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Keywords:

  • apoptosis;
  • Crohn's disease;
  • polymorphonuclear neutrophils;
  • Bcl-2 family protein expression

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Background: The etiology of Crohn's disease (CD) remains unknown, and the defective function of neutrophils appears to be associated with this pathology. Neutrophils undergo spontaneous apoptosis which, if not tightly regulated, can induce the development of chronic inflammatory disease. The Bcl-2 protein family is also involved in the regulation of neutrophil apoptosis.

Methods: This study investigated the apoptosis and expression of some regulatory factors in CD patient and control polymorphonuclear neutrophils (PMN) in suspension and in adhesion on fibronectin, an extracellular matrix protein. These 2 conditions mimic circulating neutrophils before they are recruited at the intestinal levels, and their adhesion to tissue.

Results: Apoptosis in CD patient PMN was delayed in suspension and accelerated in adhesion, which is the opposite of what happens in controls. Higher levels of Bax, Bcl-2, and Mcl-1 proteins were registered in freshly isolated CD patient PMN, in contrast to controls, in which Bcl-2 protein was undetectable. Among the studied pro- and antiapoptotic factors, Bax levels seem to be mainly related to the difference in apoptosis between PMN of CD patients and controls.

Conclusions: For the first time it has been demonstrated by direct experimental evidence that apoptosis in CD patient PMN is regulated differently from that of control PMN. Abnormal expression of regulating apoptosis proteins is shown in CD patient PMN. These data suggest that the defective functionality of neutrophils can be the early event responsible for the altered mucosal immune response in CD, and that neutrophil apoptosis may offer a new target for specific drugs and therapy tools.

(Inflamm Bowel Dis 2008)

Crohn's disease (CD) is a chronic inflammatory disorder of the gastrointestinal tract characterized by recurrent remissions and relapses and by a complex interaction of environmental, genetic, and immunoregulatory factors.1 The exact CD etiology remains unknown, but excessive and abnormal mucosal immune responses to components of the microflora are considered the prime cause of this pathology.2 In fact, the deregulated local immune defense and the constant influx of leukocytes from the circulation toward the intestinal epithelium are the basis for a continuous inflammatory state.3 The migration and accumulation of large numbers of neutrophils within the intestinal lumen are evident and may be involved in CD pathogenesis.4 Indeed, this pathology is associated with defective functioning of neutrophils,5 and some of the literature attributes the pathological persistence of the inflammatory state in the gastrointestinal tract to the delayed neutrophil apoptosis.6, 7 Neutrophils undergo spontaneous apoptosis; this is the preferred mechanism for inducing controlled cell death and prevents the release of toxic mediators, in contrast to necrosis. It also promotes the resolution of inflammation through the phagocytic elimination of neutrophils from inflamed tissue. Nevertheless, apoptosis closes down several activities of the neutrophils, impairing their functions8 and, if not tightly regulated, can harm the release of reactive oxygen species (ROS) and proteases, promoting the development of chronic inflammatory disease.9 When the neutrophils are recruited to the infected or inflamed site they survive longer10 and the induction or prevention of neutrophil apoptosis by local inflammatory mediators may be important in the control of inflammation.11, 12 Members of the Bcl-2 protein family, including antiapoptotic (Bcl-2, Bcl-XL, Mcl-1) and proapoptotic (Bax, Bad) factors, are involved in the regulation of neutrophil apoptosis13 and may contribute to the pathogenesis of many diseases.14 In fact, the shift of equilibrium between factors that suppress or activate apoptosis can promote or hinder human neutrophil apoptosis.15

The aim of this study was to investigate apoptosis and related factors in polymorphonuclear neutrophils (PMN) isolated from CD patients and control subjects in different experimental conditions, taking into account the literature on the neutrophils and their apoptosis in inflammatory processes. We wished to identify some aspects of the altered functionality of CD patient neutrophils, and to clarify their role in the defective immunological response at the onset of the inflammation and relapses in all intestines in CD patients.5 The few reported data on apoptosis of CD patient neutrophils are often indirect evidence, and show only a delayed apoptosis obtained in neutrophils in suspension isolated from ulcerative colitis (UC) and CD patients, without any distinction between the pathologies.6, 7 We therefore investigated the apoptosis of neutrophils from CD patients and controls cultured in suspension and in adhesion on an extracellular matrix (ECM) protein, the fibronectin: this condition mimics the adhesion of neutrophils to the tissue. The literature has data about the regulation of apoptosis in neutrophils adhering to fibronectin and other proteic substrates.16 Our previous studies have shown reduced Omath image production and adhesion in CD patient PMN plated on fibronectin after stimulation with tumor necrosis factor (TNFα), a proinflammatory cytokine largely produced in CD patients,17 suggesting cells in this condition have altered functionality. PMN apoptosis in suspension was investigated because neutrophils are in circulation before they are recruited at the intestinal level and the results can be compared with those obtained in adhesion. Finally, since no data about variations of pro- and antiapoptotic proteins related to the regulation mechanisms of apoptosis are reported in CD patient neutrophils, the levels of Bax, Bcl-2, and Mcl-1 proteins in CD patient and control subjects PMN were measured in the experimental conditions.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Patients

Twenty-five patients with CD (12 males, 13 females; 23–54 years) and 27 controls, healthy individuals or patients without intestinal or immune pathologies (13 males, 14 females; 26–57 years) participated in this study and blood was drawn after receiving informed consent. We chose a group of CD patients similar to the controls with regard to sex and age range and, for homogeneity, all CD patients suffered from ileal disease, had received bowel resections, and were in the active phase of the disease as documented both through clinical assessment and the Crohn's Disease Activity Index (CDAI, ranging from 220–400). Patients with a score below 150 are considered to be in clinical remission and scores above 450 reflect severe active CD.18 Patients included in this study were treated with drugs prevalently used in CD pathology and we excluded patients submitted to anti-TNF or immunosuppressive therapy and no patients had received steroids for at least 1 month before blood samples were taken.

PMN Preparation

PMN were isolated from peripheral venous blood in the presence of EDTA in sterile conditions by centrifugation on Ficoll-hypaque gradient as previously reported.17 PMN were resuspended in RPMI 1640 supplemented with 2 mM L-glutamine, 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin (complete medium) at a concentration of 2 × 106 cell/mL. PMN viability was greater than 99% as assessed by Trypan blue exclusion test and by flow cytometry; purity was greater than 97% as analyzed by microscopy using methyl-violet 1% in isocitric acid 0.1 N staining.

Measurement of PMN Apoptosis

PMN were cultured in fibronectin-coated 96-well plates (adhesion) or in polypropylene tubes to prevent adherence (suspension) at 37°C in a 5% CO2 atmosphere at a concentration of 0.3 × 106 cells/150 μL complete medium. Apoptosis was assessed measuring the phosphatidylserine (PS) on the external membrane of apoptotic cell and the DNA fragmentation using the Guava Nexin Kit (Guava Technologies, Hayward, CA) and Cell Death Detection ELISAplus Kit (Roche Laboratories, Nutley, NJ), respectively, according to the manufacturer's instructions. Briefly, for the first method PMN adherent in wells and in suspension were centrifuged at 300g for 10 minutes at 4°C and resuspended in 40 μL of cold Nexin buffer; 5 μL of Annexin V-PE and 5 μL of Nexin 7-amino-actinomycin D (7-ADD), a compound that is excluded from live and apoptotic cells but permeates necrotic cells. Samples were incubated on ice in the dark for 20 minutes and the volume was increased to 500 μL with Nexin buffer immediately before analysis by flow cytometer. The results are expressed as the percentage of positive PMN binding Annexin V. For the second method cytoplasm fractions obtained from 1 × 106 cells (20 μL) were dispersed into microtitration wells coated with streptavidin. A mixture of anti-histone-biotin-antibody and anti-DNA-peroxidase antibody were added and wells were incubated at room temperature for 2 hours on a microplate shaker. Then the wells were washed and 100 μL peroxidase substrate solution was added to each well, after which absorbance at 405 nm was measured (reference wavelength ≈490 nm).

Data are expressed as specific enrichment of mono- and oligonucleosomes released into the cytoplasm and calculated using the following ratio = absorbance of the sample/absorbance of the corresponding negative control (apoptosis at 0 time).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

PMN Apoptosis in Adhesion and in Suspension

In the circulation neutrophils have a very short half-life (8–20 hr), which increases when they enter infected or inflamed tissue.10 In the acute phase of inflammation neutrophils are recruited from the circulation at the site of the infection, where they adhere to other cells or to ECM proteins. We determined PMN apoptosis after the adhesion of neutrophils on fibronectin and in suspension for 20 hours. Apoptosis was measured by double labeling the CD patient and control PMN with Annexin V-phycoerytrin (AnV), a calcium-dependent phospholipid binding protein with a high affinity for PS, which translocates from the internal face of the cell membrane on the surface of apoptotic cells, and also with 7-ADD, which is an indicator of the structural integrity of the membrane.20, 21 By this method 3 populations of cells were distinguished: viable nonapoptotic cells (AnV, 7-ADD); early-apoptotic cells (AnV+, 7-ADD); and late apoptotic-necrotic cells (AnV+, 7-ADD+). The quantitative estimation of PMN which had been cultured for 20 hours on fibronectin with PS in the outer leaflet of the plasma membrane demonstrated a higher percentage of CD patient apoptotic PMN compared to controls, causing a significant decrease in cell viability (Fig. 1A). Values for late apoptotic-necrotic PMN were very low in both groups of PMN, so we do not consider this effect physiologically important. These results indicate that CD patient PMN in a state of adhesion undergo programmed death more readily than the control PMN. In contrast, Figure 1B shows that apoptosis and viability in CD patient PMN maintained in suspension for 20 hours were significantly delayed and increased, respectively, relative to control PMN. This confirms published findings on late apoptosis in IBD patient PMN in suspension6, 7 and demonstrates that, in contrast to what occurs in control, apoptosis in CD patient PMN was delayed when the cells were in suspension and accelerated when they were in adhesion. Indeed, the literature reports that apoptosis of PMN in adhesion on fibronectin is delayed compared with when these cells are circulating.16

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Figure 1. Phosphatidylserine analysis in control and CD patient PMN in adhesion on fibronectin (A) or in suspension for 20 hours (B). The apoptosis was measured by the Guava Nexin Kit in PMN cultured in fibronectin-coated 96-well plates (adhesion) or in polypropylene tubes (suspension) for 20 hours resuspended in cold Nexin buffer containing Annexin V-PE and Nexin 7-ADD. Data acquired by the Flow Cytometer Guava PcA system are displayed on the computer screen in a dot-plot format with a user-controlled quadrant marker and 3 populations of cells can be distinguished as viable nonapoptotic, AnV/7ADD; early-apoptotic, AnV+/7ADD; late-apoptotic and necrotic, AnV+/7ADD+. Data are expressed as the percent ± SD of 6 separate experiments performed in triplicate. In parentheses the variation percentage with respect to control is reported. *P ≤ 0.005 compared with the respective values of control PMN; °P ≤ 0.001 compared with the respective values obtained in PMN in suspension.

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Since it is very important when studying apoptosis to use complementary techniques to assess cell death, we also measured DNA fragmentation in PMN in the same experimental conditions. Figure 2 shows that these results were in agreement with those previously observed (Fig. 1).

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Figure 2. DNA fragmentation analysis in control and CD patient PMN in adhesion on fibronectin or in suspension for 20 hours. The apoptosis was measured by determination of cytoplasmic histone-associated-DNA-fragment using the Cell Death Detection ELISAplus kit. PMN were cultured in fibronectin-coated 96-well plates (adhesion) or in polypropylene tubes (suspension) for 20 hours. Cytoplasmic fractions obtained from 106 cells (20 μL) were dispersed into microtitration wells coated with streptavidin and a mixture of anti-histone-biotin-Ab and anti-DNA-peroxidase Ab was added. Data are expressed as enrichment factor of mono- and oligonucleosomes released into cytoplasm calculated using the ratio between optical density of the sample and that of the corresponding negative control (apoptosis at 0 time). Values are the means ± SD of 6 separate experiments performed in triplicate. *P ≤ 0.001 compared with the respective values of control PMN.

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Expression of BCl-2 and Bax Proteins in PMN

To identify each of the target proteins involved in the different apoptotic processes in control and in CD patient PMN in suspension and in adhesion, we found the expression of Bcl-2 and Bax proteins, which have opposite effects on apoptosis. In fact, since Bcl-2 represses and Bax promotes apoptosis, it has been suggested that one inhibits the activity of the other,22 and that the shift of equilibrium between pro- and antiapoptotic factors can promote or hinder the apoptosis of human neutrophils.15 The expression of Bax and Bcl-2 was examined in freshly isolated PMN (0 time) or cultured for 6 and 20 hours on fibronectin and in suspension by Western blot analysis using antibodies specific to each protein. Figure 3 shows typical immunoblots of Bcl-2, Bax, and actin, and relative densitometric analysis from 6 independent experiments. Figure 3A shows that Bax expression in CD patient PMN at 0 time was higher than that at 0 time in control PMN, and this did not change in either group of PMN after 6 and 20 hours of adhesion. Similarly, high levels of Bcl-2 protein were measured in CD patient PMN at 0 time: these decreased significantly after 6 and 20 hours of adhesion. On the contrary, Bcl-2 expression in control PMN at 0 time was undetectable and then increased (Fig. 3B). Contradictory data about Bcl-2 expression in neutrophils are reported in the literature; we also detected this protein in peripheral blood mononuclear cells (PBMC) used as a positive control (data not shown). These results suggest that in CD patient PMN in adhesion, downregulation of Bcl-2, and maintenance of high levels of Bax may play a role in increased apoptosis, as opposed to the results for control PMN, in which these proteins exhibited the reverse behavior.

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Figure 3. Bax and Bcl-2 expression in control and CD patient PMN in adhesion on fibronectin or in suspension. The expression of Bax and Bcl-2 was measured in freshly isolated PMN (0 time), in PBMC (as positive control), and after adhesion on fibronectin for 6 and 20 hours or in suspension for 20 hours (20S). Western blot with specific Ab of cell lysates was performed using anti-Bax Ab and, after stripping procedure, the same filter was reprobed with anti-Bcl-2 Ab and anti-actin Ab. The blot is representative of 6 separate experiments (A). The normalized values of Bax (B) and Bcl-2 (C), obtained by densitometric analysis, and reported as arbitrary unity, represent the mean ± SD of 6 separate experiments. *P ≤ 0.005 compared with the respective values of control PMN; °P ≤ 0.001 compared with the respective values in PMN in adhesion; §P ≤ 0.005 compared with values of CD patient PMN at 0 time and after 20 hours in suspension.

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In Figure 3A,B the expression of Bax and Bcl-2 in PMN in suspension is shown only after 20 hours, since no significant variation was observed after 6 hours, compared with freshly isolated PMN (data not shown). Figure 3A shows the remarkable increase in Bax levels in control PMN after 20 hours in suspension compared to Bax levels in PMN in adhesion for 20 hours and to Bax expression measured in CD patient PMN in suspension; this last value was significantly reduced in comparison with Bax values in the same cells in adhesion. Bcl-2 values in control PMN in suspension also increased, but these values were similar to those measured in the same condition in CD patient PMN: in fact, Bcl-2 remained high and unchanged in these cells compared with 0 time values, in contrast to that observed in Bax (Fig. 3B). Since no significant change was observed in Bcl-2 values in 2 groups of PMN after 20 hours of adhesion or suspension, the variations of Bax expression (a protein that promotes programmed cell death) appear to be the main cause of the different apoptosis values in CD patient PMN than in controls. Some data suggest that the interaction between Bcl-2 and Bax is in part necessary for their function.22, 23 In fact, the Bax/Bcl-2 ratio can be considered critical in determining susceptibility to apoptosis in many cell systems19, 24 and it is more important than the levels of expression of individual proteins.25 We therefore determined the ratio between the band density of Bax and Bcl-2 (Bax/Bcl-2 ratio) obtained by Western blot analysis.

Figure 4 shows that the Bax/Bcl-2 ratio in CD patient PMN after 20 hours of adhesion was higher than that registered in control cells. This seems to be due mainly to the higher Bax values in CD patient PMN compared with the control, given that following the changes of Bcl-2 expression in adhesion its values after 20 hours were similar in both PMN groups (see Fig. 3). On the other hand, in CD patient PMN in suspension for 20 hours the Bax/Bcl-2 ratio was lower than that measured in control PMN, and this too is related mainly to the different values of Bax in suspension in the 2 groups (see Fig. 3). Thus, we note that the different Bax/Bcl-2 ratios after 20 hours in both conditions in 2 groups of PMN are related to the different apoptosis values in PMN in the same conditions (Fig. 4). However, the tendency of PMN to undergo apoptosis in both conditions seemed unrelated to the Bax/Bcl-2 ratio, since the values of this ratio at 0 time were higher than those measured at other times (data not shown); this indicates that these proteins affect PMN apoptosis independently.

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Figure 4. Bax/Bcl-2 ratio in control and CD patient PMN in adhesion on fibronectin or in suspension. Bax/Bcl-2 ratio in PMN cultured in adhesion or suspension for 20 hours was calculated using the normalized values of Bax or Bcl-2 band density obtained by Western blot densitometric analysis of 6 separate experiments and measured as reported in Figure 3. In parentheses apoptosis percentage in control and CD patient PMN obtained from Figure 1 is reported. *P ≤ 0.001 compared with the respective in PMN in adhesion.

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Expression of Mcl-1 Protein in PMN

Mcl-1 is an antiapoptotic protein, structurally related to Bcl-2, rapidly inducible, and with a high turnover rate which plays an important role in the regulation of apoptosis.26 Figure 5 shows that the expression of Mcl-1 protein is significantly higher in freshly isolated PMN from CD patients than in control PMN. In both PMN groups its expression was unchanged after 6 hours of adhesion, whereas after 20 hours it had decreased in CD patient PMN and increased in control PMN, similar to Bcl-2 expression in the same condition. In both groups of PMN in suspension, Mcl-1 expression decreased compared to the values measured at 0 time. The pattern of Mcl-1 expression in control PMN in suspension and in adhesion confirms the data reported in the literature27–29 and seems to be correlated with the apoptotic processes observed in these conditions. However, this relationship is not found in CD patient PMN, although Mcl-1 levels in both conditions were similarly downregulated.

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Figure 5. Mcl-1 expression in control and CD patient PMN in adhesion on fibronectin or in suspension. The expression of Mcl-1 was measured in freshly isolated PMN (0 time) and after adhesion on fibronectin for 6 and 20 hours or in suspension for 20 hours (20S). Western blot with specific Ab of cell lysates was performed using anti-Mcl-1 Ab and, after stripping procedure, the same filter was reprobed with anti-actin Ab. The blot is representative of 6 separate experiments (A). The normalized values of Mcl-1, obtained by densitometric analysis, and reported as arbitrary unity, represent the mean ± SD of 6 separate experiments (B). *P ≤ 0.001 compared with the respective values of control PMN; °P ≤ 0.001 compared with the respective values in PMN at 0 time; §P ≤ 0.001 compared with the respective values in PMN in adhesion.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Using 2 different methods, this study demonstrates for the first time that apoptosis in CD patient PMN in suspension or in adhesion on fibronectin for 20 hours is regulated differently from that of control PMN. In fact, adhesion induces a decrease in the apoptotic process in control PMN and an increase in CD patient PMN, and the opposite results in suspension. The apoptotic behavior of control PMN in the 2 conditions confirms the data in the literature,16 but our findings on apoptosis of CD patient PMN constitute the first direct experimental evidence. Other authors have found decreased values in the apoptosis of neutrophils in suspension obtained from patients affected by IBD compared with controls,6, 7 as we observed in CD patient PMN, but those authors do not distinguish between the data obtained from CD and UC patient neutrophils.

The adherence of PMN to ECM proteins affects the functions of PMN, such as respiratory burst, degranulation, and phagocytosis,30 and inadequate or inappropriate cell-ECM interactions may induce PMN apoptosis.31 β2 integrins, leukocyte-specific integrins required for neutrophil adhesion, have recently been implicated in the regulation of neutrophil apoptosis, and a model has been suggested whereby the engagement of β2 integrin Mac-1, through its link to ECM proteins, extends the survival of neutrophils.16 Mac-1 is mainly involved in leukocyte activation after adhesion,32 and the increased life span of neutrophils after binding of Mac-1 to ECM is attributed to the phosphatidylinositol 3-kinase (PI3K)/Akt pathway.16 In several cell types Akt, after its activation by phosphatidylinositol 3-kinase, inhibits the apoptotic mitochondrial pathway33 and modulates the expression of Bcl-2 family proteins.34, 35 The increased apoptosis that occurs in CD patient PMN may therefore be due to impaired Mac-1 signaling, even if we have previously observed similar adhesion on fibronectin of untreated CD patient and control PMN.17 Moreover, the neutrophil constitutive apoptosis involves mitochondria36 and is accelerated or delayed by modulation of the cellular survival or death factors, such as Bcl-2 family proteins.29, 37, 38 These are very important in the control of apoptosis, which they regulate in part by manipulating the integrity of these organelles and affecting the release of cytochrome C.39 Human PMN express numerous proteins of the Bcl-2 family, including Bax and Mcl-1,40, 41 whereas numerous studies show that the antiapoptotic Bcl-2 is not expressed in freshly isolated human neutrophils.19, 42 We too detected a very low expression of Bcl-2 in freshly isolated control PMN (0 time). However, we found significant expression of Bcl-2 in CD patient PMN at time 0; both the antiapoptotic Mcl-1 and the proapoptotic Bax proteins are prevalent in these cells relative to control PMN in the same condition. This may allow CD patient PMN to undertake a minor or major apoptosis, depending on the conditions. In fact, the difference in the changes of Bax, Bcl-2, and MCl-1 expression in the 2 groups of PMN, in adhesion and in suspension, can explain their opposite behavior in apoptosis. The increase in these proteins in freshly isolated CD patient PMN can be related to the high levels in these patients of factors such as growth factors or interleukin, which modulate the concentration of some proteins of the Bcl-2 family.29

The downregulation of antiapoptotic factors (Bcl-2 and Mcl-1) in CD patient PMN that we observe after 20 hours in adhesion disagrees with the literature, which reports the upregulation of the expression of antiapoptotic proteins in neutrophils in adhesion.28, 43 Effectively, we too detected upregulation of Bcl-2 and Mcl-1 in control PMN in adhesion after 20 hours. Therefore, since the activation of the phosphoinositide 3-kinase/Akt signaling pathway induces an increase in antiapoptotic proteins such as Mcl-1,34 the different results obtained in CD patient PMN compared with controls support the hypothesis that there is defective activation in CD patient neutrophils of this signaling pathway involving the integrin Mac-1.16 This may be consistent with the defective Omath image production and adhesion process we identified previously in CD patient PMN after stimulation with TNFα.17

In particular, the differing modulation of the apoptotic mechanism in control and CD patient PMN in both conditions seems to be due mainly to the regulation of proapoptotic Bax expression, rather than to Bcl-2 or Mcl-1 level changes. In fact, some data show the role of Bax protein as an important regulatory factor in neutrophil apoptosis.15, 19 Moreover, even if a correlation is found between Bax/Bcl-2 ratios and PMN apoptosis values after 20 hours in adhesion and in suspension, these proteins regulate apoptosis independently.44

In conclusion, this study provides the first direct evidence of the difference in apoptotic behavior of CD patient PMN in suspension or in adhesion compared with control PMN, and demonstrates that the expression of pro- and antiapoptotic factors is also altered. These findings as a whole indicate that the delayed apoptosis and long life of circulating CD patient PMN can be responsible for their excessive transmigration at inflamed intestinal sites. However, our data suggest that, contrary to what other authors propose,6, 7 this is not the main cause of the persistent inflammatory state that characterizes CD; rather, it is the increased apoptosis and reduced viability found in CD patient PMN in adhesion. This may in fact cause the inefficiency of the neutrophils to end the acute inflammation, increasing the recruitment of cells responsible for chronic inflammation. The increased apoptosis of neutrophils when adhering on tissue, along with the defective apoptosis of lymphocytes and monocytes shown by others in this pathology,45 can therefore constitute an important pathogenic mechanism in CD.

Moreover, given that the resolution of the inflammatory process is due to phagocytosis following programmed cell death46 and that various factors involved in inflammatory chronic diseases inhibit the ability of macrophages to phagocytate apoptotic neutrophils,47 the abnormal apoptosis of CD patient PMN adhering to the intestinal mucosa can exacerbate the injury to the tissue. This is because neutrophils release histotoxic granular content, which is prompted by the secondary necrosis that the apoptotic cells undertake.48

Other investigations are needed to identify specific factors related to the changes in the rate of apoptosis of CD patient PMN with respect to controls. However, these data suggest that some aspects of the altered immunological response may start the inflammatory process and its chronicization in the intestine of CD patients. Moreover, this study suggests that neutrophils and the factors involved in the regulation of their vitality and apoptosis are important targets for specific drugs, and may help in the evaluation of new biological therapies for treating this pathology.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES