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- MATERIALS AND METHODS
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.
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 O 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.
- Top of page
- MATERIALS AND METHODS
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 O 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.