A comparative macroarray analysis between healthy blood PMNs and CF blood and airway PMNs was performed on 1050 different genes. For ethical reasons, we could not obtained PMN from healthy children, but there are probably few differences between PMNs from children and young adults.
We mainly focus our discussion on genes whose expression was significantly enhanced in CF PMNs compared with healthy PMNs. Vitronectin is one of them. Vitronectin is a multifunctional glycoprotein present in blood and anchored to the extracellular matrix (19
). It promotes cell adhesion, spreading, and migration by interaction with specific integrins. Vitronectin is involved in fibrinolysis and also in the immune defense through its interaction with the terminal complex of complement and in hemostasis through its binding to heparin. If released in the airways, vitronectin can potentially regulate the proteolytic degradation of the matrix. All these properties make vitronectin an important molecule associated with the inflammatory process in the airways of CF patients.
Among other genes whose expression was significantly enhanced in CF blood PMNs, it is worth mentioning the chemokines CCL17, CCL18, XCL1, CXCL9, and CXCL10. The upregulation of two, CCL17 and CXCL10, was further confirmed by qPCR and ELISA. Interestingly, these chemokines are different from those for which gene expression was upregulated in vitro by lipopolysaccharide (LPS) (20
), after receptor-mediated phagocytosis (21
), or after bacterial-induced apoptosis (22
). Our analysis was performed in patients negative for P. aeruginosa
. Thus, the difference between CF and healthy PMNs is of interest because it suggests that the absence of the active CFTR molecule is sufficient to initiate a reprogramming of gene expression in circulating PMNs in the absence of microbial stimuli. This modification may be a direct consequence of the CFTR mutation itself (1
), or an indirect effect of the inflammatory process occurring early after birth in CF patients (2
Among the chemokines whose gene expression was upregulated, CXCL9 and CXCL10 are both induced in response to IFNγ. Two other IFNγ-related genes were also upregulated, 1-8D and interferon responsive factor-1 (IRF-1). Mainly induced by IFNγ, the function of 1-8D protein is unknown at present. IRF-1 is a transcription factor that acts as a regulator of cell cycle and apoptosis and negatively regulates cell growth. A role for IFNγ in the pathophysiology of cystic fibrosis is thus suggested by these observations. Indeed, IFNγ has been detected in sputa (23
) and circulating γδT cells of CF patients with P. aeruginosa -
Another striking finding is the upregulation of the G-CSF gene revealed by macroarray analysis and confirmed by qPCR. In addition, an increased presence of G-CSF in supernatants of CF blood neutrophils was observed. G-CSF was previously detected in the airway of CF patients (25
) and in their serum (23
). It is known that G-CSF regulates the production, maturation, function, and survival of neutrophils, and it can also exacerbate underlying inflammatory diseases (27
). Whereas many cells can produce G-CSF, it is interesting to note that neutrophils could contribute to their own activation process in an autoregulatory loop. Accordingly, G-CSF could be a natural factor present in the serum of CF patients that contributes to the activation status observed for patients’ neutrophils, even in the absence of infection.
Another interesting observation was the upregulated expression of genes coding for cytokine receptors. This was particularly the case for IL-3R. Although IL-3 is not as potent as GM-CSF to activate or prime mature PMNs (28
), it may act synergistically with other cytokines such as IFNγ (30
) or more efficiently if the number of receptors is enhanced. The upregulation of the gene expression of CXCR2 further suggests that IL-8, found in large amounts in CF sputa (31
) and in supernatants of CF airway epithelial cell/PMN coculture (12
), is acting on PMNs. This result also correlates nicely with the fact that CF blood PMNs showed significantly increased migration to IL-8 (32
). Among cytokine receptors, IL-10Rα gene expression was found to be upregulated by both macro-array and qPCR. It is worth mentioning that blood PMNs from CF patients are responsive to the anti-inflammatory effects of IL-10, which can inhibit IL-8 production in vitro in response to bacterial LPS or peptidoglycan (33
We also found an upregulation of the expression of several genes regulating the NF-κB transcription factor in both blood and airway PMNs of CF patients. In unstimulated cells, NF-κB dimers are maintained in the cytoplasm through interaction with inhibitory proteins, the IκBs. In response to cell stimulation, a multisubunit protein kinase, IκB kinase (IKK), is rapidly activated and phosphorylates two critical serines in the N-terminal regulatory domain of the IκBs. IKK complex, which consists of two catalytic subunits, IKKα and IKKβ, and a regulatory subunit, IKKγ (NEMO) (34
), is the master regulator of NF-κB–mediated innate immune and inflammatory responses. The reported upregulation of the gene coding for IKKγ further illustrates that circulating PMNs are activated. Upregulated expression of the gene coding for IKK
was also observed. IKK
and TBK1 (TANK-binding kinase 1) synergize with TANK (TRAF family member-associated NF-κB activator) to promote their interaction with the IKKs, allowing IKK
and another kinase (TBK1) to modulate NF-κB activation (35
). In addition, IKK
is also a key element in the signaling pathway downstream of the Toll/interleukin-1 receptor domain-containing adapter protein (TRIF) and TBK1. This pathway leads to IFNβ production through activation of IFN regulatory factor (IRF)-3 that is directly phosphorylated by TBK1 and IKK
). Our data on the upregulated genes coding for IKKγ and IKK
in CF blood PMNs are in good agreement with recent observations by Srivastava et al. (37
) that show differentially overexpressed proteins of the TNF-α/NF-κB signaling pathway (IKKα, I-TRAF, and IKK
) in sera of CF patients. According to those authors, pooled sera from CF patients, characterized by a CF versus non-CF serum proteomic signature using an antibody microarray platform, are enriched in protein mediators of inflammation that may be selectively expressed in CF-affected tissues such as lung.
Among other genes that give evidence that CF blood PMNs are activated is the up-regulation of the gene coding for DP1. DP1 is a binding partner for E2F transcription factors. Target genes include those involved in DNA synthesis, cell cycle, and apoptosis (38
). Of course, the macroarray approach does not allow measurement of some other aspects of cell signaling, where activation is related to protein phosphorylation or cleavage.
We did not find any modulation of the genes coding for α-integrin (-2, -3, -4, -5, -6, -8, -9), β-integrin (-1, -2, -3, -5, -7, -8), or other adhesion molecules (ICAM-1, ICAM-2, CD11a). This is consistent with the literature, where a very limited modulation of these genes has been reported, if one ignores the downregulation of α-5 integrin by LPS (39
) and the upregulation of α-3 integrin, α-7 integrin, and ICAM-1 by receptor-mediated phagocytosis (21
). Among cell adhesion molecules, we found increased expression of thrombospondin-1 that was confirmed by qPCR. Except for caspase 1, we failed to find major modulation of apoptosis-related genes (bcl-2 and bcl-2 family members, caspase-2, -3, -4, -6, -7, -8, -9, -10). In the literature, the gene expression of caspase-1 was shown to be upregulated by phagocytosis, and the gene expression of caspase-3, -8, and -9 was shown to be downregulated by LPS or phagocytosis (21
We also compared blood PMNs to those prepared from sputum of CF patients. There was a limited difference between CF blood- and airway-derived PMNs. In spite of different CFTR mutations, we could find important homogeneity in terms of gene expression in the neutrophils from these two compartments. Among the 1050 studied genes, only six genes were significantly more reduced and two genes more upregulated among CF airway PMNs compared with CF blood PMNs, including amphiregulin. Although we failed to detect amphiregulin in PMN culture supernatants, we found significant amounts of amphiregulin in crude sputum from six of seven patients. Amphiregulin is an epidermal growth factor receptor ligand that activates epithelial cells and contributes to TNF-induced IL-8 release by human airway epithelial cells (41
). It also activates human lung fibroblasts and favors their proliferation (42
). Furthermore, amphiregulin enhances transmigration of human neutrophils through epithelial cell monolayers after alteration of E-cadherin–dependent tight junctions (43
). Our findings and all these properties make amphiregulin a new marker of CF-associated lung inflammation and an interesting putative target molecule. Thus, a greater understanding of molecular mechanisms by which CF airway PMNs upregulated amphiregulin production will identify novel therapeutic windows of opportunity. Altogether, this study clearly demonstrates that PMNs from cystic fibrosis patients display a profound modification of their gene expression profile associated with the disease. Most interestingly, the nature of the expressed mRNA of blood PMNs, in the absence of obvious interaction with inflammatory and/or infectious foci, was altered as compared to blood PMNs from healthy donors, and there was a very limited difference between the PMNs isolated from blood and airway in terms of gene expression.