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Published ahead of print on August 4, 2005, doi:10.1165/rcmb.2005-0204OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 33, pp. 463-469, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2005-0204OC

Requirement for Tumor Necrosis Factor-Receptor 2 in Alveolar Chemokine Expression Depends upon the Form of the Ligand

Jun Liu, Min Q. Zhao, Lumei Xu, C. V. Ramana, Wim Declercq, Peter Vandenabeele and Richard I. Enelow

Department of Medicine, Yale University School of Medicine, New Haven, Connecticut; Departments of Surgery and Pathology, University of Virginia School of Medicine, Charlottesville, Virginia; and Department of Molecular Biomedical Research, VIB, Ghent University, Ghent, Belgium

Correspondence and requests for reprints should be addressed to Richard I. Enelow, VA Connecticut Healthcare System/111A, 950 Campbell Ave., West Haven, CT 06516. E-mail: richard.enelow{at}yale.edu


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Respiratory virus infection evokes a potent T-cell response that may result in a considerable insult to the structural and functional integrity of the gas exchange units of the lung. Alveolar antigen recognition by CD8+ T lymphocytes results in significant injury that is critically dependent upon tumor necrosis factor (TNF)-{alpha} expressed by the CD8+ T cells and is largely dependent upon TNF-receptor 1 expression on the alveolar epithelial target cells. TNF-receptor 2 (TNF-R2)-deficient mice were used to demonstrate that CD8+ T-cell–mediated lung injury associated with clearance of experimental influenza requires TNF-R2 for full expression of immunopathology. In vitro analysis indicates that alveolar cell expression of TNF-R2 is critical in the induction of epithelial monocyte chemoattractant protein (MCP)-1 expression specifically in response to soluble TNF-{alpha}, suggesting an important role for this receptor in bystander lung injury. However, TNF-R2 was dispensable for induction of alveolar MCP-1 expression in response to transmembrane TNF-{alpha} expressed by antigen-specific CD8+ T cells, and the effects of the two receptors seem to be additive. Because TNF-R2 may be rapidly shed as part of feedback inhibition of bystander inflammation, this suggests a mechanism by which immunopathology in respiratory virus infection may be regulated and by which T-cell receptor–dependent TNF-{alpha} activity might bypass such negative regulation for contact-dependent antiviral activities.

Key Words: chemokines • inflammation • lung • T cells


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
A potent CD8+ T-cell recall response can result in complete protection from an otherwise lethal respiratory virus infection. This has been effectively modeled in murine influenza pneumonia with adoptive transfer of CD8+ effector cells (1, 2). CD8+ T lymphocytes have several important antiviral effector activities at their disposal, including the perforin/granzyme system, interferon (IFN)-{gamma}, tumor necrosis factor (TNF)-{beta}, and TNF-{alpha} (3). CD8+ T cells express transmembrane TNF (tmTNF) in an antigen-dependent fashion (4), and this is processed to soluble TNF (sTNF), although the degree to which this occurs and the factors that regulate it are not clear. Notwithstanding its importance in respiratory virus clearance, CD8+ T-cell antigen recognition in the alveolar space exacts a significant cost to the structural and functional integrity of the lung. The lung injury associated with respiratory virus infection seems to result primarily from T-cell responses and activities, and in their absence injury is considerably abrogated (5). We have shown that lung injury induced by CD8+ T-cell recognition of alveolar antigen is critically dependent upon TNF-{alpha} expressed by the CD8+ T cells and TNF receptor-1 (TNF-R1) expressed on the target cells (6). We have also shown that the evolution of the immunopathology is largely independent of continued T-cell effector activity (or even T-cell presence) in the lung parenchyma after initial antigen recognition (7). The inflammatory process thereafter is largely mediated by the alveolar epithelial target cells, which survive the encounter with the T cells without evidence of direct cytotoxicity (7, 8). Epithelial chemokine expression leads to the massive recruitment of host inflammatory cells, which is associated with the observed morbidity and mortality. Neutralization of one important chemokine, monocyte chemoattractant protein (MCP)-1 (or CCL2), significantly abrogates the inflammation and injury associated with CD8+ T-cell recognition of alveolar antigen (8).

TNF-{alpha} is a pleiotropic cytokine critical for inflammation, maintenance of lymphoid organ structure, and host defense against various pathogens (9). Dysregulated TNF production can be deleterious and has been associated with a variety of inflammatory diseases, such as rheumatoid arthritis, septic shock, and inflammatory bowel disease (10–12). For many years, TNF was viewed solely as a soluble proinflammatory cytokine produced by innate immunocytes, such as macrophages and neutrophils (13). However, TNF is expressed in soluble and transmembrane forms by various cell types with distinct homing and migratory properties, such as T lymphocytes (14, 15). TNF is first produced as a bioactive 26-kD transmembrane molecule (tmTNF), which is cleaved by the metalloproteinase-disintegrin TACE (TNF converting enzyme/ADAM17) (16) to generate a soluble 17 kD molecule (sTNF). The membrane and soluble species interact with two TNF receptors, TNF-R1 and TNF receptor-2 (TNF-R2) (17). Generally, sTNF has been regarded as the primary ligand for TNF-R1, whereas evidence suggests that TNF-R2 may be preferentially activated by tmTNF (18, 19).

Our data indicate that, although TNF-R1 is necessary and sufficient to mediate induction of alveolar epithelial chemokine expression in response to tmTNF-{alpha}, TNF-R2 is required for induction of alveolar MCP-1 expression triggered specifically by sTNF-{alpha}. Furthermore, deficiency of TNF-R2 results in a considerable abrogation of lung injury associated with CD8+ T cell–mediated clearance of influenza infection. These findings suggest an important contribution of TNF proteolytic processing by CD8+ T cells in the immunopathology associated with respiratory virus infection.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Mice
TNF-R2–deficient, TNF-deficient, and C57BL/6 mice were obtained from Jackson Laboratories (Bar Harbor, ME). For infection and transfer experiments, animals were used at 12–16 wk of age (18–24 g). On Day 5 after stimulation, CD8+ T-cell clones were separated from stimulators by density gradient centrifugation and injected via the tail vein into appropriate recipient animals as described (8, 20). Simultaneous infection was performed by subjecting mice to light methoxyflurane anesthesia and instilling 50 µl of diluted A/Japan/57 allantoic fluid. Mice were weighed daily, and, at appropriate times after adoptive transfer, animals were killed, their trachea exposed, and their airways perfused with 10% formalin. Sections were cut and stained with hematoxylin-eosin. All animal procedures were approved by the institutional animal care and use committee.

T-Lymphocyte Lines and Clones
CD8+ T-cell clones (wild type [WT] and mutant) specific for Kd- and Db-restricted epitopes of A/Japan/57 HA and NP, respectively, were generated as previously described (8, 20). CD4+ TNF-deficient T-cell lines allospecific for H-2q were developed by harvesting splenocytes from TNF-deficient mice (H-2b; Jackson Laboratories) followed by stimulation in vitro with irradiated splenocytes harvested from FVB/n mice (Jackson Laboratories).

Alveolar Epithelial Cells
For analysis of chemokine expression, 2 x 106 mouse lung epithelial (MLE)-Kd cells (MLE-15 cells transfected with the class I MHC molecule, Kd) (20, 21) were plated in 24-well plates and allowed to adhere overnight. On the following day, sTNF-{alpha} (R&D Systems, Minneapolis, MN) or CD8+ T cells were added to the culture (with HA210–219 peptide where appropriate). In some cases, IFN-{gamma} (R&D Systems) was added. After incubation for a specified period, the supernatants were removed and analyzed by enzyme-linked immunosorbent assay (ELISA) for MCP-1 (BD Biosciences, Franklin Lakes, NJ). Where appropriate, the matrix metalloproteinase inhibitor (MPI) KB8301 (BD Biosciences) was added at a final concentration of 5 µM for the duration of the incubation period. Trypsin/ethylenediaminetetraacetic acid was added to the remaining (adherent) cells, and these were removed and centrifuged. The cells were collected for total RNA extraction with Trizol (Gibco, Grand Island, NY). RNAse protection assays were performed using 32P-labeled probes (BD Biosciences) in accordance with the manufacturer's instructions. Blocking antibodies included anti–TNF-R1 and anti–TNF-R2 (Stratagene, La Jolla, CA) (22) and anti–TNF-{alpha} (R&D Systems). For primary alveolar type II cell isolation, animals were anesthetized and exsanguinated, and their lungs were perfused via the pulmonary artery. Isolation of primary alveolar type II cells was performed as described (23). Briefly, Dispase (Collaborative Research, Inc., Bedford, MA) with 0.1 mM actinomycin D (Sigma, St. Louis, MO) was instilled via the trachea, followed by 1% low-melt agarose. After disruption of the lungs, cell suspensions were negatively selected with anti-CD45 and anti-CD32 (BD Biosciences), resulting in a type II cell purity of 90–95% (23). Cells were stained for flow cytometry using biotinylated goat anti–TNF-R1 and goat anti–TNF-R2 (R&D Systems) and avidin-fluorescein-isothiocyanate (Vector Laboratories, Burlingame, CA).

Constructs and Transfection
Murine CD40 cDNA (ATCC, Manassas, VA) and murine p55 (TNF-R1) cDNA (Belgian Coordinated Collections of Microorganisms, Ghent, Belgium) were used as templates for generation of the CD40/p55 chimeric receptor. CD40 signal peptide, extracellular and transmembrane domains, and the cytoplasmic domain of TNF-R1 were amplified using oligonucleotide primer sets 5'-AAGCTTGGGCATGGTGTCTTTGCCTCG-3' (sense)/5'-GGTACCCCGACCGGTCGTAGAGA (antisense), and 5'-ACCGGTCGCCCCGGTGGAGGCCCGAAGT-3' (sense)/5'-TCTAGAGCTTATCGCGGGAGGCGGGT-3' (antisense), respectively. Polymerase chain reaction–amplified products were cloned into pCR-XL-TOPO vector (Invitrogen, Carlsbad, CA) and then subcloned into the expression vector pcDNA3.1a (Invitrogen). Other constructs used included pSV25S-hTNF-R75 and pSV25S-hTNF-R75 m5 (human TNF-R2, WT and with a deletion in the terminal 37 amino acids, respectively) (24) MLE-Kd cells were transfected using Lipofectamine 2,000 (Invitrogen), and, 24 h later, the cells were harvested, counted, and plated for assay.

Statistical Analysis
Significant differences were determined by analysis of variance followed by Tukey's test or the Mann-Whitney U test.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Lung Injury Accompanying T-Cell Clearance of Respiratory Virus Is Abrogated in the Absence of TNF-R2
TNF-{alpha} expressed by the CD8+ T cell is required for lung injury occuring upon alveolar antigen recognition and in the absence of replicating virus. Because deficiency of TNF-R1 in recipients abrogates this process (6), we explored the role of TNF-R2 in immunopathology triggered by T-cell recognition of alveolar antigen. TNF-R2–deficient and WT mice were infected with A/Japan/57 influenza followed by adoptive transfer of activated HA-specific CD8+ T cells. An intranasal dose of 10LD50 A/Japan/57 influenza resulted in progressive weight loss and death in mice that received no T-cell transfer (Figure 1). WT mice (C57BL/6) that received 107 CD8+ T cells (specific for the Db-restricted epitope of the nucleoprotein) by tail vein in conjunction with intranasal influenza exhibited a significant weight loss, which returned to baseline after peaking 2 d after infection. In contrast, TNF-R2–deficient influenza-infected T-cell recipients showed no significant weight loss. This was performed with several doses of influenza, and no differences in virus titers were observed at multiple time points (not shown). WT and mutant animals that received a lower dose of virus (1LD50) followed by T-cell transfer did not become clinically ill but exhibited different histopathologic abnormalities after being killed 8 d postinfection and transfer. Figure 2A demonstrates the diffuse alveolar inflammatory infiltration in the C57BL/6 lungs at Day 8 (a time when no detectable virus was evident). In contrast, the TNF-R2–deficient lungs (Figure 2B) were nearly free of alveolar infiltration, with the exception of scattered small nodular foci of inflammatory cells. No other abnormalities were present. These findings suggest that TNF-R2 plays an important role in the lung injury associated with T-cell clearance of respiratory virus.



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  		Figure 1. Weight loss associated with T-cell–mediated virus clearance is abrogated in TNF-R2–deficient mice. Mutant (squares) and WT (C57BL/6) (circles) mice were infected intranasally with 10LD50 A/Japan/57 influenza followed by adoptive transfer of 107 NP-specific CD8+ T cells (compared with infected animals that received no T cells; triangles). n = 6 per group; *P < 0.05. Data are representative of two experiments with similar results.

 


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  		Figure 2. TNF-R2 deficiency abrogates immunopathology associated with T-cell–mediated virus clearance. Representative sections of lungs harvested on Day 8 from WT (A) versus TNF-R2–deficient (B) recipients of 1LD50 A/Japan/57 influenza followed by 107 NP-specific CD8+ T cells.

 
Alveolar Epithelial-Derived Cells Express Functional TNF-R2
We have previously shown that transcriptional activation of type II cells is an important contributor to the inflammatory infiltration that occurs after T-cell recognition of alveolar antigen (8), and because the expression patterns of the TNF receptors on each of the cellular components of the respiratory tract has not been clearly delineated, we addressed the question of whether type II cells express TNF-R2. We initially analyzed the MLE-Kd cell line, which structurally and functionally reproduces the characteristics of alveolar type II cells with exceptional fidelity (21, 23). TNF-R2 (p75) and TNF-R1 (p55) message was detectable in MLE-Kd cells by RNAse protection assay, although baseline expression of TNF-R2 message was faint (Figure 3A). Expression of both receptors was enhanced by incubation with TNF-{alpha}, although no further induction was evident after incubation with TNF-{alpha} and IFN-{gamma}, despite the presence of an IFN-{gamma}–responsive element in the p75 promoter region (25). To confirm protein expression, MLE-Kd cells were stained for flow cytometry with antireceptor antibodies, and significant surface expression of TNF-R1 and TNF-R2 was evident at baseline (Figure 3B) without stimulation with TNF or IFN-{gamma}. TNF-R2 expression was somewhat lower than TNF-R1, although not as low as might be predicted on the basis of the RNA levels. To ensure that this was not unique to the MLE-Kd cell line, primary alveolar type II cells were isolated and stained in a similar fashion for both TNF receptors, demonstrating that the level of expression of both TNF receptors was comparable to that observed on the cell line (Figure 3C).



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  		Figure 3. TNF-R1 and TNF-R2 mRNA are expressed by alveolar epithelial cells. (A) RNAse protection assay performed on RNA extracted from MLE-Kd cells at baseline and after 4 h of stimulation with 100 ng/ml TNF-{alpha} or 100 ng/ml TNF-{alpha} and 100 ng/ml IFN-{gamma} (performed twice with similar results). (B) Flow cytometric analysis of TNF-R1 (dark line) and TNF-R2 (light line) on MLE-Kd cells. (C) Primary type II cells (2). Dotted line represents isotype control. Data are representative of two experiments with similar results.

 
It has been proposed that TNF-R2 may be preferentially triggered by tmTNF-{alpha} (19) and that CD8+ T lymphocytes express transmembrane and soluble (cleaved) TNF-{alpha} (14, 26). To assess the contribution of tmTNF versus sTNF expressed by T cells on the antigen-specific induction of epithelial MCP-1 expression, a hydroxamic acid MPI was used to block cleavage of the TNF transmembrane species by TNF-{alpha} converting enzyme (16, 27). We found that inhibition of cleavage resulted in no sTNF in the supernatants (Figure 4A), and a significant (though incomplete) reduction in epithelial MCP-1 expression (Figure 4B). We have previously shown that CD8+ T cells do not express MCP-1 (8). The degree of inhibition was maintained at the higher effector/target (E:T) ratio, indicating that qualitatively distinct signaling occurred despite a considerable difference in cytotoxicity (which increases directly with E:T ratio) (28). In all cases, the induction of epithelial MCP-1 was inhibited by antibody to TNF and was dependent on the presence of cognate peptide. This suggests that sTNF cleaved from the surface of CD8+ T cells during antigen recognition also induced expression of MCP-1 by the target epithelial cells and that the contributions of the soluble and transmembrane ligands were additive. We further confirmed expression of tmTNF on T cells that were subjected to MPI treatment by flow cytometric analysis on CD8+ T cells in the presence and absence of the MPI after 4 h stimulation with PMA/ionomycin. Although tmTNF expression on TACE-inhibited T cells was somewhat lower than on untreated cells, significant expression was apparent (Figure 4C). Because there was no detectable sTNF in the MPI-treated culture and because the induction of MCP-1 was inhibited by anti-TNF, we concluded that the epithelial MCP-1 expression in the MPI-treated cultures was induced by tmTNF. Based upon the results of this experiment alone, we cannot exclude the possibility that sTNF had no effect on MCP-1 expression and that the decrease of MCP-1 expression in the presence of the MPI was solely due to the quantitative diminution of tmTNF expression. We therefore explored the specific role of sTNF in the induction of epithelial MCP-1 expression using several different strategies.



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  		Figure 4. Transmembrane and sTNF each trigger alveolar cell MCP-1 expression upon antigen recognition by T cells in an additive fashion. (A) ELISA for TNF-{alpha} from supernatants of MLE-Kd cells co-cultured with CD8+ T cells (with or without 10–8 M HA210 peptide) in the presence (gray bars) or absence (white bars) of MPI KB8301 (MPI) at E:T ratios of 5:1 and 1:1. (B) Induction of epithelial MCP-1 production in the identical cultures in the presence of neutralizing antibody to TNF-{alpha} (black bars) or an isotype control (gray bars). No induction of MCP-1 was observed in the absence of peptide (not shown). (C) Flow cytometric analysis of surface expression of TNF-{alpha} in the presence (light line) or absence (dark line) of KB8301 (MPI) after stimulation with PMA/ionomycin (isotype control is the filled tracing). Data are representative of two experiments with similar results.

 
TNF-R2 Is Required for Induction of Alveolar Chemokine Expression by sTNF-{alpha}
Because TNF-R1 is required for alveolar cell MCP-1 induction by antigen-specific T cells (6, 8), we explored in further detail the contribution of TNF-R2 to alveolar cell activation by sTNF-{alpha}. We performed a dose-response analysis of MCP-1 production using anti–TNF-R2 upon treatment of MLE-Kd cells with sTNF-{alpha} at a saturating concentration (8, 23). TNF-R2 receptor blockade almost entirely abrogated induction of MCP-1 expression by sTNF-{alpha} (Figure 5A). To confirm this requirement, we took advantage of the ability of human TNF-{alpha} to ligate murine TNF-R1 but not murine TNF-R2 (17). Human TNF-{alpha} did not induce expression of appreciable MCP-1 by mock-transfected MLK-Kd cells, but this was reconstituted by transient transfection with full-length human TNF-R2 (Figure 5B). Transfection with a human p75 with a deletion in the C-terminal amino acids in the cytoplasmic tail, which disrupts the TRAF2-binding domain (24), did not reconstitute induction of MCP-1 expression by human TNF despite equivalent transfection efficiencies (not shown). This suggests that the mechanism of p75 involvement in this process depends, at least in part, upon its ability to transduce a signal upon ligation. Finally, to confirm that this phenotype is reflective of the behavior of bone fide alveolar epithelial cells, primary type II cells were isolated from WT, p55-deficient and p75-deficient mice. They were treated with murine TNF for 6 h, and the supernatants were analyzed by ELISA. Absence of either of the TNF receptors ablated the induction of MCP-1 in primary cells by sTNF (Figure 6).



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  		Figure 5. Induction of MCP-1 production by alveolar epithelial cells triggered by sTNF-{alpha} requires TNF-R2 expression. (A) ELISA for MCP-1 production on supernatants from MLE-Kd cells treated with 100 ng/ml TNF-{alpha} with or without antagonistic antibody to TNF-R2 (**P < 0.05; *P < 0.01). Data are representative of four experiments with similar results. (B) ELISA for MCP-1 production on supernatants from MLE-Kd cells treated with 100 ng/ml human (or murine) TNF-{alpha} and transfected with human TNF-R2 (R75) or mutant human TNF-R2, with a deletion in the TRAF-2 binding domain (R75M5) (*P < 0.05; **P < 0.01). Data are representative of two experiments with similar results.

 


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  		Figure 6. ELISA for MCP-1 production on supernatants from primary alveolar type II cells isolated from WT, TNF-R1–deficient, or TNF-R2–deficient mice after 6 h of treatment with 100 ng/ml of murine TNF-{alpha} (gray bars) versus untreated (black bars) (*P < 0.01 versus untreated). Data are representative of two experiments with similar results.

 
TNF-R2 Is Dispensable for the Induction of Alveolar Chemokine Expression by tmTNF-{alpha}
Because TNF-R2 is required for the induction of alveolar MCP-1 expression by sTNF, we examined the requirement for TNF-R2 in alveolar cell activation by tmTNF-{alpha}. To circumvent the difficulties associated with antibody blockade, we adopted an alternative strategy using TNF-deficient T cells and taking advantage of the antigen-specific T-cell expression of CD40L (CD154), a TNF-homolog that is regulated in a similar fashion to TNF-{alpha} on T cells (except that the murine transmembrane form is not processed to a soluble species). To test the effect of exclusive oligomerization of TNF-R1 by a transmembrane ligand in the absence of any involvement of TNF-R2, we generated a chimeric CD40 receptor in which the extracellular region was derived from murine CD40 (a member of the TNF receptor family) and the transmembrane and intracellular domains derived from the murine TNF-R1 receptor. Ligation of this receptor was achieved through antigen-specific CD40L expression by TNF-deficient CD4+ T cells allospecific for I-Aq, the native haplotype of the MLE-Kd cell (endogenous CD40 was not detected on MLE-Kd cells; not shown). TNF-deficient CD4+ T cells co-cultured with transfected MLE-Kd cells triggered a significant induction of MCP-1, compared with co-culture of mock transfected MLE-Kd cells, indicating that oligomerization of the TNF-R1 intracellular domains by a transmembrane ligand was sufficient to induce expression of MCP-1 without a contribution from TNF-R2 (Figure 7). Co-culture of mock transfected target cells with WT CD8+ T cells (in the presence of HA210–219 peptide) resulted in epithelial MCP-1 production as well but to a lesser degree than did ligation of the chimeric receptor by CD40L expressed on TNF-deficient T cells. The relatively exuberant expression triggered by ligation of the chimeric receptor by CD40L expressed by the CD4+ (TNF-deficient) T cells compared with that triggered by ligation of native TNF-R1 (and presumably TNF-R2) by TNF-{alpha} expressed by CD8+ T cells may reflect a greater level of cytotoxicity of MLE-Kd cells in response to CD8+ T-cell recognition (28). These data suggest that higher-order clustering of TNF-R1 by a transmembrane ligand is sufficient to induce MCP-1 expression by an epithelial cell upon recognition by a T cell.



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  		Figure 7. TNF-R1 is sufficient for induction by a transmembrane ligand of alveolar epithelial-derived cell expression of MCP-1. ELISA for MCP-1 production in co-cultures. MLE-Kd cells were transiently transfected with a CD40:TNF-R1 chimeric receptor (or empty vector) and co-cultured with allospecific TNF-deficient CD4+ T cells at an E:T of 1:1 for 6 h. As a positive control, HA-specific CD8+ T cells (plus 10–8 M HA210 peptide) were added to mock-transfected MLE-Kd cells at an E:T ratio of 1:1. Data are representative of two experiments with similar results.

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
TNF-{alpha} expressed by the CD8+ T cell is required for lung injury occuring upon alveolar antigen recognition, and deficiency of TNF-R1 in the recipients abrogates T-cell–mediated immunopathology (6). In this study, we demonstrate that TNF-R2 plays a role in this immunopathology and in the epithelial transcriptional responses to T-cell recognition, which are critical amplifiers of injury. The nature of the contribution of TNF-R2 to induction of epithelial chemokine expression in response to TNF expressed by the T cell depends upon the form of the ligand. TNF-{beta} exhibits certain overlapping properties with TNF-{alpha} and is expressed by CD8+ T cells, but we have not found a role for this molecule in immunopathology or viral clearance in our system. The biology of the TNF receptors has been extensively studied, and the function of TNF-R1 in induction of apoptosis and in induction of inflammatory gene transcription has been clearly defined (29). In contrast, the function of TNF-R2 is much less clear and remains controversial. TNF-R2 expression and function has been largely characterized on cells of hematopoietic origin, in which TNF-R2 is expressed at a much higher level than TNF-R1, and the specific function may vary with the regulated expression of the individual receptors and with that of intracellular adaptor molecules, such as RIP (22, 30). Its role on other cell types is less clear. Although numerous studies have demonstrated distinct respiratory phenotypes in the absence of TNF-R1 or TNF-R2, the roles of the two receptors on alveolar type II cells have not been examined.

In several cell types examined, there seems to be cooperativity between the two receptors in transducing signals triggered by sTNF-{alpha}, although the mechanisms involved are still a matter of debate. Proposed mechanisms include "ligand passing," in which the higher affinity of TNF-R2 for the ligand allows for amplification of signaling through TNF-R1 and the clustering of cytoplasmic domains and the adaptor molecules associated therewith (31, 32). However, Grell and colleagues (19) suggested that the transmembrane form of TNF-{alpha} is the primary ligand for TNF-R2, although this study used target cells expressing much higher levels of TNF-R2 than TNF-R1. Furthermore, these investigators used CHO cells transfected with TNF to demonstrate that ligation of TNF-R1 and TNF-R2 was required to induce the biologic effect (i.e., cytotoxicity) on these target cells. We would argue that the biology of a T-cell recognition event is likely to be fundamentally different, with highly regulated, focused, and directional expression of effector function (i.e., TNF expression) upon antigen recognition, and this may be an important factor obviating the need for TNF-R2 ligation to induce a biologic effect (in our studies, MCP-1 expression). Our findings may reflect unique properties of type II cells as targets and may not be generalizable to readouts other than MCP-1 expression. Our data indicate that TNF-R2 is not required for alveolar epithelial MCP-1 expression triggered by tmTNF, whereas both receptors are required for induction of MCP-1 expression by sTNF-{alpha}. This may reflect a requirement for "ligand-passing" of sTNF, the need for which is obviated by tmTNF-{alpha} and the higher-order oligomerization of TNF-R1 which may occur as a result of oligomerization of tmTNF on the T-cell surface. It has been demonstrated that other members of the TNF receptor family require higher-order multimerization for signaling than is possible with a soluble ligand (33–35). However, our data also suggest that the role of TNF-R2 in MCP-1 expression in response to sTNF involves a direct signal transduction event mediated by the cytoplasmic tail of TNF-R2 (the TRAF-2 binding domain), though these are not mutually exclusive possibilities. Depending upon the cell type studied, TNF-R2 signaling seems to mediate activation of NF-{kappa}B and c-Jun NH2-terminal kinase, and these functions may require TRAF-2, although this is not entirely clear (31, 36, 37). It does seem, at least in some cell types, that the TRAF-2 binding domain of TNF-R2 is required for cooperation between the two TNF receptors in the induction of apoptosis (24). Although other cell types have been shown to demonstrate enhancement of TNF-R1–mediated effects by TNF-R2 (and vice-versa), alveolar epithelial cells may be unique in their absolute requirement for both TNF receptors to induce a response triggered by sTNF-{alpha}. The reasons for this are unclear but may reflect the need to tightly regulate inflammatory responses in the alveolus, where excessive inflammation may disrupt critical gas exchange function.

A corollary to these observations is that sTNF may be required for the full expression of the inflammatory phenotype in the lung, as has been observed in other model systems (38). We have previously shown TNF message in the lungs of HA-transgenic CD8+ T-cell recipients for as long as 4 d after transfer, a point at which the transferred T cells are undetectable in the parenchyma and the infiltrates are rich in host macrophages, an important source of sTNF (7). This suggests that the timing of sTNF production may have an important impact on the inflammatory phenotype. Our data indicate that the presence of sTNF in the lung in the first 24 h after antigen recognition results in a brief burst of MIP-2 expression and neutrophil influx, neither of which is observed in the absence of the early sTNF production by the T cells (L. Xu and R. I. Enelow, unpublished observations). This may be a vulnerable period in which the alveolar epithelium is particularly susceptible to the effects of sTNF because TNF-R2 seems to be required for this response and because TNF-R2 is rapidly shed in an inflammatory milieu (39).

Although TNF-{alpha} may be an important participant in host defense against influenza (40) and other infections (15), under these experimental conditions there was no diminution of antiviral activity in the absence of TNF-R2. It is possible that this may be more evident in infections with other strains of virus (41) or in a natural infection in which the T-cell responses (primary or memory) may lag behind viral replication by several days. Nevertheless, TNF-R2 may be worth exploring as a therapeutic target in severe respiratory virus infection because it may provide a strategy to ablate the inflammatory effects of TNF in the lung without impeding the potential antiviral effects of tmTNF mediated through TNF-R1.

Received in original form May 31, 2005

Received in final form July 29, 2005


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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