Introduction

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Tumor necrosis factor (TNF)- was discovered as a factor that induced the death of tumor cells and caused tumor- related cachexia. The cytokine is now known to be highly pleiotropic and pathogenic in systemic inflammatory response syndromes such as septic shock, and in chronic inflammatory diseases such as idiopathic pulmonary fibrosis. The cellular effects of TNF- are mediated by two receptors, TNF receptor (TNFR)I (55 kD) and TNFRII (75 kD), members of the nerve growth factor/TNFR superfamily of proteins characterized by their conserved, extracellular, cysteine-rich repeats (1). The intracellular domains of these receptors have low homology and lack kinase domains. Transduction of TNFR type-dependent responses occurs by recruitment of secondary proteins. Several proteins that interact directly or cooperatively with the TNFR have been identified by immunoprecipitation and yeast two-hybrid systems (2). TNFR-associated factors 1 and 2 (TRAF1 and TRAF2) were detected by their association as a heterodimer with TNFRII through homologous C-terminal "TRAF" domains. TRAF1 and TRAF2 also associate with TNFRI indirectly through the TNFR-associated death domain (TRADD) protein (5). Unlike TRAF2 and TRADD, which are ubiquitously expressed, TRAF1 was found to be expressed only in the spleen, testis, and lung, suggesting a unique function in those tissues (6).

Recently, TRAF1 was shown to prevent TNF--induced DNA fragmentation and caspase 8 activation in conjunction with the mammalian inhibitor of apoptosis proteins (IAPs) homologs B (cIAP1) and C (cIAP2) (7). IAPs were first described in baculovirus as proteins induced by TNF- that prevented cytokine-induced apoptosis of baculovirus-infected cells (4). Several homologous mammalian IAP proteins were subsequently identified that contain N-terminal baculovirus IAP repeat motifs and a C-terminal RING finger. TRAF and IAP proteins function interdependently. For example, the association of the IAPs with TNFRII required the heterodimer of TRAF1 and TRAF2 (8). TRAF1 was also required for cIAP1 or cIAP2 protein to inhibit TNF--induced cell death (7).

TNF- is highly regulated and active in the lung. The cytokine was elevated in bronchoalveolar lavage of adults with pneumonia, acute respiratory distress syndrome, or chronic pulmonary fibrosis, and in infants with neonatal respiratory distress syndrome or chronic bronchopulmonary dysplasia (BPD). Specifically in pulmonary epithelial cells, TNF- stimulated nuclear binding activity of the transcription factor nuclear factor (NF)-B (9), inhibited the synthesis of surfactant specific phospholipids and proteins (9), and induced the antioxidant manganese superoxide dismutase and the intracellular adhesion molecule-1 (12, 13). TNF also induced proliferation of lung mesothelial cells and fibroblasts while causing apoptosis of, for example, polymorphonuclear cells (14).

Signal transduction pathways by which TNF- induces phenotypic changes in lung cells are incompletely understood. Regulation of TNFR proximal proteins, TRAF, and IAP gene expression has not been previously studied in lung cells. The current study demonstrates dose-dependent regulation of TRAF1 and cIAP2 by TNF- in H441 and A549 pulmonary epithelial cell lines as well as induction of TRAF1 protein in whole murine lung exposed to intratracheal recombinant murine TNF- (rmTNF-). Relevance to human lung disease is demonstrated by immunohistochemical localization of TRAF1 in lung tissue of human neonates dying with inflammatory lung disease.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Cell Culture

The NCI H441-4 and U937 cell lines (American Type Culture Collection, Manassas, VA), were cultured in a 37°C, 5% CO₂, humidified incubator in RPMI 1640 medium with 10% fetal bovine serum (FBS) (Invitrogen, San Diego, CA) and 1% antibiotic/antimycotic solution (abx) (GIBCO BRL, Gaithersburg, MD). The A549 cells were similarly cultured in Dulbecco's modified Eagle's medium with 10% FBS and 1% abx. Recombinant human TNF- (rhTNF-) and rmTNF- (R&D Systems, Minneapolis, MN) were diluted in sterile phosphate-buffered saline (PBS), pH 7.4, to stock solutions of 50 µg/ml and 5 µg/50 µl, respectively. Annexin V and propidium iodide staining was performed with commercial reagents and protocol (PharMingen, Inc., San Diego, CA) and analyzed on a Becton Dickinson FACSCalibur flow cytometer with CELLQuest software (PharMingen).

Intratracheal TNF-

rmTNF- or an equal volume of sterile saline diluent (50 µl) was administered into the trachea of pathogen-free, 10-wk-old, C57BL/6J male mice (Jackson Laboratories, Bar Harbor, ME) by blunt-needle intubation under light isoflurane anesthesia. The University Committee on Animal Research approved all methods. Previous instillation studies with vital dye demonstrated diffuse delivery to both lungs in greater than 95% of mice treated (data not shown). The animals were allowed to recover for 18 h with food and water ad lib before death. After appropriate euthanasia (intraperitoneal pentobarbital, 150 µg/kg) and exsanguination by transection of the abdominal aorta, the lungs were exposed, the trachea was cannulated, the right bronchus ligated, and the left lung inflated in situ with 2% glutaraldehyde in 0.1 M cacodylic acid fixative at 10 cm H₂O pressure. The left lung, with the trachea ligated, was stored in the same fixative for 18 h before being washed in cacodylic acid for at least 24 h, dehydrated in ethanol, and embedded in paraffin.

Neonatal Lung Tissue

Lung tissue was harvested, after parental consent to autopsy, within 6 h of death from human neonates whose death was due to congenital, nonrespiratory disease (n = 3); bronchopulmonary dysplasia (n = 1); necrotizing enterocolitis (n = 1); or necrotizing, aspiration pneumonia (n = 1). The infants were 40 ± 2 wk gestation at the time of death. The right middle or lower lung lobe was inflation-fixed in 10% buffered formalin at 25 cm H₂O pressure for 24 h before being dehydrated and paraffin-embedded.

Immunohistochemistry

Murine and human lung sections (4 µm) were deparaffinized and hydrated before blocking of endogenous peroxidase with hydrogen peroxide. Sections were then digested for 30 min with bovine testicular hyaluronidase (1 mg/ml in 0.1 M sodium acetate, pH 5.5, 0.85% NaCl) and blocked with 1.5% normal serum-1.0% bovine serum albumin (BSA), followed by a 30-min incubation with polyclonal rabbit anti-TRAF1 antibody (1 µg/ml in PBS/1% BSA; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Nonspecific binding was removed by washing extensively in PBS before incubation with biotinylated secondary antirabbit antibody and avidin-enzyme complex. Sections were reacted with 3,3'-diaminobenzidine (Sigma Chemical Co., St. Louis, MO) and counterstained with methyl green.

Western Analysis

Protein electrophoresis and transblotting were performed by modification of a Santa Cruz Biotechnology protocol. After the indicated treatment, cells were lysed and isolated in 1× PBS, 1% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1.0 mM sodium orthovanadate, 100 µg/ml phenylmethylsulfonyl fluoride, and 30 µl/ml aprotinin, #A6279 (Sigma). Total protein was analyzed by bicinchoninic acid assay using commercial reagents and protocol (Pierce, Inc., Rockford, IL) (17). Proteins (80 µg) in Laemmli buffer were separated on polyacrylamide-SDS gels and transferred to polyvinylidene difluoride membrane. Membranes were blocked overnight in PBS with 5% nonfat dry milk (NFDM) at 4°C before a 1-h incubation in a rabbit TRAF1-specific polyclonal immunoglobulin G antibody (1 µg/ml in PBS, 5% NFDM, and 0.05% Tween 20). The antibody cross-reacts with human and murine TRAF1 (SC S-19; Santa Cruz Biotechnology). The membranes were washed in PBS with 0.05% Tween-20 before incubation in peroxidase-conjugated antirabbit secondary antibody (1:2,000; Santa Cruz Biotechnology). After extensive washing, immunodetection was performed using enhanced chemiluminescence protocols modified according to the reagent distributor (ECLplus; Amersham, Arlington Heights, IL).

Ribonuclease Protection Assay

Cells were lysed in situ in 4 M guanidinium isothiocyanate (Kodak Chemical Co., Rochester, NY), 0.5% N-lauryl sarcosine, and 25 mM sodium citrate (Sigma), and stored at 80°C. Total cell RNA was extracted by Phase Lock Gel II columns (5Prime-3Prime, Boulder, CO) as previously described (9). The ribonuclease protection assays (RPAs) were performed with commercial reagents and protocols (Riboquant; PharMingen). Radiolabeled, single-strand RNA probes for X chromosome-linked IAP, MIHA (XIAP); TRAF1; TRAF2; cysteine-rich domain associated with RING and TRAF protein, TRAF4; neuronal apoptosis protein (NAIP); MIHC (cIAP2); MIHB (cIAP1); CD40 receptor- associated factor 1, TRAF3; clusterin, apolipoprotein J, complement-associated protein SP-40 (TRMP2); TNFRI; TNFRII; ribosome-associated protein L32 (rpL32); and glyceraldehyde phosphate dehydrogenase were synthesized at room temperature using [³²P]uridine triphosphate (3,000 Ci/mmol, EasyTides; Dupont, New England Nuclear, Boston, MA) and T7 polymerase. RNA samples (5 µg by absorbance at 260 nm), including human RNA and yeast transfer RNA (2 µg) as positive and negative controls, were dried, then resuspended in 8 µl of hybridization buffer and radiolabeled probe (2 µl, 3 × 10⁵ cpm/µl). The samples were overlaid with mineral oil, denatured at 90°C, and incubated overnight at 56°C. After incubation, single-stranded RNA was digested in an RNase A/T1 cocktail, followed by proteinase K digestion. The remaining radiolabeled RNA fragments, protected from digestion by endogenous messenger RNAs (mRNAs), were resolved on a 6% acrylamide/urea gel (GIBCO BRL), using radiolabeled probe (1,000 to 2,000 cpm) as size markers. The gels were dried and analyzed by phosphorimaging (Molecular Dynamics, Sunnyvale, CA).

Statistical Analysis

Quantitative RPA data were analyzed by single-factor analysis of variance and Fischer's Protected Least Significant Difference statistic, using Statview 4.0 statistical analysis software (Abacus Concepts, Berkeley, CA).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

No gross cytotoxicity was detected by light microscopy and trypan blue staining of H441 or A549 cells after 24 h of the doses of TNF- used in this study. Annexin V binding to exteriorized phosphatidylserine, an early event in apoptosis, was assayed, in conjunction with propidium iodide vital stain, by flow cytometry as a measure of apoptosis. A maximum of 25% of U937 and only 1 to 2% of A549 cells became apoptotic after 4 h exposure to 25 ng/ml rhTNF-. Cell counts were performed by hemocytometer in conjunction with trypan blue exclusion assay. The number of viable H441 cells in plates treated with TNF- for 24 h was 140% ± 15% (mean ± standard error of the mean [SEM], n = 6) of the number on control plates, whereas U937s were approximately 80% of control.

Expression of mRNA for TNFRs in Whole Lung and in the Tumor Cell Lines

To determine whether TNF- receptor expression in the pulmonary adenocarcinoma cell lines reflects expression in lung cells in vivo, the TNFR mRNA levels in whole lung and in each of the tumor lines were determined. H441 and A549 cells expressed only TNFRI mRNA, whereas U937 cells expressed both TNFRI and TNFRII (Figure 1). The ratio of TNFRI to TNFRII mRNA did not change with exposure to TNF-. TNFRII mRNA was detectable but markedly less abundant than TNFRI mRNA in whole adult murine lung RNA (data not shown) and human lung RNA obtained at autopsy within 6 h of death at 40 ± 2 wk gestation (Figure 1).

View larger version (55K): [in this window] [in a new window]   Figure 1.   Human lung and cell type-specific TNFR mRNA expression. Total U937, H441, and A549 RNA (5 µg) were analyzed for TNFRI and TNFRII mRNA by RPA 24 h after addition of control RPMI medium (C) or rhTNF- (25 ng/ml). RNA was also isolated from a human term newborn lung within 6 h of death due to congenital heart disease and analyzed for TNFR mRNA (5 µg, Lung).

Cell-Specific Regulation of TRAF1 and cIAP2 mRNA

To determine which proximal TNF-/TNFR signaling proteins are expressed in pulmonary epithelial cells, H441 and A549 cells were analyzed by RPA for TNFR-associated factors TRAF1 through TRAF4 and for inhibition of apoptosis proteins XIAP, NAIP, cIAP2, and cIAP1, and for TRMP2. TRAF3 and TRAF4 do not interact with TNFR1 or TNFR2 but bind to the cytosolic domains of other members of the TNFR superfamily (18, 19). In both A549 and H441 cells, only TRAF1 and cIAP2 mRNA levels were altered by TNF- (25 ng/ml) (Figure 2a). NAIP mRNA was not detected in the epithelial cell lines. XIAP, TRAF2, TRAF3, TRAF4, and cIAP1 mRNA were constitutively expressed and not significantly induced by TNF-. TRMP2 mRNA was not detected in H441 cells but was constitutively expressed in A549 cells. The extent of TRAF1 and cIAP2 mRNA induction was similar in A549 cells compared with H441 cells (Figures 2b and 2c). Induction of TRAF1 and cIAP2 mRNA by TNF- in H441 cells was dose-dependent, reaching statistical significance at 5.0 ng/ml (Figure 3).

View larger version (49K): [in this window] [in a new window]   Figure 2.   Cell type-specific induction of TRAF1 and cIAP2 mRNA by TNF-. Total RNA was isolated from human H441 and A549, epithelial, and U937 monocytic cells 24 h after treatment with control RPMI medium (A lanes or open bars) or rhTNF- (25 ng/ml; B lanes or filled bars) was analyzed by RPA for TRAF and IAP mRNAs. (a) Representative RPA. Arrows mark RNAs induced by TNF-. The RPAs were analyzed by phosphorimaging and cumulative data, normalized to rpL32 mRNA levels as internal control, and presented as (b) TRAF1 mRNA and (c) cIAP2 mRNA levels relative to medium-treated, cell-specific controls. Bars indicate means ± standard deviation, n = 7-9 experiments. *P < 0.001 versus cell type control.

View larger version (46K): [in this window] [in a new window]   Figure 3.   Dose-dependent induction of TRAF1 and cIAP2 mRNA by TNF-. Total RNA was isolated from H441 cells 24 h after culture in increasing concentrations of rhTNF-. The indicated mRNA were detected by RPA and imaged and quantified by phosphorimaging. (a) Representative RPA. Arrows mark RNAs induced by TNF-. The cumulative data, normalized to rpL32 mRNA levels as internal control, are presented as (b) TRAF1 mRNA and (c) cIAP2 mRNA levels relative to medium-treated, cell-specific controls. *P < 0.01 compared with control (mean ± SEM, n = 3).

Monocytic U937 cells constitutively expressed XIAP, TRAF2, TRAF3, TRMP2, and TRAF4, and very low levels of TRAF1, NAIP, and cIAP1 mRNA (Figure 2a). In contrast to the pulmonary adenocarcinoma cell lines, TRAF1 and cIAP2 mRNA were not significantly increased in U937 cells after TNF- exposure (Figures 2b and 2c).

Induction of TRAF1 Protein

Western blot analysis and immunohistochemistry were used to determine whether TNF--dependent changes in TRAF1 and cIAP2 mRNA were reflected in increased protein levels of the factors. The commercially available cIAP2 antibody failed to detect a protein band in the tumor cell lysates and was unsatisfactory in immunohistochemistry. TRAF2 protein was constitutively expressed in H441 cells and demonstrated little or no change in expression after treatment with TNF-, consistent with the RPA results (data not shown). In contrast, TRAF1 protein was barely detectable in control, medium-treated H441 cells but was markedly induced within 6 h of TNF- administration (Figure 4a). The abundance of TRAF1 protein continued to increase over 24 h of exposure. The protein band induced by TNF- was competed by TRAF1 control peptide, confirming antibody specificity (Figure 4b). As in H441 cells, TRAF1 protein was markedly induced in A549 cells treated with TNF- (Figure 5). U937 cells contained an immunoreactive protein of the appropriate size for TRAF1 but it was constitutively expressed and not altered by TNF- (Figure 5).

View larger version (40K): [in this window] [in a new window]   Figure 4.   Time course of TRAF1 protein induction by TNF-. (a) Western analysis of TRAF1 protein in 80 µg of total cell lysate prepared from H441 cells treated with RPMI medium () or rhTNF- (+; 25 ng/ml) for the indicated times. (b) Anti-TRAF1 antibody was incubated (A) without or (B) with TRAF1 control peptide (1 µg/ml) 1 h before use in Western analysis of protein of cells treated for 24 h with RPMI medium () or TNF- (25 ng/ ml). Arrows mark expected location of TRAF1 protein.

View larger version (41K): [in this window] [in a new window]   Figure 5.   Cell type-specific induction of TRAF1 protein. Representative Western analyses for TRAF1 protein levels in 80 µg protein lysate harvested from U937 monocytic cells in comparison with A549 and H441 cells after 24 h culture in RPMI medium (C lanes) or rhTNF- (25 ng/ml). Arrow marks the expected location of TRAF1 protein.

Induction of TRAF1 Protein In Vivo

To determine whether TNF- regulation of TRAF1 protein in the adenocarcinoma cell lines in vitro is reflective of regulation of the protein in vivo, TRAF1 protein was analyzed by immunohistochemistry in whole mouse lung 18 h after intratracheal instillation of rmTNF-. A small amount of TRAF1 antibody-related staining was noted in lung cells of control mice treated intratracheally with sterile saline (Figure 6a), primarily in cells in the alveolar space consistent with alveolar macrophages. In lung tissue of mice exposed to TNF-, individual cells of the alveolar wall were TRAF1-positive, many of which occupied corner sites or jutted into the alveolar space, morphology consistent with type II alveolar epithelial cells (Figure 6b). In addition, occasional cells in the alveolar space consistent with alveolar macrophages were immunostained.

View larger version (104K): [in this window] [in a new window]   Figure 6.   Immunohistochemistry for TRAF1 in murine and human lung. Immunohistochemistry was performed on 4-µm sections of lung tissue of C57BL/6J mice 18 h after intratracheal treatment with (a) control saline or (b) rmTNF- (5 µg/mouse), or of human neonates dying of (c and e) nonrespiratory disease, (d) BPD, or ( f ) necrotizing aspiration pneumonia. Brown diaminobenzidine tetrahydrochloride stain indicates TRAF1 protein.

To determine whether TRAF1 was also expressed in human lung, immunohistochemistry with TRAF1 antibody was performed on lung samples of neonates dying of the chronic, fibrotic disease of prematurely born infants BPD or acute pneumonia, diseases associated with TNF- release. Lung tissue of neonates who died of nonrespiratory, noninflammatory disease demonstrated minimal staining in the parenchymal tissue, airways, and vessels (Figures 6c and 6e). Lungs of infants dying with BPD (Figure 6d) or necrotizing pneumonia (Figure 6f) had intense staining for TRAF1 protein in scattered cells of the alveolar septum, the intra-alveolar space, and the walls of vessels consistent with bronchiolar arteries. By location and morphology these TRAF1-positive cells are consistent with alveolar epithelial type II cells, alveolar macrophages, and smooth-muscle cells of vessel walls, respectively.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The cytokine TNF- is produced by alveolar macrophages and type II epithelial cells of the lung in response to many stimuli, including endotoxin, hyperoxia, environmental pollutants, and cytotoxic drugs such as bleomycin. It is known that TNF- production in the lung results in abnormal surfactant synthesis, inflammation, and fibrosis, yet the receptor proximal signal transduction pathways by which pulmonary cells respond to the cytokine are not understood. TNF- alters cellular phenotype by binding to either TNFRI (p55) or TNFRII (p75). The ligand-bound TNFRs, in turn, recruit intracellular proteins into a signaling complex with several diverse potential functions, including activation of transcription factors NF-B and activator protein (AP)-1, initiation of apoptosis by activation of caspase 8, or induction of proteins that prevent apoptosis. The TNF- effect that predominates is likely to be dependent on the availability of secondary factors for recruitment to the TNFR. Regulation of TNFR-associated factors and IAP has previously not been studied in lung cells. The current study demonstrates that TNFRI mRNA is the predominant TNFR mRNA present in the lung as a whole and in the pulmonary adenocarcinoma cell lines A549 and H441. By RPA, TRAFs 2, 3, and 4, and IAPs XIAP and cIAP1 mRNA are consistently expressed in the epithelial cell lines. Only TRAF1 and cIAP2 mRNA are induced in a time- and dose-dependent manner by exposure of the cells to TNF-.

Human neonatal lung and adult mouse lung (data not shown) contain primarily TNFRI and little TNFRII mRNA, suggesting that, like the A549 and H441 cell lines, the majority of the cells of the lung express predominantly TNFRI. This finding is consistent with a previous study by Shimomoto and colleagues that described TNFRII only in alveolar macrophages whereas TNFRI was detected on alveolar macrophages, bronchiolar epithelium, and some small airways (20). Because, like the majority of the lung, the H441 and A549 cells express the TNFRI but not the TNFRII receptors, they are appropriate representative cells in which to study pulmonary TNF- signal transduction. The absence of TNFRII mRNA in the cell lines suggests that TRAF1 expression is regulated by TNFRI. Previous studies suggest that traf1 gene transcription is regulated by the transcription factor NF-B (7, 21). We previously demonstrated NF-B activation by TNF- in H441 cells (9). NF-B was also induced by TNF- treatment in U937 cells but TRAF1 was not, suggesting that induction of TRAF1 requires factors in addition to NF-B or that U937 cells contain an inhibitor of TRAF1 induction (22). U937 cells express both TNFRI and TNFRII mRNA. Further experiments are needed to determine whether the failure of TNF- to induce TRAF1 and cIAP2 mRNA in the U937 cells is due to the presence of both receptors.

The current study was performed in adenocarcinoma cell lines due to the difficulty in isolating and maintaining the phenotype of primary lung epithelial cells. To demonstrate the relevance of the in vitro studies to gene regulation in whole lung, TNF--induced expression of TRAF1 in whole murine lung tissue is also demonstrated. The histology of the cells that were TRAF1-positive by immunohistochemistry support the hypothesis that TRAF1 gene expression is inducible by TNF-, at least in distal pulmonary epithelial cells. TRAF1 protein was also detected in human lung tissue. Although the current studies involved only a small number of human samples, TRAF1 appeared abundant in the neonates with the chronic fibrotic disease BPD, and with acute pneumonia in comparison with neonates without inflammatory lung disease. TRAF1 was also abundant in the lung of an infant dying of necrotizing enterocolitis, a disease of the immature bowel associated with increased TNF- levels and a systemic inflammatory response (data not shown). The role of TRAF1 and cIAP2 in the pathogenesis or resolution of these and other TNF--related inflammatory lung diseases remains to be determined.

The physiologic relevance of TRAF1 induction and association with TNFRI has not yet been demonstrated. Transfected TRAF1 coimmunoprecipitated with TNFRI in the presence of TRADD, suggesting recruitment of TRAF1 to the TNFRI complex (23). TRAF1 also associates with other members of the TNFR superfamily, including CD30 and 4-1BB (24), receptors believed to be T cell- specific and unlikely to be present in pulmonary epithelial cells. Similar as-yet-unidentified proteins may exist in epithelial cells and interact with TRAF1. TRAF1 induction may be part of a signaling cascade initiated by TNF- specifically in lung cells. We previously demonstrated that TNF- induces NF-B and AP-1 DNA binding activity, enhances MnSOD gene expression, and inhibits surfactant protein (SP)-A and SP-B expression in H441 cells (9). Further studies will determine the role of TRAF1 in these epithelial cell responses to TNF-.

Recent in vitro studies suggest that TRAF1, TRAF2, cIAP1, and cIAP2 proteins act in concert to prevent TNF-- induced apoptosis (7). Additional overexpression studies suggest that cIAP2, in conjunction with TRAF1, inhibits apoptosis induced by TNF- but not that induced by overexpression of interleukin-1 -converting enzyme precursor protein or Fas-associated death domain (FADD) protein (8, 25). Protection of T lymphocytes from antigen-induced apoptosis has also been demonstrated in a transgenic mouse model of TRAF1 overexpression (26). In the current study, the pattern of gene expression in the epithelial cell lines differed from that seen in the monocytic U937 cells. Previously, by Northern analysis, expression of XIAP, cIAP1, and cIAP2 mRNA was not detected in the monocytic cell line (27). In the current study using a potentially more sensitive RPA, U937 cells were found to constitutively express XIAP and TRAF2-4 mRNAs, barely detectable TRAF1 and cIAP1, and essentially no detectable cIAP2. No increase in XIAP, cIAP1, or cIAP2 mRNA was detected in U937s treated with TNF-, consistent with the previous study (27). In contrast, in A549 and H441 cells, XIAP, TRAF2 through TRAF4, and cIAP1 mRNA was constitutively expressed, yet TRAF1 and cIAP2 mRNA levels were significantly induced by TNF-, suggesting cell-specific regulation of these mRNAs. These cell types also differ in their sensitivity to TNF-. A portion of U937 cells undergo apoptosis after brief exposure to TNF- (27). It is conceivable that cell type-specific responses to the TNF/TNFR superfamily of ligand/receptor proteins are determined by the relative expression levels of the factors available for recruitment to the receptors. The U937 cells undergo apoptosis after exposure to increasing doses of TNF-, whereas H441 cells demonstrated a trend toward increase in cell number in response to TNF- (28). We speculate that the coordinated induction of TRAF1 and cIAP2 by TNF- protects pulmonary epithelial cells, unlike the U937 cells, from apoptosis and may contribute to the net increase in H441 cell number in response to TNF-.

Future studies will address the hypothesis that induction of TRAF1 and cIAP2 proteins protects certain cells of the lung from apoptosis, contributes to cell type-specific responses of pulmonary cells in TNF--mediated lung diseases, and potentially, adds to the insensitivity of some lung tumors to TNF--induced apoptosis.