosphate, 5.5 mmol/liter glucose, and 0.5% [wt/vol] HSA, pH 7.4) and incubated for 15 min at room temperature in the dark. Annexin V (AV) fluorescence emission was detected in the FL-1 channel, and PI was detected in FL-2 channel. Autofluorescence was determined with the unstained PMN sample. The AV-labeled cell population was evaluated to establish the optimal compensation, and the PI-labeled population was analyzed on these settings and the population recompensed to subtract the overlapping PI fluorescence. PI staining is a dye-exclusion assay that discriminates between cells with intact membranes (PI-) and permeabilized membranes (PI+). The AV-/PI- cell population was regarded as alive, and AV+/PI- was considered as an early apoptotic population. Late stage apoptotic or necrotic cells were represented by the AV+/PI+ population.

DNA Content
Endonucleases activated during apoptosis target internucleosomal DNA sections and cause extensive DNA fragmentation. The fragmented low molecular weight DNA can be extracted from the cells following their fixation in precipitating fixatives as ethanol. As a result of DNA loss, apoptotic cells end up with fractional DNA content. When stained with a DNA fluorochrome, they can be recognized by flow cytometry as the cells having less DNA than G1 cells ("sub-G1" peak).

The method of Nicoletti and coworkers (22) was used with minor changes to evaluate PI incorporation. Briefly, 2.4 x 105 PMN were centrifuged at 4°C, 200 x g for 10 min, and then washed in PBS. Cells were resuspended in 500 µl PBS and were added dropwise to 4.5 ml ice-cold 70% ethanol while vortexing. After washing, the pellet was resuspended in 500 µl PBS and 5 ml of DNA extraction buffer (0.2 M Na2HPO4; 0.1 M citric acid; pH 7.8). After 5 min incubation, cells were washed and suspended in 140 µl RNase A (500 µg/ml) and 140 µl PI (100 µg/ml) and incubated 30 min at room temperature in the dark. Samples were washed in PBS before analysis by flow cytometry. Ten thousand events were collected in each sample, and a 488-nm laser line was used for excitation.

Microscopy Assessment of PMN Apoptosis
Cytospin preparations were fixed in methanol, stained with May-Grünwald-Giemsa (Merck, Rahway, NJ), and examined by oil-immersion light microscopy at a final magnification of x1,000. The percentage of apoptotic neutrophils 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, 300–400 PMN per slide were counted by two different operators without prior knowledge of the sample.

Cell Staining
Cell surface expression of CD11a/CD18, CD11b/CD18, Fas/APO-1, and Fc{gamma}RIIIb (CD16) was evaluated by direct immunofluorescence using saturating concentrations of monoclonal mouse antihuman-CD11a-PE, -CD11b-PE, -Fas/APO-1-PE (Ancell, Bayport, MN) and -CD16-FITC (Immunotech, Marseilles, France)-conjugated antibodies. Briefly, 5 x 105 cells were incubated with the antibody for 20 min on ice. Cells were washed and fixed in 500 µl of 1% paraformaldehyde. Using a FACScan (Becton-Dickinson Immunocytometry Systems, San Jose, CA), 10,000 events were collected in linear mode for forward scatter (FSC) and side scatter (SSC), and log amplification for FL-1 and FL-2. Analysis was performed using the CellQuest software (Becton-Dickinson), and isotype-matched controls were used to determine autofluorescence and nonspecific staining. Results were expressed as percentages of positive cells and as mean fluorescence intensity (MFI).

The cytoplasmic protein bcl-2 was measured by using a Fix and Perm kit (Caltag, Burlingame, CA). Washed cells were resuspended in 100 µl solution A (fixation) for 15 min at room temperature. After washing with PBS containing 1% azide and 5% FCS, cells were resuspended in solution B (permeabilization) and the mouse anti-human bcl-2 IgG1-FITC–conjugated antibody (Ancell). After a 20-min incubation on ice in the dark, cells were washed, resuspended in isoflow, and analyzed in the same manner as mentioned above.

Cytokine Determination
As indicated above, 3 x 106 PMN were cultured with or without heated MTB (1 x 106 bacteria) in 1 ml of CM. After 3 and 18 h of culture, supernatants were harvested, centrifuged, and kept frozen (-70°C) until used. The concentration of IL-8 in cell-free supernatants was determined using a commercial specific enzyme-linked immunosorbent assay (ELISA) kit (Immunotech) and processed according to the manufacturer's specifications. Cytokine levels were expressed as pg/ml of protein and the detection limit of the assay was 8 pg/ml.

Statistical Analysis
All values are presented as the mean ± SEM of a number of independent experiments The data were evaluated statistically using the paired or unpaired Student t tests. The relationship between percent of apoptosis and days of treatment was sought by Spearman's rank correlation test. A P value < 0.05 was considered as significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Spontaneous Apoptosis Is Enhanced in TB-PMN
As shown in Figure 1, recently isolated PMN from patients with TB patients (TB-PMN) and those from normal control subjects (N-PMN) did not exhibit apoptosis, as evaluated by morphologic features and DNA content.



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  		Figure 1. Apoptosis in N-PMN and TB-PMN in freshly isolated PMN. Cytospin preparations of N-PMN (n = 9) (A) and TB-PMN (n = 9) (B) were stained with May-Grünwald-Giemsa and photographed at a x1,000 microscope magnification. DNA content was evaluated in N-PMN (C) and TB-PMN (D) treated with 70% ethanol and resuspended in DNA extraction buffer. Cells were incubated 30 min with propidium iodide (PI) in the presence of RNase A before analysis. Mean fluorescence values are shown and this figure is representative of nine separate experiments.

 
As previously demonstrated by Homburg and colleagues (11), PMN that had been isolated from blood and aged in tissue culture showed higher morphologic changes in apoptosis, and this was time-related. Results in Table 1 show that after 3 h in culture, early apoptotic cells (AV+/PI-) as well as late apoptotic cells (AV+/PI+) were observed in both TB-PMN and N-PMN. In patients with TB, the percentage of total apoptotic PMN (AV+ = AV+/PI+ + AV+/PI- populations) and that of early apoptotic cells were significantly higher than those observed in N-PMN. However, after 18 h of culture, apoptosis increased to the same extent (P < 0.02), both in TB-PMN and N-PMN. Therefore, TB-PMN showed an accelerated rate of apoptosis in the absence of exogenous stimuli.


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  		TABLE 1 Percentage of early and total apoptotic cells incubated with medium alone, MTB or TNF-{alpha}

 
It is well known that the process of apoptosis is highly regulated by several genes in which the Fas/APO-1 receptor acts as an inducer, and that the bcl-2 molecule belongs to the survival-related genes. Fas receptor, a member of the TNF receptor (TNF-R) family, may be responsible for the induction of spontaneous apoptosis in PMN cultured overnight (14). Although the bcl-2 molcule is related to survival in many types of cells, its presence in mature PMN is under discussion (23). Therefore, to evaluate whether these molecules could be involved in the apoptosis observed in TB-PMN, we determined Fas expression on the surface membrane and bcl-2 expression in the cytoplasm by immunofluorescence analysis in recently isolated PMN. As can be observed in Figure 2, Fas was expressed by both N- and TB-PMN, but the MFI observed in TB-PMN was much higher (P < 0.03, n = 7; Mann-Whitney). On the other hand, the percentage of bcl-2–positive cells was lower in TB-PMN (P < 0.02, n = 7; Mann-Whitney), with a lower bcl-2 expression (P < 0.03, n = 7; Mann-Whitney).



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  		Figure 2. Differential expression of apoptosis-related molecules in freshly isolated PMN. (A) Fas/APO-1 expression and (B) the cytoplasmic protein bcl-2 expression on N-PMN (dotted lines) and TB-PMN (solid lines). Control isotype is represented by the filled histograms. One experiment representative of seven performed is shown.

 
Apoptosis Induced by MTB or TNF-{alpha} Is Differentially Regulated in TB and N-PMN
Bacterial compounds represent classical activating signals for PMN. This led us to investigate whether MTB could affect PMN survival. TNF-{alpha}, a proapoptotic cytokine present in TB lesions, was also studied. As shown in Table 1, culture of N-PMN for 3 h in the presence of MTB resulted in a higher percentage of total apoptotic cells (AV+) compared with control cultures (P < 0.02). In contrast, in patients with TB no differences were observed in the percentage of total (AV+) or early apoptotic cells (AV+PI-) when MTB-induced and control cells were compared.

After 18 h of culture, MTB induced a significant increase in the percentage of total apoptotic cells (AV+) in TB-PMN when compared with N-PMN (P < 0.05) or their own control cells (P < 0.02). In N-PMN, no differences in the percentage of AV+ cells were observed with respect to control.

Stimulated neutrophils undergo transient shape change, which can be detected by analyzing variations in their light-scattering properties by flow cytometry (24). As shown in Figure 3, the addition of MTB modified the FSC/SSC patterns observed in PMN in such a way that two regions could be defined: one of them corresponded to a lower FSC PMN population (R1), and the second one to a higher FSC cell population (R2). Moreover, cells could be observed in the R2 region in N-PMN after 3 h of stimulation with MTB, whereas in TB-PMN cells were present in this region even in the absence of antigen. Therefore, to evaluate whether the enhancement in MTB-induced apoptosis in TB-PMN at 18 h might be due to cell activation, we analyzed the presence of apoptotic cells in the R2 region. As shown in Figure 4, MTB-induced apoptosis was significantly increased in the R2 region of TB-PMN when compared with N-PMN after 18 h of culture (P < 0.02).



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  		Figure 3. FSC/SSC parameters of MTB- and control PMN. FSC/SSC parameters were analyzed in control and MTB-stimulated PMN from normal control subjects (N-PMN, n = 15) and from patients with tuberculosis (TB-PMN, n = 15) upon 3 (A) and 18 h (B) of culture. Two regions were defined: region 1 (R1) and region 2 (R2). Results are expressed as the percentage of PMN in the R2. This figure is representative of 15 experiments.

 


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  		Figure 4. Measurement of apoptosis in PMN with high FCS/SSC (region R2). PMN from patients with TB (shaded bars; n = 10) and from normal individuals (open bars; n = 8) were cultured during 3 and 18 h without stimuli (Control) or in the presence of MTB. PMN apoptosis was analyzed in R2 based on AV-FITC and PI binding (AV+). Results are expressed as percentage of AV+ cells (mean ± SEM). Asterisk indicates P < 0.04 for MTB-stimulated TB-PMN versus N-PMN or versus nonstimulated TB-PMN.

 
When TNF-{alpha} was assessed, we observed a higher percentage of total apoptotic cells (AV+) in N-PMN (P < 0.05) at 3 h, but there were no differences in the apoptosis of TB-PMN or of N-PMN at 18 h, even in the R2 region (data not shown). The increased apoptosis in TB-PMN was not due to the antituberculosis drug regime, because no differences were observed in the rate of apoptosis at 0 or 18 h among the patients in spite of the extent of treatment (Figure 5). Even more, patients studied before and after treatment did not show differences in the rate of apoptosis induced by MTB (data not shown).



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  		Figure 5. Correlation between percent of apoptosis and days of antituberculosis therapy. Freshly isolated TB-PMN (n = 12) (A), 18 h-cultured TB-PMN without stimulation (B) or in the presence of MTB (C) (n = 12). PMN apoptosis was analyzed based on AV-FITC and PI binding (AV+).

 
Taken together, these findings suggest that MTB-induced activation could be the reason for the increase in apoptosis observed in vitro and, likewise, that the accelerated spontaneous apoptosis of TB-PMN at 3 h could be the result of in vivo activation of peripheral TB-PMN.

Differential Fc{gamma}RIIIb (CD16) and CD11b Expression in Freshly Isolated and Cultured TB-PMN
CD16 is a low-affinity receptor for IgG (Fc{gamma}RIIIb), which is expressed on the surface of PMN. It has been demonstrated that the loss of this receptor correlates with other parameters of apoptosis in PMN cultured overnight (11, 13). Thus, we evaluated the CD16 expression in order to discriminate nonapoptotic (CD16 high expressive) from apoptotic (low expressive) neutrophils. In addition, expression of the ß2 integrins CD11a and CD11b, the latter overexpressed on the surface of activated leukocytes (5), was also evaluated.

As shown in Table 2, the percentage of CD16+ cells decreased over the culture period both in N-PMN and TB-PMN. At 3 h of incubation, a significantly lower MFI for CD16 was observed in control cultures (P < 0.04) from N-PMN and TB-PMN. TNF-{alpha} induced an increase in the CD16- population in N-PMN (P < 0.02) that correlated with the expected TNF-{alpha}–induced apoptosis. On the contrary, TNF-{alpha} did not modify CD16 expression in TB-PMN. Thus, the loss of CD16 expression in TB-PMN was partially prevented in the presence of MTB, suggesting that PMN from patients with TB are somehow sensitive to the bacterial challenge.


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  		TABLE 2 Cell surface expression of CD11b and CD16 in N-PMN and TB-PMN

 
In recently isolated PMN, the expression of CD11b in the CD16+ population was higher in TB- than in N-PMN (P < 0.04) (Table 2), and no differences in CD11a expression were observed (data not shown). After 3 h in culture, a spontaneous increase in the MFI of CD11b was observed in TB-PMN (P < 0.05) and, whereas MTB tended to enhance it, TNF-{alpha} did not modify CD11b expression. In N-PMN, both MTB (P < 0.04) and TNF-{alpha} (P < 0.05) enhanced the CD11b expression. After 18 h in culture, both TB- and N-PMN exposed to MTB overexpress CD11b with the highest MFI in TB-PMN (P < 0.04). Even in cells that underwent apoptosis (CD16-), a higher MFI for CD11b was found in the presence of MTB or TNF-{alpha} (Table 2). Furthermore, as shown in Figure 6, a population that corresponds to the R2 region, with a high CD11b expression, was induced by MTB both in N- and TB-PMN, these cells being larger and having a higher MFI in TB-PMN. Therefore, we can conclude that the rate of apoptosis is related to MTB-mediated activation, which might be differentially regulated in N and TB-PMN.



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  		Figure 6. CD11b expression on TB-PMN and N-PMN. PMN from patients with TB and from normal individuals were cultured during 18 h with medium alone (control) (A) or medium plus MTB (B). Flow cytometry analysis of CD11b-FSC was performed. Inserted histograms show the MFI and percentage of CD11b positive cells. A representative experiment of seven is shown.

 
IL-8 Production by TB-PMN
Soluble factors that can act as antiapoptotic signals could account for the unchanged rate of apoptosis observed in TB-PMN after 3 h in culture in the presence of MTB or TNF-{alpha}. PMN produce IL-8, a cytokine that is known to delay apoptosis (25). We determined IL-8 release in supernatants from TB- and N-PMN cultured with or without MTB for 3 h and 18 h. We observed that TB-PMN secreted higher amounts of IL-8 (332.4 ± 170.1 pg/ml, n = 8) than N-PMN (37.6 ± 19.1 pg/ml, n = 5) after 3 h in culture in the presence of MTB, whereas no IL-8 was detected in control supernatants from TB- and N-PMN. A high spontaneous secretion of IL-8 by TB-PMN was also observed after 18 h of culture (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Neutrophils are essential mediators of local and systemic inflammation. Although early PMN activation is required for wound healing and to limit microbial invasion, massive or prolonged activation may be detrimental to the host. PMN constitute one of the major cell populations observed during the inflammatory and exudative stages of TB (1). Their presence in the lung of an infected host is due to the release of chemotactic factors from alveolar macrophages and pulmonary epithelial cells (26, 27). PMN precede monocytes in areas of granulomatous inflammation and, in turn, release chemokines that attract monocytes (28). The PMN-mediated inflammatory response is regulated by activation of a constitutively expressed cell death program (apoptosis) that represents an effective mechanism to control their destructive potential. However, even though PMN phagocytose mycobacteria, they are generally believed to be unable to kill them efficiently and to be replaced by mononuclear cells at the mycobacteria-induced inflammatory site. Although the role played by PMN in the chronic stages of TB is still not clear, they can evoke a specific response against MTB.

In this study we demonstrate that recently isolated PMN from patients with TB do not present signs of apoptosis, although they show activation parameters as we have already shown (19). The fact that TB-PMN did not exhibit apoptosis might be due to the presence in circulation of antiapoptotic cytokines such as IFN-{gamma} (29). Moreover, the high CD11b expression observed in circulating TB-PMN could be explained by the in vivo presence of cytokines or mycobacterial products that, upon activation, provoke a rapid translocation of CD11b from intracellular granules to the plasma membrane (5). Taking into account that after migration into tissues, PMN either undergo spontaneous or activation-induced death by apoptosis, we evaluated their in vitro apoptosis. Our results indicate that TB-PMN showed an accelerated spontaneous apoptosis compared with PMN from normal individuals at 3 h. At this time, early apoptotic cells (AV+/PI-) account for the accelerated rate in spontaneous apoptosis. Besides, the higher Fas expression and the lower bcl-2 expression in TB-PMN could contribute to the accelerated rate of spontaneous apoptosis that we observed, as it was already described by Majewska and coworkers (30). Moreover, enhanced superoxide anion generation, high TNF-R55 expression, and MTB-induced TNF-{alpha} production (19) could make TB-PMN prone to undergo spontaneous apoptosis. Therefore, even in the absence of in vitro stimulation, the apoptotic process might be accelerated by the in vivo activation of TB-PMN.

It is well known that several PMN functions that depend on the activation state lead to changes in second messenger systems that are associated with the induction of apoptosis in these cells (31). We determined that PMN from patients with TB and from control subjects were activated in vitro by MTB and TNF-{alpha}, as demonstrated by the upregulation of the CD11b expression. Besides, CD11b is an important receptor that mediates phagocytosis of opsonized mycobacteria through the complement fragment iC3b (32) and also serves in nonopsonic recognition of microbes (33). Although opsonization of mycobacteria in human serum increases the percentage of cells with engulfed bacteria (33), nonopsonic recognition may confer an advantage in the alveolar space, where serum opsonins are limited.

Activation via phagocytosis of MTB by human PMN triggers the tyrosine phosphorylation of phospholipases involved in ROI production by activating p38 MAPK (34). Even though phagocytosis of the mycobacteria might be involved in PMN activation, this should be ruled out in our study because of the low ratio of bacteria to PMN employed (1:3). On the other hand, TB-PMN can also be activated by TNF-{alpha}–triggered signaling pathways, via the TNF-R55, which is overexpressed in TB-PMN (19). This could ultimately lead to apoptosis or stimulate the activation of p38, which in turn activates, directly or indirectly, nuclear factor (NF)-{kappa}B, inducing the secretion of cytokines. Therefore, it is likely that both the cytokine milieu and the activation status of the cells could influence whether TNF-{alpha} activates NF-{kappa}B or the apoptotic cascade (35).

Despite the fact that CD11b was upregulated, the degree of in vitro apoptosis triggered by MTB or TNF-{alpha} was different in TB- and N-PMN. The stimulus-induced apoptosis at 3 h of culture was not higher than spontaneous apoptosis in TB-PMN, indicating a delay in this process. In contrast, in N-PMN, MTB and TNF-{alpha}–induced apoptosis steadily increased with the time of culture. Moreover, N-PMN underwent TNF-{alpha}–induced apoptosis and presented an increased CD11b expression at 3 h, whereas TB-PMN did not. After 18 h in culture both N-and TB-PMN exhibit the same percentage of apoptotic cells, indicating that the delay in apoptosis could be overcome in TB-PMN. It is well known that IL-8 delays apoptosis (25), generating survival signals than can override the p38 MAPK death signals (36), and that apoptosis mediated by Fas and TNF-R can also be delayed by IL-8 (25). The high IL-8 secretion that we have observed in vitro in MTB-treated TB-PMN at 3 h of culture could partially explain the delay observed in apoptosis at early time points (Table 1). It has been demonstrated that in the presence of MTB, PMN from PPD-positive healthy contacts, produced IL-8 (37) and a synergistic effect on its release by TNF-{alpha} was observed before apoptosis occurred (38). Thus, certain cytokines and mycobacterial products can prolong PMN survival by interfering with the physiologic process of apoptosis. Even more, whereas N-PMN became apoptotic upon MTB stimulation for 3 h and reduced their CD16 expression, the decrease of apoptosis in TB-PMN occurred with preservation of the CD16 expression. Although it has been demonstrated that apoptotic PMN undergo a general loss in cellular functions (39), our findings suggest that TB-PMN could still be sensitive to stimuli triggering effector functions through CD16 at the site of infection. Therefore, the reshuffling of molecules such as CD11b and CD16 on the TB-PMN membrane seems to be a specific response to MTB. Hence, activation signals triggered by the mycobacterium could override the death signals making TB-PMN less sensitive to undergo apoptosis in vitro, after 3 h of stimulation.

It is well known that the CD11b receptor is dramatically upregulated on the surface of activated PMN that extravasate through the vessels and migrate into the site of infection, enhancing effector functions of these cells such as chemoattractant-inducing adhesion to endothelium, phagocytosis, spreading, and generation of oxidative burst. Besides, increased levels of CD11b may be brought about by certain cytokines such as TNF-{alpha}, IL-1, and IFN-{gamma} (40). At 18 h of culture MTB-induced apoptosis was accelerated in TB-PMN and was associated with the loss of CD16 and a high expression of CD11b (Tables 1 and 2). Even more, the PMN population defined by the R2 region (Figure 3) and with the highest CD11b expression (Figure 6) accounts for the increase in apoptosis. Hence, our findings demonstrate that activation-induced apoptosis is associated with the high expression of CD11b, although it is known that spontaneous apoptosis of circulating PMN occurs via a CD11b-independent mechanism (41). Therefore, increased CD11b expression as well as apoptosis in TB-PMN may be ascribed to the activation state of the cells.

Apoptotic effects of MTB have been described in different cell types. It is well known that infection with MTB makes macrophages and {gamma}{delta} T cells prone to undergo apoptosis. Although virulent MTB strains evade apoptosis of infected alveolar macrophages (42), no differences in apoptosis were found in chronically activated {gamma}{delta} T cells that were challenged with H37Rv, H37Ra, or LAM-deprived antigens secreted by growing mycobacteria (43). In this context, whereas the apoptosis of macrophages may contribute to the host defense by preventing the spread of infection (44), the role of mycobacteria-induced apoptosis in {gamma}{delta} T cells has been ascribed to the control of acute inflammatory response (45). Thus, as in {gamma}{delta} T cells, the role of MTB-induced apoptosis of PMN may be a mechanism that helps to control the inflammatory response. In the context of pulmonary inflammatory reactions with massive PMN recruitment, there is considerable potential for incidental tissue damage to occur. Hence, for resolution of inflammation, it is necessary both to limit leukocyte influx and to clear the redundant cells from the tissues. Our findings have potential clinical relevance because PMN that have migrated to a site of inflammation are most likely to be either primed or activated before encountering mycobacteria. The fact that MTB or TNF-{alpha}, both present in the affected lungs (46), did not increase apoptosis at short culture times in TB-PMN as expected, together with the preservation of CD16, suggests that PMN would be able to perform their effector functions at the site of infection. Prolongation of survival may be important for the regulation of host resistance and inflammation, and may represent a crucial step for certain cytokines that activate PMN functions. Likewise, later in time, MTB-induced activation might lead to accelerated apoptosis of PMN, thus avoiding tissue damage. It will therefore be important to identify the signaling pathways that are triggered in PMN, because neutrophil apoptosis and survival might be involved in the pathology of TB.


   Acknowledgments
 
This work was supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, PIP 0711/98), Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT, 05-04816), and Fundación Alberto J. Roemmers. M.A. is recipient of an ANPCyT fellowship. The authors thank Dr. Susana Fink for critically reading the manuscript and Dr. Lucía Barrera for kindly providing the M. tuberculosis H37-Rv strain. The authors also thank the medical staff of División Tisioneumonología of Hospital F. J. Muñiz for their great help in providing clinical samples from patients with tuberculosis.

Received in original form April 2, 2002

Received in final form June 6, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Murphy, S., and A. L. Florman. 1983. Lung defences against infection: a clinical correlation. Pediatrics 72:1–15.[Abstract/Free Full Text]
  2. Appelberg, R. 1992. Mycobacterial infection primes T cells and macrophages for enhanced recruitment of neutrophils. J. Leukoc. Biol. 57:472–477.
  3. Condos, R., W. N. Rom, Y. M. Liu, and N. W. Schluger. 1998. Local immune responses correlate with presentation and outcome in tuberculosis. Am. J. Respir. Crit. Care Med. 157:729–735.[Abstract/Free Full Text]
  4. Ozaky, T., S. Nakahira, K. Tani, F. Ogushi, S. Yasuoka, and T. Ogura. 1992. Differential cell analysis in bronchoalveolar lavage fluid of patients with tuberculosis. Chest 102:54–59.[Abstract/Free Full Text]
  5. Timothy, M. C., and J. M. Harlan. 1994. Leukocyte-endothelial adhesion molecules. Blood 84:2068–2101.[Abstract/Free Full Text]
  6. Jones, G. S., H. J. Amirault, and B. R. Andersen. 1990. Killing of Mycobacterium tuberculosis by neutrophils: a nonoxidative process. J. Infect. Dis. 162:700–704.[Medline]
  7. May, M. E., and P. J. Spagnuolo. 1987. Evidence for activation of a respiratory burst in the interaction of human neutrophils with Mycobacterium tuberculosis. Infect. Immun. 55:2304–2307.[Abstract/Free Full Text]
  8. Brown, A. E., T. J. Holzer, and B. R. Andersen. 1987. Capacity of human neutrophils to kill Mycobacterium tuberculosis. J. Infect. Dis. 156:985–989.[Medline]
  9. Babior, B. M. 1978. Oxygen-dependent microbial killing by phagocytes. N. Engl. J. Med. 298:659–668.[Medline]
  10. Cox, G., J. Crossley, and Z. Xing. 1995. Macrophage engulfment of apoptotic neutrophils contributes to the resolution of acute pulmonary inflammation in vivo. Am. J. Respir. Cell Mol. Biol. 12:232–237.[Abstract]
  11. Homburg, C. H. E., M. Haas, A. E. G. von dem Borne, A. J. Verhoeven, C. P. M. Reutelingsperger, and D. Roos. 1995. Human neutrophils lose their surface Fc{gamma}RIII and acquire annexin V binding sites during apoptosis. Blood 85:532–540.[Abstract/Free Full Text]
  12. Savill, J. S., A. H. Wyllie, J. E. Henson, M. J. Walport, P. M. Henson, and C. Haslett. 1989. Macrophage phagocytosis of aging neutrophils in inflammation: programmed cell death in the neutrophil leads to its recognition by macrophages. J. Clin. Invest. 83:865–867.[Medline]
  13. Dransfield, I., A. M. Buckle, J. S. Savill, A. McDowall, C. Haslett, and N. Hogg. 1994. Neutrophil apoptosis is associated with a reduction in CD16 (Fc{gamma}III) expression. J. Immunol. 153:1254–1263.[Abstract]
  14. Liles, W. C., P. A. Kiener, J. A. Ledbetter, A. Aruffo, and S. J. Klebanoff. 1996. Differential expression of Fas (CD95) and Fas ligand on normal human phagocytes: implications for the regulation of apoptosis in neutrophils. J. Exp. Med. 184:429–440.[Abstract/Free Full Text]
  15. Colotta, F., F. Re, S. Polentarutti, S. Sozzani, and A. Mantovani. 1992. Modulation of granulocyte survival and programmed cell death by cytokines and bacterial products. Blood 80:2012–2020.[Abstract/Free Full Text]
  16. Takeda, Y., H. Watanabe, S. Yonehara, T. Yamashita, S. Saito, and F. Sendo. 1993. Rapid acceleration of neutrophil apoptosis by tumor necrosis factor-alpha. Int. Immunol. 5:691–694.[Abstract/Free Full Text]
  17. Watson, R. W. G., H. P. Redmond, J. H. Wang, C. Condron, and D. Bouchier-Hayes. 1996. Neutrophils undergo apoptosis following ingestion of Escherichia coli. J. Immunol. 156:3986–3992.[Abstract]
  18. Yousefi, S., D. R. Green, K. Blaser, and H. U. Simon. 1994. Protein-tyrosin phosphorylation regulates apoptosis in human eosinophils and neutrophils. Proc. Natl. Acad. Sci. USA 91:10868–10872.[Abstract/Free Full Text]
  19. Aleman, M., M. Beigier-Bompadre, C. Borghetti, S. de la Barrera, E. Abbate, M. Isturiz, and M. C. Sasiain. 2001. Activation of peripheral blood neutrophils from patients with active advanced tuberculosis. Clin. Immunol. 100:87–95.[Medline]
  20. Boyum, A. 1968. Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Invest. 97:77–89.
  21. Vermes, I., C. Haanen, H. Steffens-Nakken, and C. Reutelingsperger. 1995. A novel assay for apoptosis: flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labeled Annexin V. J. Immunol. Methods 184:39–51.[Medline]
  22. Nicoletti, I., G. Migliorati, M. C. Pagliacci, F. Grignani, and C. Riccardi. 1991. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J. Immunol. Methods 139:271–279.[Medline]
  23. Akgul, C., D. A. Moulding, and S. W. Edwards. 2001. Molecular control of neutrophil apoptosis. FEBS Lett. 487:318–322.[Medline]
  24. Stocks, S. C., A. K. Michael, C. Haslett, and I. Dransfield. 1995. CD66-dependent neutrophil activation: a possible mechanism for vascular selectin-mediated regulation of neutrophil adhesion. J. Leukoc. Biol. 58:40–48.[Abstract]
  25. Kettritz, R., M. L. Gaido, H. Haller, F. C. Luft, C. J. Jennette, and R. J. Falk. 1998. Interleukin-8 delays spontaneous and tumor necrosis factor-{alpha} apoptosis of human neutrophil. Kidney Int. 53:84–91.[Medline]
  26. Sadek, M. I., E. Sada, Z. Toossi, S. K. Schwander, and E. A. Rich. 1998. Chemokines induced by infection of mononuclear phagocytes with Mycobacteria and present in lung alveoli during active pulmonary tuberculosis. Am. J. Respir. Cell Mol. Biol. 19:513–521.[Abstract/Free Full Text]
  27. Kurashima, K., N. Mukaida, M. Fujimura, M. Yasui, Y. Nakazumi, T. Matsuda, and K. Matsushima. 1997. Elevated chemokine levels in bronchoalveolar lavage fluid of tuberculosis patients. Am. J. Respir. Crit. Care Med. 155:1474–1477.[Abstract]
  28. Antony, V. B., S. A. Sahn, R. N. Harada, and J. E. Repine. 1983. Lung repair and granuloma formation: tubercle bacilli stimulated neutrophils release chemotactic factors for monocytes. Chest 83(Suppl. 5):95S–96S.
  29. Verbon, A., N. P. Juffermans, S. J. van Deventer, P. Speelman, H. van Deutekom, and T. van der Poll. 1999. Serum concentration of cytokines in patients with active tuberculosis (TB) and after treatment. Clin. Exp. Immunol. 115:110–113.[Medline]
  30. Majewska, E., Z. Sulowska, and Z. Baj. 2000. Spontaneous apoptosis of neutrophils in whole blood and its relation to apoptosis gene proteins. Scand. J. Immunol. 52:496–501.[Medline]
  31. Watson, R. W. G., H. P. Redmond, J. H. Wang, and D. Bouchier-Hayes. 1996. Bacterial ingestion, tumor necrosis factor-alpha, and heat induce programmed cell death in activated neutrophils. Shock 5:47–51.[Medline]
  32. Sengelov, H. 1995. Complement receptors in neutrophils. Crit. Rev. Immunol. 15:107–131.[Medline]
  33. N'Diaye, E. N., X. Darzacq, C. Astaire-Dequeker, M. Daffe, J. Calafat, and I. Maridonneau-Parini. 1998. Fusion of azurophil granules with phagosomes and activation of the tyrosine kinase Hck are specifically inhibited during phagocytosis of mycobacteria by human neutrophils. J. Immunol. 161:4983–4991.[Abstract/Free Full Text]
  34. Perskvist, N., L. Zheng, and O. Stendahl. 2000. Activation of human neutrophils by Mycobacterium tuberculosis H37Ra involves phospholipase C{gamma}2, Shc adapter protein, and p38 mitogen-activated protein kinase. J. Immunol. 164:959–965.[Abstract/Free Full Text]
  35. Beg, A. A., and O. Baltimore. 1996. An essential role for NF{kappa}B in preventing TNF-{alpha}-induced cell death. Science 274:782–784.[Abstract/Free Full Text]
  36. Frasch, S., J. Nick, V. Fadok, D. Bratton, and G. Worthen. 1998. p38-Mitogen-activated protein kinase-dependent and independent intracellular signal transduction pathways leading to apoptosis in human neutrophils. J. Biol. Chem. 273:8589–8597.
  37. Riedel, D. D., and S. H. E. Kaufmann. 1997. Chemokine secretion by human polymorphonuclear granulocytes after stimulation with Mycobacterium tuberculosis and lipoarabinomannan. Infect. Immun. 65:4620–4623.[Abstract]
  38. Kasahara, K., I. Sato., K. Ogura, H. Takeuchi, K. Kobayashi, and M. Adachi. 1998. Expression of chemokines and induction of rapid cell death in human blood neutrophils by Mycobacterium tuberculosis. J. Infec. Dis. 178:127–137.[Medline]
  39. Whyte, M. K. B., L. C. Meagher, J. MacDermot, and C. Haslett. 1993. Impairment of function in aging neutrophils is associated with apoptosis. J. Immunol. 150:5124–5134.[Abstract]
  40. Limb, G., A. Hamblin, R. Wolstencroft, and D. Dumonde. 1991. Rapid upregulation of granulocyte integrins and complement receptor 1 by cytokines. Immunol. 74:696–702.[Medline]
  41. Coxon, A., P. Rieu, F. J. Barkalow, S. Askari, A. H. Sharpe, U. H. von Andrian, M. A. Amin-Arnaout, and T. M. Mayadas. 1996. A novel role for the ß2 integrin CD11b/CD18 in neutrophil apoptosis: a homeostatic mechanism in inflammation. Immunity 5:653–666.[Medline]
  42. Keane, J., H. G. Remold, and H. Kornfeld. 2000. Virulent Mycobacterium tuberculosis strains evade apoptosis of infected alveolar macrophages. J. Immunol. 164:2016–2020.[Abstract/Free Full Text]
  43. Manfredi, A. A., S. Heltai, P. Rovere, C. Sciorati, C. Paolucci, G. Galati, C. Rugarli, R. Vaiani, E. Clementi, and M. Ferrarini. 1998. Mycobacterium tuberculosis exploits the CD95/CD95 ligand system of {gamma}{delta} T cells to cause apoptosis. Eur. J. Immunol. 28:1798–1806.[Medline]
  44. Fratazzi, C., R. D. Arbeit, C. Carini, M. K. Balcewicz-Sablinska, J. Keane, H. Kornfeld, and H. G. Remold. 1999. Macrophage apoptosis in mycobacterial infections. J. Leukoc. Biol. 66:763–764.[Abstract]
  45. D'Souza, C. D., A. M. Cooper, A. A. Frank, R. J. Mazzaccaro, B. R. Bloom, and I. M. Orme. 1997. An anti-inflammatory role for gamma delta T lymphocytes in acquired immunity to Mycobacterium tuberculosis. J. Immunol. 158:1217–1221.[Abstract]
  46. Barnes, P. F., S. J. Fong, P. J. Brennan, P. E. Twomey, A. Mazumder, and R. L. Modlin. 1990. Local production of tumor necrosis factor and IFN-gamma in tuberculous pleuritis. J. Immunol. 145:149–154.[Abstract]



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