TNFα Production, Receptors, and Biological Activity

TNFα is primarily produced by macrophages in response to tissue injury or infection. ^{Reference Aggarwal, Gupta and Kim11,Reference Zelová and Hošek12} Synthesized as a transmembrane homotrimer consisting of three 26 KDa subunits (mTNFα), it is cleaved by TNFα-converting enzyme (TACE) to soluble TNFα (sTNFα), a homotrimer consisting of three 17 KDa subunits. ^{Reference Horiuchi, Mitoma and Harashima13} The activity of both mTNFα and sTNFα is mediated by binding to cell surface receptors identified as type 1 (TNFR1) and type 2 (TNFR2) (Figure 1). TNFR1 is expressed on the surface of most cell types, whereas TNFR2 is largely restricted to immune cells and endothelial cells. ^{Reference Zelová and Hošek12} Both forms of TNFα bind to TNFR1 and TNFR2. However, TNFR2 binds to TNFα with lower affinity and may easily dissociate from the ligand. ^{Reference Zelová and Hošek12,Reference Tracey, Klareskog and Sasso14} Thus, it appears that the biological activity of TNFα mainly involves TNFR1 signaling. ^{Reference Jang, Lee and Shin10,Reference Aggarwal15,Reference Grell, Douni and Wajant16} Reports also suggest that signaling pathways activated by these 2 receptors overlap or they transduce signaling cooperatively as genetic deletion of either receptor blocks signaling initiated by TNFα. ^{Reference Aggarwal15,Reference Mukhopadhyay, Suttles, Stout and Aggarwal17} Ligand binding to TNFR1 initiates signaling, resulting in the activation of mitogen activated protein (MAP) kinases and transcription factors, including AP-1 and nuclear factor-kappa B (NF-κB), which regulate genes involved in inflammation, cell proliferation, and differentiation. ^{Reference Jang, Lee and Shin10,Reference Aggarwal15} TNFR1 also recruits TNFR1-associated death domain (TRADD) protein, which promotes cell death. TNFR2 recruits TNFR-associated factor (TRAF)-1 and TRAF-2 proteins, resulting in the activation of MAP kinases, NF-κB, and protein kinase B. ^{Reference Jang, Lee and Shin10,Reference Aggarwal15} Functionally, TNFR1 activation is associated with the induction of cytotoxic and proinflammatory responses of TNFα, whereas TNFR2 mediates homeostatic bioactivities, including tissue regeneration, cell proliferation, and cell survival. ^{Reference Aggarwal15,Reference Probert18}

Figure 1. Tumor necrosis factor (TNF)α signaling. Binding of soluble or membrane bound TNFα to TNFR1 and TNFR2 initiates signaling events associated with apoptosis or activation of transcription factors, NFκB and AP-1. TNFα binding to TNFR1 and/or TNFR2 can also activate protein kinase B/Akt, which leads to prolonged NF-κB activation. Together, these responses contribute to inflammation, leukocyte trafficking, cell death, cell proliferation, and tissue remodeling.

AP-1, activator protein-1; IKK, IκB kinase; MAPKs, mitogen-associated protein kinases; mTNF, membrane-bound TNFα; NFκB, nuclear factor kappa B; sTNF, soluble TNFα; TACE, TNFα-converting enzyme; TNFR1, TNFα receptor 1; TNFR2, TNFα receptor 2; TRADD, TNFR associated death domain; TRAF1, TNFR associated factor 1; TRAF2, TNFR associated factor 2.

TNFα is a master regulator of inflammation generated early after injury or infection in response to bacterially derived lipopolysaccharide, as well as interleukin (IL)-1, interferon-γ, granulocyte macrophage colony stimulating factor, platelet derived growth factor, and TNFα itself. ^{Reference Malaviya, Laskin and Laskin19–Reference Sabio and Davis21} The biological actions of TNFα are varied and summarized in Table 1. TNFα promotes inflammation by upregulating adhesion molecules important in leukocyte trafficking to inflammatory sites, including intracellular leukocyte adhesion molecule, endothelial leukocyte adhesion molecule-1, and vascular cell adhesion molecule-1, and by stimulating the release of macrophage and neutrophil chemokines, such as CXCL8 (IL-8), CCL2 (MCP)-1, interferon-inducible protein 10 (IP-10) (CXCL10), as well as bioactive lipids (eg, eicosanoids and platelet activating factor), which promote vasodilatation, leukocyte adhesion, and chemotaxis. ^{Reference Zelová and Hošek12,Reference Semenzato22–Reference Kelly, Hwang and Kubes25} TNFα also stimulates phagocytic cells to release proinflammatory cytokines (eg, IL-1, IL-6, IL-12, IL-15, IL-23, and TNFα) and reactive oxygen and nitrogen species. In the lung, these cytotoxic/proinflammatory mediators cause alveolar epithelial cell injury, denudation of the basement membrane, hyalin membrane formation, impaired surfactant activity, and altered pulmonary functioning. ^{Reference Malaviya, Laskin and Laskin19,Reference Shimabukuro, Sawa and Gropper26,Reference Laskin, Malaviya and Laskin27}

Table 1. Biological activities of tumor necrosis factor (TNF)α in mustard or radiation-induced lung injury

Arg, arginase; CCL, C-C chemokine ligand; CCR, C-C chemokine receptor; COX, cyclooxygenase; CTGF, connective tissue growth factor; CXCL, C-X-C chemokine ligand; CXCR, C-X-C chemokine receptor; Fas L, Fas ligand; Fas R, Fas receptor; FGF, fibroblast growth factor; γH2A.X, histone variant H2A.X; HMGB, high mobility group box; HO, heme oxygenase; H₂O₂, hydrogen peroxide; IFN, interferon; IL, interleukin; iNOS, inducible nitric oxide synthase; KC/GRO, keratinocyte chemoattractant/human growth-regulated oncogenes; LC3B, light chain 3B; MMP, matrix metalloproteinase; MR, mannose receptor; Gal, galactin; NO, nitric oxide; PARP, poly (ADP-ribose) polymerase; PCNA, proliferating cell nuclear antigen; PDGF, platelet-derived growth factor; PGI₂, prostacyclin; SMA, smooth muscle actin; SOD, superoxide dismutase; sRAGE, soluble receptor for advanced glycation end product; TGF, tumor growth factor; TIMP, tissue inhibitors of metalloproteinases; TNF, tumor necrosis factor; TXA, thromboxane A.

TNFα is known to cause oxidative and nitrosative stress. ^{Reference Rahman28,Reference Blaser, Dostert, Mak and Brenner29} It also depletes intracellular glutathione, which contributes to its pro-oxidant actions. ^{Reference Rahman28,Reference Obrador, Navarro and Mompo30} Oxidative stress is associated with activation of redox sensitive transcription factors, including NF-κB and AP-1 that upregulate proinflammatory genes, further contributing to inflammation and tissue injury. ^{Reference Aggarwal, Gupta and Kim11} TNFα is also a potent mitogen, stimulating epithelial cell proliferation. ^{Reference Lu, Beuerman and Zhao31} This is thought to be due in part to activation of AP-1 and upregulation of cyclin-D1, a cell cycle regulatory protein. ^{Reference Rahman28,Reference Mukhopadhyay, Mukherjee and Stone32} TNFα-induced proliferation leads to epithelial thickening and pulmonary fibrosis. ^{Reference Allen and Spiteri33,Reference Sasaki, Kashima and Ito34} TNFα also promotes fibrosis by inducing focal accumulation of fibroblasts and collagen deposition and by stimulating the production of matrix metalloproteinases (MMPs) and transforming growth factor (TGF)β. ^{Reference Sasaki, Kashima and Ito34–Reference Oikonomou, Harokopos and Zalevsky37} In humans, circulating levels of MMP-9 and TGFβ correlate with the extent of fibrosis. ^{Reference Kim, Min and Cho38} Collectively, these data suggest that blocking TNFα may be efficacious in mitigating mustard or radiation-induced acute lung injury and inflammation, as well as their long-term pulmonary complications.

Role of TNFα in Mustard-Induced Lung Injury

Mustard vesicants, including sulfur mustard (SM) and nitrogen mustard (NM), are cytotoxic alkylating agents that cause incapacitating injury to the respiratory tract. ^{Reference Kristinsson and Johannesson39} Toxicity is largely due to its lipophilic nature that allows it to rapidly penetrate tissues and cells, and alkylate and cross-link cellular macromolecules, including nucleic acids, lipids, and proteins. ^{Reference Malaviya, Laskin and Laskin40} Both conducting and respiratory airways are affected by mustards. ^{Reference Graham and Schoneboom1,Reference Ghabili, Agutter and Ghanei41} Early symptoms include cough, hoarseness, sore throat, mucus discharge, loss of smell and taste, and irritation of the nasal mucosa. ^{Reference Graham and Schoneboom1,Reference Kehe, Thiermann and Balszuweit42–Reference Sezigen, Ivelik and Ortatatli44} Pulmonary edema and damage to the pharynx induced by acute mustard inhalation result in an inability to speak, moist rales, tachypnea, and tachycardia. ^{Reference Sohrabpour45,Reference Kehe and Szinicz46} At high doses, necrosis of the respiratory epithelium, epithelial sloughing, pseudo-membrane formation, lung lobe collapse, and death occur. ^{Reference Ghabili, Agutter and Ghanei41,Reference Kehe, Balszuweit and Emmler43,Reference Wang and Xia47,Reference Rancourt, Veress and Ahmad48}

Chronic clinical and pathological manifestations of mustard exposure have been observed in survivors of chemical attacks and in manufacturing plant workers. The most common symptoms in long-term survivors of mustard gas exposure are chronic cough, dyspnea, increases in sputum and hemoptysis (airway bleeding), progressive airway deterioration, hyperreactivity, and stenosis of the conducting airways. ^{Reference Graham and Schoneboom1,Reference Malaviya, Laskin and Laskin40,Reference Bijani and Moghadamnia49,Reference Weinberger, Laskin and Sunil50} Common pathologies in victims of the Iran–Iraq war include asthma, bronchitis, bronchiectasis, airway narrowing, COPD, and pulmonary fibrosis. ^{Reference Malaviya, Laskin and Laskin40,Reference Weinberger, Laskin and Sunil50} Emphysema, bronchiectasis, centrilobular nodules, bronchial wall thickening, reticular opacity, ground glass opacity, consolidation, honeycombing, and other respiratory pathologies have similarly been reported in survivors of mustard gas exposure in a manufacturing factory. ^{Reference Malaviya, Laskin and Laskin40,Reference Nishimura, Iwamoto and Ishikawa51–Reference Mukaida, Hattori and Iwamoto53}

Pulmonary injury from mustard exposure is associated with an accumulation of large numbers of inflammatory cells, including macrophages, neutrophils, and eosinophils at sites of injury in the lung, as well as oxidative stress and production of cytotoxic/proinflammatory cytokines, including TNFα. ^{Reference Weinberger, Malaviya and Sunil4,Reference Malaviya, Laskin and Laskin40,Reference Macit, Yaren and Aydin54,Reference Das, Mukherjee, Smith and Chatterjee55} Levels of TNFR1 are also upregulated, suggesting a role of TNFα signaling through this receptor in the pathogenic response to mustards. ^{Reference Sunil, Patel-Vayas and Shen56} Accumulating evidence described below provides support for this activity.

Rodent Models of Mustard Lung Injury Used to Investigate the Impact of Targeting TNFα

In initial mechanistic studies, 2-chloroethyl ethyl sulfide (CEES), a monofunctional analog of SM and NM, was used as a model for mustard lung toxicity. In rodents, CEES causes injury to the alveolar epithelial barrier as measured by increases in cells, protein, and IgM in bronchoalveolar lavage fluid (BAL); fibrinogen and prothrombin levels also increase, a response associated with impairment of fibrin-degrading activity in the lung. ^{Reference Sunil, Patel-Vayas and Shen56,Reference Rancourt, Ahmad and Veress57} Proliferating cell nuclear antigen (PCNA), a marker of cellular proliferation in response to injury, ^{Reference Arbel, Choudhary, Tfilin and Kupiec58} is upregulated after CEES administration to rodents, along with cyclin D1. ^{Reference Mukhopadhyay, Mukherjee, Smith and Das59} CEES also causes pulmonary oxidative stress, characterized by increases in superoxide dismutase (SOD), Ym-1, and lipid peroxidation end products and decreases in intracellular glutathione levels. ^{Reference Ng, Sim and Loke60–Reference Sunil, Shen and Patel-Vayas62} Inflammatory proteins, including inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, TNFα, TNFR1, and CCR2 are also increased in the lung after CEES. ^{Reference Sunil, Patel-Vayas and Shen56,Reference Sawale, Ambhore and Pawar61,Reference Sunil, Shen and Patel-Vayas62} These responses are linked to functional alterations, including decreases in lung compliance and increases in elastance. ^{Reference Sunil, Patel-Vayas and Shen56,Reference Sunil, Shen and Patel-Vayas62} Additionally, methacholine-induced alterations in total lung resistance and central airway resistance are dampened following CEES exposure.

More recently, rodent models of lung injury and chronic disease using NM and SM have been developed as they more closely reflect pulmonary responses in humans. ^{Reference Weinberger, Malaviya and Sunil4,Reference Tang and Loke63} In general, injury, oxidative stress, and inflammatory responses are similar to CEES, appearing early (1–3 days) after exposure; however, they are more severe and prolonged. Additionally, pulmonary fibrosis is observed, typically within 28 days of exposure. The increased pathologic response to SM and NM when compared to CEES is likely due to the fact that they are bifunctional alkylating agents, allowing them to induce DNA intrastrand and interstrand cross-links, as well as DNA-protein cross-links. ^{Reference Jan, Heck, Laskin and Laskin64} These cross-links can alter the structure of DNA and interfere with replication and transcription. In contrast, monofunctional alkylation of DNA and proteins caused by CEES can more readily be repaired. ^{Reference Abbotts and Wilson65}

Owing to high reactivity of SM and NM with mucosal surfaces of the upper respiratory track, rodent models of lung injury involve intratracheal exposure to ensure delivery to the lower lungs, where most damage occurs in humans. ^{Reference Malaviya, Sunil and Venosa66–Reference Anderson, Byers and Vesely69} A single exposure of rats to SM or NM causes dose and time-related histopathological changes in the lungs, including multifocal lesions comprising perivascular and peribronchial edema, blood vessel hemorrhage, patchy mild thickening of alveolar septa, increased numbers of alveolar macrophages and neutrophils, and luminal accumulation of cellular debris and fibrin. ^{Reference Malaviya, Sunil and Venosa66,Reference Malaviya, Abramova and Rancourt67,Reference Xiaoji, Xiao and Rui70–Reference Sunil, Patel and Shen73} Bronchiolization of alveolar walls, indicating type I epithelial cell damage and repair by type II epithelial cells, hyperplasia, and hypertrophy of the bronchial epithelium leading to piling of bronchiolar epithelial cells have also been noted. SM also causes severe ulceration of the proximal bronchioles and deposits of fibrillar membranes in bronchiolar lumen, suggesting apoptosis and necrosis. ^{Reference Malaviya, Abramova and Rancourt67,Reference Anderson, Yourick and Moeller74} Consistent with early SM-induced histopathologic evidence of acute lung injury and bronchiolar epithelial denudation, proteins involved in cell apoptosis and autophagy, including caspase-3, caspase-6, caspase-8, caspase-9, poly (ADP-ribose) polymerase (PARP)-1 and LC3BI and LC3BII, are upregulated in the lung; terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positive cells are also present. ^{Reference McGraw, Rioux and Garlick75–Reference Andres, Keyser and Melber77}

The chronic phase of respiratory injury caused by SM and NM in rats (beginning 28 days post-exposure) is characterized by a predominance of fibroblasts, neutrophils, lymphocytes, and enlarged foamy macrophages in the alveoli and/or alveolar septal walls, multifocal fibrotic lesions, and collagen deposition. ^{Reference Malaviya, Laskin and Laskin40,Reference Malaviya, Sunil and Venosa78–Reference Solopov, Biancatelli and Marinova80} Fibroplasia, squamous metaplasia of the bronchial wall, honeycombing, and emphysema-like changes in alveolar regions of the lung are also evident. ^{Reference Malaviya, Laskin and Laskin40,Reference Malaviya, Abramova and Rancourt67,Reference McGraw, Dysart and Hendry-Hofer68,Reference Mishra, Rir-Sima-Ah and Grotendorst81} Hyperplasia and squamous metaplasia in ulcerated proximal bronchiolar epithelium are also observed after SM exposure, indicative of aberrant wound repair. ^{Reference Malaviya, Abramova and Rancourt67} These structural changes are correlated with impaired pulmonary functioning. ^{Reference Solopov, Biancatelli and Marinova80,Reference Sunil, Vayas and Abramova82} As observed with CEES, NM- and SM-induced lung injury and apoptosis/necrosis of epithelial cells are associated with increases in BAL cells, protein, IgM, fibrinogen, and total phospholipids. ^{Reference Malaviya, Sunil and Venosa66,Reference Malaviya, Abramova and Rancourt67,Reference Sunil, Vayas and Abramova82} Levels of fibrinogen/fibrin and surfactant protein (SP)-D are also increased in the lung and/or BAL. This is evident early after exposure and remains elevated up to 28 days. ^{Reference Malaviya, Abramova and Rancourt67,Reference Sunil, Vayas and Abramova82,Reference McGraw, Osborne and Mastej83}

Lung injury induced by SM and NM is associated with oxidative stress characterized by increases in 8-hydroxy-2-deoxyguanosine (8OHdG), 2-deoxyguanosine, malondialdehyde and 4-hydroxynonenal, and decreases in glutathione levels. ^{Reference Malaviya, Sunil and Venosa66,Reference McElroy, Min and Huang84,Reference Yaren, Mollaoglu and Kurt85} Nitrates and nitrites also increase in BAL and urine. ^{Reference McElroy, Min and Huang84,Reference Yaren, Mollaoglu and Kurt85} Additionally, antioxidants such as heme oxygenase (HO)-1, lipocalin-2, Ym-1, and Mn-SOD are upregulated, a response that persists for at least 28 days post-SM or NM exposure. ^{Reference Malaviya, Sunil and Venosa66,Reference Malaviya, Abramova and Rancourt67,Reference Malaviya, Bellomo and Abramova86} A marker of DNA damage, γH2A.X, and PCNA are also detectable in the lungs. ^{Reference Malaviya, Abramova and Rancourt67,Reference Venosa, Smith and Gow87,Reference Malaviya, Venosa and Hall88} Whereas γH2A.X increases 1–3 days after mustard exposure, PCNA is increased 3–28 days in bronchiolar epithelium, alveolar epithelial cells, interstitial cells, and in focal areas exhibiting honeycombing and/or fibrosis.

Inflammatory genes/proteins, including IL-1, IL-2, IL-6, TNFα, KC/GRO, CCR2, CCR5, CCL2, CCL3, CCL5, CCL11, CX3CR1, CX3CL1, high mobility group box (HMGB)1, and MMP-9, are evident in the lungs and/or BAL fluid from mustard-treated rodents within 1–3 days post-exposure. ^{Reference Malaviya, Abramova and Rancourt67,Reference Venosa, Malaviya and Choi79,Reference Mishra, Rir-Sima-Ah and Grotendorst81,Reference McElroy, Min and Huang84} Proinflammatory macrophages expressing TNFα (Figure 2), iNOS, MMP-9, HMGB1, or COX-2 are also present at this time. ^{Reference Malaviya, Abramova and Rancourt67,Reference Sunil, Vayas and Abramova82,Reference Malaviya, Sunil and Venosa89} Whereas NM-induced increases in expression of inflammatory markers are maximum at 3 days post-exposure, persisting at lower levels up to 28 days, the response to SM is biphasic. ^{Reference Malaviya, Abramova and Rancourt67,Reference Malaviya, Venosa and Hall88} Thus, SM exposure causes an early increase in inflammatory markers at 1–3 days, which is followed by a decrease at 7–16 days, and then a generally more robust increase at 28 days. Antiinflammatory/profibrotic genes (IL-10, pentraxin-2, connective tissue growth factor [CTGF], ApoE) have also been identified in the lungs; however, the timing of their appearance varies with the gene. ^{Reference Venosa, Malaviya and Choi79} Antiinflammatory macrophages characterized by expression of CD206 (mannose receptor), CD68, CD163, galectin-3, and arginase-II are also present in histologic lung sections most prominently at later time points. ^{Reference Malaviya, Abramova and Rancourt67,Reference Malaviya, Sunil and Venosa78,Reference Venosa, Malaviya and Choi79} This is correlated with the upregulation of α-smooth muscle actin, TGFβ, platelet-derived growth factor (PDGF), PDGF receptor-α, and CTGF. ^{Reference McGraw, Dysart and Hendry-Hofer68,Reference Malaviya, Sunil and Venosa78,Reference Mishra, Rir-Sima-Ah and Grotendorst81}

Figure 2. Effects of sulfur mustard on TNFα expression in the lung. Rats were exposed by intratracheal inhalation to air (CTL) or sulfur mustard (SM, 0.4 mg/kg) as previously described. ^{Reference Malaviya, Abramova and Rancourt67} Lung sections were prepared 3, 7, 16, and 28 days later and immunostained with anti-TNFα antibody. Binding was visualized using a Vectastain kit. Original magnification, 600X. Representative images from 8–9 rats/group are shown.

Role of TNFα in Radiation-Induced Lung Injury

Radiation exposure causes acute lung injury, which progresses to pneumonitis within weeks to months and fibrosis within months to years. ^{Reference Giuranno, Ient, De Ruysscher and Vooijs102} Early injury is characterized by damage to the alveolar wall, interstitial edema, and an accumulation of proteins and inflammatory cells in the lung lining fluid; this is followed by thickening of alveolar walls, solid lesions with collagen deposits, and bronchiectasis as the pathology develops. Mechanistically, radiation-induced lung injury involves DNA damage and the generation of cytotoxic reactive oxygen and nitrogen species. ^{Reference Azzam, Jay-Gerin and Pain103,Reference Beach, Groves, Williams and Finkelstein104} This is associated with loss of epithelial and endothelial barrier function, an accumulation of inflammatory cells in the lung that produce mediators such as IL-1, IL-6, IL-13, IL-17, TNFα, and TGFβ that can further damage the tissue and/or contribute to tissue remodeling and fibrogenesis. ^{Reference Giuranno, Ient, De Ruysscher and Vooijs102,Reference Darby and Hewitson105–Reference Arroyo-Hernández, Maldonado and Lozano-Ruiz110} Radiation also affects pulmonary endothelial cell function as measured by decreases in the activity of angiotensin converting enzyme (ACE) and plasminogen activator (PLA). ^{Reference Ward, Kim and Molteni111} This is accompanied by increases in lung wet weight, protein and hydroxyproline content, and eicosanoids.

Radiation-induced lung injury is characterized by increases in TNFα and TNFR1. ^{Reference Przybyszewska, Miloszewska and Rzonca112–Reference Malaviya, Gow and Francis114} This is observed early (1–3 hours) after exposure to a single dose of radiation ^{Reference Rübe, Wilfert and Palm6,Reference Zhang, Qian and Xing113} and aligned with increases in numbers of TUNEL-positive epithelial cells and upregulation of cleaved capase-3, markers of apoptosis. Radiation-induced increases in TNFα persist in the lung up to 24 hours post-exposure; subsequently, TNF levels return to baseline. This is followed by secondary, more exaggerated increases 2–24 weeks post-exposure coinciding with radiation-induced histopathological changes in the lung, including diffuse alveolitis, inflammatory cell accumulation, thickening of alveolar walls, depletion of type II epithelial cells, fibroblast proliferation, and interstitial and alveolar deposition of extracellular matrix. ^{Reference Rübe, Wilfert and Palm6,Reference Roy, Salerno and Citrin106,Reference Johnston, Piedboeuf and Rubin108,Reference Malaviya, Gow and Francis114–Reference Osterreicher, Mokry and Navrátil118} Fractionated radiation exposure (single high dose divided into low dose radiation over several days) is also associated with increases in TNF levels at early times but at reduced levels when compared to a single high dose. Cumulative TNFα levels after fractionated radiation, however, are greater and more persistent. ^{Reference Zhang, Qian and Xing113} Of note, early increases in TNFα precede radiation-induced increases in IL-1α and IL-6, suggesting that TNFα plays a role in the initiation of the inflammatory cytokine cascade. ^{Reference Rübe, Wilfert and Palm6}