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The master cytokine, IFN-γ possesses a wide spectrum of biological effects and is crucial for development of the highly activated macrophage phenotype characteristically found during inflammation. However, no data exists regarding the potential influence of cigarette smoke on the status of the expression of the cell surface receptor for IFN-γ (IFN-γR) on alveolar macrophages (AM) of smokers. Here in, we report reduction in the expression of the IFN-γR α-chain on AM of cigarette smokers, when compared with nonsmokers. Ensuing from the loss of receptor expression on the AM of smokers there was a decrease in IFN-γ-mediated cell signaling. This included a decrease in the phosphorylation of signal transducer and activator of transcription (STAT)-1 and induction of interferon regulatory factor (IRF)-1. Further, diminished activation/induction of transcription factors did not appear to result from induction of known members of the ‘suppressors of cytokine signaling (SOCS)’ family. Decreased IFN-γ signal transduction in AM from smokers may have an important implication regarding the use of therapeutic IFN-γ in the lungs ofpatients that develop respiratory disorders as a result of tobacco use.
Smoking-related morbidity encompasses many organ systems, but none more so than the lung. The acute and chronic impairment of immune function associated with smoking has been well described, yet there continues to be no cohesive model that explains precisely how smoking causes disease within the human lung.
Multiple defense mechanisms, directed against infection and malignancy, are operative throughout the human lung, with the alveolar macrophages (AM) playing a sentinel role in immune surveillance within the alveolar space. However, significant changes have been found to occur in AM isolated from smokers, when compared with those isolated from non-smokers (Harris et al., 1970; Barbers et al., 1987; Janoff et al., 1987; Brown et al., 1989). The numbers of AM have been reported to increase in smokers (Barbers et al., 1987; Janoff et al., 1987) and these macrophages show: a) changes in redox status (Rahman and MacNee, 1996); b) an accumulation of iron (McGowan and Henley, 1988; Thompson et al., 1991; Ghio et al., 1997) and ; c) an increased rate of glucose utilization (Harris et al., 1970). In addition, previous studies from our lab and by others on AM from smokers suggest attenuated secretion of IL-1 (Brown et al., 1989; Yamaguchi et al., 1989; O'Brien-Ladner et al., 1998), TNF-α (Yamaguchi et al., 1993; O'Brien-Ladner et al., 1998), and IL-6 (Soliman and Twigg, 1992), upon stimulation with LPS compared with AM isolated from non-smokers.
The responsiveness of macrophages to LPS, can be greatly enhanced by their prior or simultaneous exposure to interferon-γ (IFN-γ)(Gifford and Lohmann-Matthes, 1987; Scheibenbogen and Andreesen, 1991). This cytokine possesses a wide spectrum of biological effects and is crucial for development of the highly activated macrophage phenotype characteristically found during inflammation (Murray, 1988; Williams et al., 1999). Activation of the IFN-γ-signaling pathway within the macrophage begins by binding of this cytokine to the cell surface IFN-γ receptor (IFN-γR). The receptor is constitutively present on virtually every nucleated cell, but several studies have shown that the usual constitutive expression of this protein can be increased after addition of external stimuli, such as IL-1 (Krakauer and Oppenheim, 1993), TNF-α (Sanceau et al., 1992; Krakauer and Oppenheim, 1993) or IL-6 (Braun et al., 1998). The IFN-γR complex consists of two subunits: a ligand-binding protein, the alpha (α)-chain; and a species-specific accessory factor, or beta (β)-chain (Bach et al., 1997). All of the physiologic and pathogenic effects of IFN-γ are dependant upon its binding to α-chain of the receptor complex. The extracellular domain of IFN-γ α-chain binds the IFN-γ whereas the intracellular domain has the constitutive binding site for Janus kinase (JAK)1 and the IFN-γ-dependent tyrosine phosphorylation site for docking of transcription factor, signal transducer and activator of transcription-1 (STAT1)(Greenlund et al., 1994). IFN-γR β chain does not directly interact with ligand but is known to increase the association of IFN-γ with the α-chain and is required to confer responsiveness to IFN-γ (Skrenta et al., 2000) through the binding site for JAK2 tyrosine kinase on its intracellular domain (Sakatsume et al., 1995). Direct binding of IFN-γ to IFN-γR α chain on cell surface results in dimerization of α and β chains. This binding of IFN-γ to its receptor results in transphosphorylation and activation of JAK kinases followed by phosphorylation of STAT1 (Darnell, 1998) which is then known to regulate immune/inflammatory responses, cell proliferation, apoptosis and antiviral/anti-bacterial responses (Chesler and Reiss, 2002; Bruder et al., 2006; Gattoni et al., 2006, b; Gattoni et al., 2006a). The complete activation of Stat 1 by IFN-γ requires phosphorylation of both: a) Tyrosine 701 and, b) Serine 727 (Bach et al., 1997; Stark et al., 1998). While two reports from the same laboratory demonstrated that exposure of a mouse macrophage cell line to cigarette smoke constituents down regulated their responsiveness to added IFN-γ (Braun et al., 1998; Edwards et al., 1999) nothing is known regarding the in-vivo status of IFN-signaling in cigarette smokers. In the present study we evaluated the potential influence of cigarette smoke on the status of expression of IFN-γR and the related downstream signal transduction in AM isolated from smokers.
We show here that chronic exposure of the alveolar space to cigarette smoke may lead to a loss of IFN-γR α-chain expression on AM and a consequent down regulation of IFN-γ-mediated signaling. These findings indicate that the augmentative role played by IFN-γ during immune surveillance may be significantly curtailed in the lungs of smokers. This observation may have widespread significance with respect to infection, the development of cancers, and the treatment of such, in the lungs of smokers.
Eight healthy, smoking volunteers and four nonsmoking volunteers underwent bronchoalveolar lavage for the isolation of AM. Mean ages were 39.5±3.0 years for smokers and 40±4.7 for non-smokers. Smokers reported an average consumption of 39±13.8 pack-years [pack-years = (number of pack/day) x (number of years smoked)]. None of the subjects used for this study were taking medication, had a history of pulmonary disease, or had a recent upper respiratory tract infection. Subjects were given physical exams, each of which proved normal, and no subject showed evidence of lung disease, as judged by pulmonary function tests. All gave informed consent according to a protocol that had received prior approval by the institutional human subject’s committee.
AM were recovered from BAL as described previously (O'Brien-Ladner et al., 1998). Lavage fluid was filtered through four layers of sterile gauze and cells were pelleted by centrifugation (400 × g for 10 min). The cell pellet was washed three times with RPMI-1640 followed by resuspension in RPMI-1640 medium supplemented with 100 mg/ml streptomycin, 100 U/ml penicillin and 10% heat inactivated fetal bovine serum. Cell viability was determined by trypan blue exclusion, and cells were counted in a hemocytometer. A differential cell count and purity of AM was determined using a cytospin slide preparation stained with Diff-Quik. Approximately 2.5 × 106 AM were re-suspended into 10 ml of RPMI medium and then seeded onto 100 mm tissue culture dishes and allowed to adhere for 1h. AM isolated from either smokers or from non-smokers, had no difference in their ability to adhere to the surface. Dishes with 80–90% confluent adherent cells were then subsequently incubated for various time intervals either in the presence of medium alone or medium that contained 200 U/ml human IFN-γ (Upstate Biotechnology, Lake Placid, NY).
After 1 hour adherence to assure macrophage purification, AM isolated from both smokers and non-smokers, were used for soluble extract (SE) preparation. Briefly, AM were washed twice with ice cold phosphate buffered saline (PBS) followed by resuspension of cells in SE buffer, which consisted of PBS containing 1% Triton X-100, 1 mM PMSF, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 2 mM EDTA. The lysate was incubated on ice for 30 min and then centrifuged at 100,000 × g for 30 min at 4oC. The SE was then aliquoted and stored at −70oC until used for analysis.
Whole cell extracts (WCE) were prepared from cultured AM that were incubated in the presence or absence of IFN-γ. Extracts were prepared as described by Gao et al (Gao et al., 1998) and then aliquoted and stored at −70oC until used.
Protein extracts of mouse brain were used as a positive control for the binding of antibodies raised against ‘suppressor of cytokine signaling-1’ (SOCS-1) and SOCS-3, (Santa Cruz Biotech, Santa Cruz, CA).
Equal amount of protein (25μg) samples from SE or WCE, were run on a 12% SDS-polyacrylamide gel in reducing conditions followed by transfer onto a PVDF membrane as described by Gao et al (Gao et al., 1998). The blots were blocked with 5% non-fat dry milk in phosphate buffered saline. Western blots were probed with anti-human-IFN-γR α- and β-chain antibodies (Santa Cruz Biotech), anti-p84/p91 antibodies (anti-total ‘signal transducer and activator of transcription 1’ (Stat1) protein), anti-tyrosine-phosphorylated Stat1 (Zymed Laboratories, Inc., South San Francisco, CA), anti-serine-phosphorylated Stat1 antibodies (Zymed Laboratories, Inc.), anti-‘interferon regulatory factor-1’ (IRF-1), and anti-SOCS-1 and anti-SOCS-3 proteins (each from Santa Cruz Biotech). The secondary antibodies used were horseradish peroxidase-conjugated anti mouse or anti rabbit (1:5000, Pierce Chemical Co., Rockford, IL) and detection was performed using the enhanced chemiluminescence system (Pierce Chemical Co.). Where appropriate, membranes were stripped, reblocked, and sequentially reprobed with subsequent antibodies. Autoradiographic images were densitometrically scanned using a Molecular Dynamics (Sunnyvale, CA) Personal Densitometer SI and analyzed with the ImageQuaNT software package (Molecular Dynamics).
In some experiments, SE were first treated with peptide N-glycosidase F (PNGase F) prior to western blot analysis to remove N-linked oligosaccharides from the peptide backbone (Fischer et al., 1990). Breifly, cellular lysate was denatured by boiling for 10 min in extraction buffer supplemented with 0.5% SDS and 1% β-mercaptoethanol, cooled and mixed with 50 mM sodium phosphate buffer (pH 7.5) and 1%NP-40. Samples were then digested with PNGase F (New England Biolabs, Beverly, MA) at 4 × 105 U/ml for 1h at 37oC followed by western blot analysis.
Statistical analysis was performed using one-way or two-way analysis of variance with a post hoc Student t test. Results were judged statistically significant if p < 0.05 by analysis of variance.
In order to examine the effects of cigarette smoking on expression of the IFN-γR α-chain on the surfaces of AM, western blot analysis was performed using soluble extract prepared from cells isolated from 4 non-smokers and 8 smokers (Figure 1). The receptor protein displayed a diffuse banding pattern due to the heterogeneous addition of carbohydrate moieties to the polypeptide backbone. Nonetheless, the diffuse staining pattern of around 90kDa, evident in extracts prepared from AM of non-smokers, was considerably reduced or absent in extracts prepared from smokers’ AM (Figure 1A). Densitometric analysis of band intensities revealed a significant decrease (p value=0.005) in IFN-γR α-chain/ β-actin ratio obtained from smokers compared with non-smokers as shown in figure 1A, lower panel.
Further, to confirm the cell surface nature of the broad bands detected in Figure 1A, and to sharpen the resultant banding pattern, we treated protein extracts with PNGase F prior to western blot analysis. PNGase F is an endoglycosidase that removed the majority of N-linked polysaccharide from the IFN-γR α protein backbone. Figure 1B shows a representative blot of SE from AM of 3 non-smokers and 3 smokers treated with or without PNGase F. It can be readily seen in the figure that the broad banding pattern of IFN-γR α-chain observed without treatment were converted to sharper, major band of around 70kDa as reported earlier (Fischer et al., 1990). A comparison of PNGase F-treated and untreated extracts derived from smokers’ AM revealed very little difference in banding patterns or band intensities. Densitometric scanning analysis of PNGase F-treated samples revealed significant decrease (p value=0.05) in expression of N-deglycosylated IFN-γR α-chain in smokers compared to non-smokers (Figure 1B, lower panel), consistent with the changes observed in the analysis of native IFN-γR.
In order to determine the alterations in the β-chain of IFN-γR of AM isolated from smokers, western blots were probed with antibodies against IFN-γR β-chain. This protein is also very heterogeneous in size and was difficult to detect in western blots of extracts, not digested with PNGase F. However, as shown in Figure 1C, a protein band of appropriate size (38kDa), was observed in PNGase F-treated samples that was not found in untreated samples. β-chain protein was found to be present in both non-smokers and smokers. Interestingly, the blots revealed upregulation of β-chain in smokers compared with non-smokers but this increase was not statistically significant when analyzed by two-tail, t-test (p value=0.06) (Figure 1C, lower panel).
The primary pathway by which IFN-γ acts as an immunomodulator is through regulation of gene expression via the JAK (Janus kinase)/STAT tyrosine kinase-dependent cascade (Darnell, 1998). To elucidate whether the loss of IFN-γR α-chain expression observed in smokers’AM resulted in a concomitant loss of IFN-γ-induced responsiveness, we investigated the activation of transcription factor Stat1. Human AM isolated from non-smokers or smokers were stimulated with IFN-γ for 0.5 and 4h followed by cell lysis and analysis of transcription factors. As shown in one of the representative western blot analysis (Figure 2A), treatment of AM with IFN-γ resulted in dramatic phosphorylation of Stat1 at Tyr701 (Stat1-PY) at both time intervals tested in non-smokers, with more phosphorylation at 0.5h compared to 4h stimulation. However, very little Tyr-phosphorylation STAT-1 was observed in AM from smokers, on stimulation with IFN-γ. To determine whether the decreased phosphorylation of Stat1 was the result of diminished accumulation of Stat1 proteins, we stripped and reprobed the blot with anti-Stat1 (p84/p91) antibodies which can detect both isoforms of Stat1; Stat1α (91kDa) and the splice variant Stat1β (84kDa). Both Stat1α and Stat1β were detectable in untreated extracts prepared from the nonsmoker (Figure 2A). This level of Stat1α/β expression was elevated after treatment of AM with IFN-γ for either 0.5h or 4h consistent with a previous report from our laboratory (Gao et al., 1998). However, the overall levels of detectable steady-state levels of Stat1α /β proteins were observed to be significantly decreased in the smokers. As shown in Figure 2B, the level of total Stat-1 protein was lower in AM from smokers than in AM from non-smokers, when normalized with β-actin (p value <0.05 non-smokers vs. smokers at 0h). While treatment of smokers’ AM with IFN-γ increased the accumulation of Stat1α and Stat1β proteins, this level of expression was less than that observed in non-smokers (p<0.01 at 0.5h and p<0.05 at 4.0h, smokers vs. non-smokers). Although, the levels of Stat-1 in response to IFN-γ stimulation were significantly decreased in AM from smokers compared to AM from non-smokers, the fold increase in Stat1α /β on IFN-γ stimulation relative to before stimulation (at 0h) within the group was almost same. We found 2.6 & 2.1 fold increase at 0.5h and 4.0h, respectively in AM from non-smokers and 2.55 & 1.93 fold increase in AM from smokers relative to un-stimulated cells.
Densitometry analysis of Stat1-PY showed significant decrease in Stat1-PY when normalized with total STAT1 at both 0.5h (p value=0.03) and 4h (p value=0.05) post-stimulation in smokers’ AM compared to an increase in density of Stat1-PY band with IFN-γ treatment within non-smokers’ AM (Figure 2C). So apart from a decrease in the amount of available total Stat1α /β there was reduced IFN signaling that resulted in diminished ability of IFN-γ to induce Stat1 phosphorylation at Tyr 701, in smokers’ AM as compared to non-smokers.
Similarly, the phosphorylation of Stat1α at Ser 727 (Stat1-PS) when normalized to total STAT1 was also increased following treatment with IFN-γ within AM acquired from non-smokers. IFN-γ also induced Stat1α phosphorylation at Ser 727 in smokers’ AM, but the extent to which Stat1-PS/total STAT1 ratio increased was greatly reduced in smokers when compared to non-smokers at corresponding post-stimulation times (Figure 2A). Densitometric analysis of scanned images revealed a significant decrease in Stat1-PS/Stat1 ratio in IFN-γ stimulated AM isolated from smokers at both the time points tested (p value<0.001) when compared with non-smokers (Figure 2D). The Stat1β protein, a truncated form of Stat1α, lacks the necessary serine residue (Schindler et al. ,even in the un-stimulated AM from non-smokers whereas Stat1-PS was observed only by IFN-γ-stimulation in AM from smokers.
Activation of Jak/Stat pathway leads to increase in expression of many IFN-responsive target genes including transcription factors like interferon regulatory factor (IRF). In order to examine the ability of IFN-γ to induce new gene expression, we assayed for the induction of IRF-1 in AM isolated from non-smokers and smokers. Consistent with previous reports (Hayes and Zoon, 1993) we observed constitutive expression of IRF-1 in untreated AM isolated from non-smokers (Figure 2A). Because the appearance of newly induced IRF-1 requires both de novo gene transcription and protein synthesis, there is little additional IRF-1 protein detected after 0.5h of IFN-γ treatment, but considerable new protein after 4h of treatment. Nevertheless, the pattern of IRF-1 expression in AM from smokers differed markedly from that observed in AM from non-smokers. In smokers, IRF-1 was virtually undetectable in both untreated AM and in AM that were treated with IFN-γ for 30 minutes. Densitometric scans of untreated AM or treated with IFN-γ for 0.5h (Figure 2E) confirmed the absence of IRF-1 in smokers. However, after AM had been treated with IFN-γ for 4 hours, differences in IRF-1 levels of smokers and non-smokers were not statistically significant (p= 0.22), presumably because of the high variability in expression of IRF-1 in smokers.
A wide array of cytokines and growth factors induce the synthesis of multiple suppressors of cytokine signaling (SOCS) proteins, which in turn inhibit the JAK /STAT signaling pathway (Starr et al., 1997). To determine whether SOCS proteins might be involved in the suppression of IFN-γ-signaling, we assayed, by western blot analysis, for the presence or absence of the two most commonly induced SOCS family members, SOCS-1 and SOCS-3. When blots of the protein extracts from AM of smokers and non-smokers were probed with anti-SOCS-1 antibody, no protein was detectable in either the non-smokers or smokers group (data not shown), although the SOCS-1 band did appear in the tissue extract from mouse brain used as a positive control. However, when blots were probed with anti-SOCS-3, it was found that AM from both non-smokers (n=4) and smokers (n=8) expressed equivalent amounts of SOCS-3 protein (Figure 3A). Analysis of densitometric scans showed no statistical significant difference between the ratio of SOCS-3/β-actin band intensities in AM extracts from smokers and non-smokers (P = 0.23) (Figure 3B). We conclude from this study that there is no difference in SOCS-3 expression between the AM of smokers and non-smokers and, consequently, that reduced IFN-γ-mediated signaling in the smokers’ AM is not due to the result of over-expression of the SOCS family of suppressors.
In this study we have shown a significant down-regulation in expression of the α-chain of the IFN-γR, on surfaces of AM acquired from smokers, when compared with non-smokers whereas no significant change was demonstrated in the expression of IFN-γR β-chain within the groups. Furthermore, signal transduction and gene induction, in response to added IFN-γ, is reduced in the AM of smokers, in comparison with non-smokers. These results are important in that they demonstrate a potential defect in the IFN-γ-directed immune defense of the smoker’s lung.
The differential regulation of the α- and β-chain of IFN-γR, in response to tobacco smoke, is likely due to the distinct organization of their promoters. The α-chain promoter contains several candidate cis-acting DNA elements that might be responsive to external stimuli. These include Sp1, AP-1, AP-2, CREB (Merlin et al., 1997). The β-chain gene, on the other hand, appears to have a more limited potential for regulation because its promoter primarily contains candidate Sp1 and AP-2 sequences, and one candidate Pu-box (Rhee et al., 1996). Thus, differences in the promoters of the receptor subunits’ genes may account, at least in some part, for the discrepancy in expression of α-chain of the IFN-γR protein as compared to the IFN-γR β-chain protein. Furthermore in CD4+T cells (Sakatsume and Finbloom, 1996), down regulation of β-chain on differentiation into the Th1 type, has been reported to be dependent on IFN-γ binding whereas down regulation of α-chain on T cell activation, has been reported to be IFN-γ independent (Skrenta et al., 2000). Similar differential regulation of α- and β-chain may exist in macrophages and the increase in β-chain of the receptor, although not significant, in AM smokers, therefore, may be the result of a feedback response of the cells to the lack of IFN-γ-signaling. However, previous studies by Celada et al. (Celada and Schreiber, 1987) demonstrated no change in the expression of IFN-γ receptor on macrophages on binding with IFN-γ. They suggested that constant presence of intracellular receptors and constitutive recycling of receptors to the cell surface, maintains same level of cell-surface receptors on macrophages in the absence or presence of IFN-γ. Furthermore, no report exist on the alteration or regulation of IFN-γ receptor in response to cigarette smoking, however, an alternative mechanism similar to that found by recent study by HuangFu et al. (HuangFu et al., 2008) on mouse fibroblasts, reporting down-regulation of IFNAR1 chain of IFNα/ β receptor on exposure to cigarette smoke through phosphorylation-dependent ubiquitination, may be possible. Due to the concern regarding risk to human subjects with bronchoscopy we limited this study after finding statistically significant changes in the protein expression of the IFN-γ receptor. Certainly, future experiments are needed to examine the molecular mechanisms involved in the down-modulation of IFN-γR-α-chain in response to cigarette smoke exposure.
IFN-γ activates the Jak/Stat pathway through, α- and β-subunits of IFN-γ receptor (Darnell, 1998; Lackmann et al., 1998). In almost all cells IFN-γ binds to the α- and β-subunits of its receptor leading to activation of Jak1 and Jak2 kinases followed by phosphorylation of Stat1 (Muller et al., 1993; Shuai et al., 1993; Darnell, 1998). The IFN-γ-induced accumulation of Stat1 a /β in AM from either smokers or non-smokers, and the decreased availability of these proteins in AM isolated from smokers in this study, is consistent with our previous observations in mouse macrophages (Gao et al., 1998). In mouse macrophages we demonstrated induction of Stat1 α /β protein synthesis by IFN-γ as well as by IFN-β and LPS. Furthermore, peritoneal macrophages prepared from mice in which the IFN-γ gene had been targeted for deletion showed considerably lower steady-state levels of Stat1α and β (Gao et al., 1998) similar to the decreased availability of Stat1α/β in AM isolated from smokers in the present study. Thus, we hypothesize that fully functional IFN-γ related receptor/ligand interactions may be necessary for the maintenance of homeostatic levels of Stat1α/β. In addition, the same levels of IFN-γ-induced increase in the expression of Stat1α/β relative to the un-stimulated levels within smokers or non-smokers may indicate that both groups were equally responsive to IFN-γ but differ in the number of functional receptors as confirmed by the decrease in α-chain of the IFN-γR in smokers. However, following treatment of smokers’ AM with IFN-γ, phosphorylation of Stat1 at Tyr 701 and at Ser 727 when normalized with total Stat1α/β was significantly lower in smokers than in non-smokers and this may be due to changes in IFN-induced activity. Absence of IRF-1 expression in un-stimulated AM from smokers and decreased induction of IRF-1 in response to IFN-γ further confirmed the defect in IFN-γ-signaling in smokers
Although this study was limited by the availability of human AM acquired by bronchoscopy, the results reported may provide insight into the variable effect of IFN-γ on both lung cancer cell lines and on AM isolated from lung cancer patients, as well as into the general failure of IFN-γ-based immunotherapy modalities for the treatment of lung cancer. IFN-γ had a variable effect on proliferation of several small cell lung cancer (SCLC) cell lines and induced the synthesis of MHC class I molecules by more than twofold in only 4 of eight of these lines (Ball et al., 1986). IFN-γ also failed to inhibit colony formation among 3-of-4 non-small cell lung cancer (NSCLC) cell lines tested by Hong et al (Hong et al., 1987). Clonogenic assays similarly showed a variable antiproliferative effect of IFN-γ on SCLC and NSCLC cell lines (Jabbar and Twentyman, 1990), with SCLC lines apparently being more susceptible to the effects of IFN-γ than were the NSCLC lines (Jabbar and Twentyman, 1990; Prior et al., 1998). Furthermore, AM isolated from 3-of-7 lung cancer patients, in the study of Halme et al. (Halme et al., 1995), failed to show an IFN-γ-induced increase in oxidative burst, over pre-IFN- treatment samples.
Clinical trials, utilizing an intravenous (Schiller et al., 1989) or subcutaneous (Jett et al., 1994; van Zandwijk et al., 1997) route of IFN-γ administration, have failed to demonstrate improved survival or time-to-progression among either SCLC (Jett et al., 1994; van Zandwijk et al., 1997) or NSCLC (Schiller et al., 1989) patients. In addition, IFN-γ failed to improve upon the efficacy of either cisplatin or etoposide in combinatory trials (Schiller et al., 1989). Interestingly, intrapleural instillation of IFN-γ showed potential as a therapeutic agent in that NSCLC cells disappeared from the pleural effusion after treatment (Schiller et al., 1989; Yanagawa et al., 1997). In none of the studies cited above was the question of expression of the receptor for IFN-γ, either on AM, lung cancer cells, or lung cancer cell lines, addressed. Because the vast majority of lung cancers arise as the result of cigarette smoking, determining the presence or absence of the IFN-γR is of paramount importance.
Although the IFN-γR is constitutively present on virtually every nucleated cell type, it is clear that expression of the receptor may be modulated negatively. Thus, the potential exists for the restoration of IFN-γR levels in the lungs of smokers and this could also lead to the development of more efficacious treatment therapies, based on the instillation of the IFN-γ into the lung. In any case, full understanding of the molecular mechanisms involved in the regulation of IFN-γR expression is essential.
The authors dedicate this paper to the fond memory of Joseph C. Cates. This work was supported, in part, by an NIH IDeA grant (P20 RR11825) (AOL), Wilkinson Endowment for Cancer Research, American Lung Association - Kansas (AOL. and WJM.), Joseph C. Cates Family Foundation (AOL), Parker B. Francis Research Fellowship (ND) and by a scholarship (BD/9586/96) from Fundação para Ciência e Tecnologia, Portugal (AC.).
Conflict of Interest statement
The authors declare that there are no conflicts of interest.
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