HSV vector-mediated GAD67 suppresses neuropathic pain induced by perineural HIV gp120 in rats through inhibition of ROS and Wnt5a.

Human immunodeficiency virus (HIV)-related neuropathic pain is a debilitating chronic condition that is severe and unrelenting. Despite the extensive research, the exact neuropathological mechanisms remain unknown, which hinders our ability to develop effective treatments. Loss of GABAergic tone may have an important role in the neuropathic pain state. Glutamic acid decarboxylase 67 (GAD67) is one of the isoforms that catalyze GABA synthesis. Here, we used recombinant herpes simplex virus (HSV-1) vectors that encode gad1 gene to evaluate the therapeutic potential of GAD67 in peripheral HIV gp120-induced neuropathic pain in rats. We found that (1) subcutaneous inoculation of the HSV vectors expressing GAD67 attenuated mechanical allodynia in the model of HIV gp120-induced neuropathic pain, (2) the anti-allodynic effect of GAD67 was reduced by GABA-A and-B receptors antagonists, (3) HSV vectors expressing GAD67 reversed the lowered GABA-IR expression and (4) the HSV vectors expressing GAD67 suppressed the upregulated mitochondrial superoxide and Wnt5a in the spinal dorsal horn. Taken together, our studies support the concept that recovering GABAergic tone by the HSV vectors may reverse HIV-associated neuropathic pain through suppressing mitochondrial superoxide and Wnt5a. Our studies provide validation of HSV-mediated GAD67 gene therapy in the treatment of HIV-related neuropathic pain.

Neuropathic ocular pain: an important yet underevaluated feature of dry eye.

Dry eye has gained recognition as a public health problem given its prevalence, morbidity, and cost implications. Dry eye can have a variety of symptoms including blurred vision, irritation, and ocular pain. Within dry eye-associated ocular pain, some patients report transient pain whereas others complain of chronic pain. In this review, we will summarize the evidence that chronicity is more likely to occur in patients with dysfunction in their ocular sensory apparatus (ie, neuropathic ocular pain). Clinical evidence of dysfunction includes the presence of spontaneous dysesthesias, allodynia, hyperalgesia, and corneal nerve morphologic and functional abnormalities. Both peripheral and central sensitizations likely play a role in generating the noted clinical characteristics. We will further discuss how evaluating for neuropathic ocular pain may affect the treatment of dry eye-associated chronic pain.

HSV-mediated p55TNFSR reduces neuropathic pain induced by HIV gp120 in rats through CXCR4 activity.

Human immunodeficiency virus (HIV)-related neuropathic pain is a debilitating chronic condition that is severe and unrelenting. Despite extensive research, the detailed neuropathological mechanisms remain unknown, which hinders our ability to develop effective treatments. In this study, we investigated the role of proinflammatory molecules, tumor necrosis factor-α (TNFα), CXCR4 and stromal-derived factor-1 α (SDF1α), in the L4/5 dorsal root ganglia (DRG) and the spinal dorsal horn in HIV gp120 protein-mediated neuropathic pain. Our results showed that the application of HIV gp120 to the sciatic nerve induced upregulation of TNFα, CXCR4 and SDF1α in both the DRG and the lumbar spinal dorsal horn. Non-replicating herpes simplex virus (HSV) vector encoding the p55TNFSR gene and producing a TNF-soluble receptor (TNFSR) to block bioactivity of TNFα reversed mechanical allodynia. Intrathecal AMD3100 (CXCR4 antagonist) increased mechanical threshold. The HSV vectors expressing p55TNFSR reversed upregulation of TNFα, CXCR4 and SDF1α induced by gp120 in the DRG and the spinal dorsal horn. These studies suggest that proinflammatory TNFα to the CXCR4/SDF1 pathway has an important role in the HIV-related neuropathic pain state and that blocking the proinflammatory cytokines or chemokines is able to reduce neuropathic pain. This work provides a novel gene therapy proof-of-concept for HIV-associated neuropathic pain.

Expression of HCLCA1 in cystic fibrosis lungs is associated with mucus overproduction.

Mucus overproduction is typical in cystic fibrosis (CF) airway disease. The human calcium-activated chloride channel, hCLCA1, has been reported to be upregulated by interleukin (IL)-9 and to regulate the expression of mucins. Therefore, the expression of IL-9, IL-9 receptor (IL-9R) and hCLCA1 between the lungs of CF patients and healthy control subjects was compared. Endoscopic biopsy samples of bronchial mucosa from 10 CF patients and six control subjects were stained with periodic acid-Schiff. IL-9, IL-9R and hCLCA1 expression was determined by immunocytochemistry. Expression of hCLCA1 mRNA was also determined by in situ hybridisation. The present study found significant increases in IL-9, IL-9R and hCLCA1 immunoreactivity, hCLCA1 mRNA expression, and numbers of mucus-producing cells in the mucosa of CF patients compared to control subjects. Positive correlations were found between IL-9R-positive-cells with IL-9-positive cells and hCLCA1-positive cells, and between PAS-positive cells with hCLCA1-positive cells and IL-9R-positive cells. Expression of hCLCA1 mRNA was colocalised with IL-9R expression and PAS-positive staining in epithelial cells. Increased expression of interleukin-9 and interleukin-9 receptor, as well as an upregulation of the human calcium-activated chloride channel, hCLCA1, in mucus-producing epithelium of cystic fibrosis patients, support the hypothesis that interleukin-9 contributes to mucus overproduction in cystic fibrosis airway disease.

Th2 cytokines and asthma. Interleukin-9 as a therapeutic target for asthma.

Asthma is a complex heritable inflammatory disorder of the airways in which the development of clinical disease depends on environmental exposure. It has been well established that T helper type 2 (TH2) lymphocytes and their cytokines have an important role in allergic asthma. Interleukin (IL)-9, a member of the TH2 cytokine family, has recently been implicated as an essential factor in determining mucosal immunity and susceptibility to atopic asthma. In this review we examine the critical experiments and observations that support this hypothesis. We also discuss these results in comparison with the experiments supporting the involvement of other T H2 cytokines such as IL-4, IL-5 and IL-13.

Characterization of a calcium-activated chloride channel as a shared target of Th2 cytokine pathways and its potential involvement in asthma.

Interleukin (IL)-9 is a T helper (Th) 2 cytokine recently implicated as an essential factor in determining susceptibility to asthma. Transgenic mice overexpressing IL-9 exhibit many features that are characteristic of human asthma. To better understand the mechanism by which IL-9 mediates the various biologic activities in asthma, we performed suppressive subtraction hybridization with whole lung from IL-9 transgenic and control mice. Here we report the identification of mCLCA3, a calcium-activated chloride channel that was specifically induced in the lung epithelium of IL-9 transgenic mice. Expression of mCLCA3 could also be induced by intratracheal administration of IL-9 or other Th2 cytokines (IL-4, IL-13), but not by interferon-gamma. Moreover, expression of mCLCA3 was induced in the lung of antigen-exposed mice, and this induction could be suppressed by neutralizing IL-9 antibody treatment, indicating IL-9 is both necessary and sufficient to induce mCLCA3 in this experimental model of asthma. Finally, we demonstrate that hCLCA1 is the human counterpart to mCLCA3 and is also induced in vitro in human primary lung cells by Th2 cytokine treatment. Together, these data strongly implicate the involvement of mCLCA3 (in mice) and hCLCA1 (in humans) in the pathogenesis of Th2 cytokine-mediated asthmatic disorders.

UGRP1, a uteroglobin/Clara cell secretory protein-related protein, is a novel lung-enriched downstream target gene for the T/EBP/NKX2.1 homeodomain transcription factor.

A novel gene that is down-regulated in lungs of T/ebp/Nkx2.1-null mouse embryos has been identified using a suppressive-subtractive hybridization method. The gene product is a secreted protein, forms a homodimer, and exhibits an amino acid sequence similar to that seen in the uteroglobin/Clara cell secretory protein family of proteins. This gene, designated Ugrp1 (uteroglobin-related protein 1), consists of three exons and two introns and produces three transcripts by alternative splicing. The Ugrp1 gene was localized by fluorescence in situ hybridization to mouse chromosome 18 at region 18C-D; this region is homologous with human 5q31-34, where one of the asthma susceptibility genes has been assigned. UGRP1 mRNA is predominantly expressed in the lung, with low levels of expression in the thyroid. Expression in the lung is detectable as early as embryonic day 12.5 and increases markedly by embryonic day 16.5. In T/ebp/Nkx2.1-null embryo lungs, UGRP1 expression was significantly reduced as assessed by RT-PCR analysis. Cotransfection assays using a T/EBP/NKX2.1 expression construct with Ugrp1 promoter-luciferase reporter constructs confirmed that T/EBP/NKX2.1 regulates Ugrp1 gene activity at the transcriptional level. Thus, Ugrp1 is a downstream target gene for the T/EBP/NKX2.1 homeodomain transcription factor. Changes in UGRP1 mRNA levels in lungs from antigen-sensitized mice suggest the possible involvement of UGRP1 in inflammation.

Interleukin 9 promotes influx and local maturation of eosinophils.

The interleukin 9 (IL-9) pathway has recently been associated with the asthmatic phenotype including an eosinophilic tissue inflammation. The mechanism by which IL-9 affects eosinophils (eos) is not known. To investigate whether this cytokine has a direct activity on the development of eos and eosinophilic inflammation, a model of thioglycolate-induced peritoneal inflammation was used in IL-9 transgenic (TG5) and background strain (FVB) mice. In this model, a transient eosinophilic infiltration in the peritoneal cavity was observed in FVB mice 12 to 24 hours after thioglycolate injection that coincided with peak IL-5 and IL-9 release. In contrast, TG5 mice developed a massive eosinophilia that persisted at high levels (81% of total cells) even 72 hours after thioglycolate injection. Release of eosinophilic major basic protein (MBP), IL-4, and IL-5 to the peritoneal cavity of these mice was significantly increased when compared with the control FVB strain. To study the mechanism by which IL-9 exerts its effect on eos, bone marrow or peritoneal cells were cultured in the presence of IL-5, IL-9, or their combination in vitro. IL-5 alone was able to generate significant numbers of eos in TG5 but not FVB mice, whereas a combination of IL-5 and IL-9 induced marked eosinophilia in both strains indicating a synergism between these 2 cytokines. These data suggest that IL-9 may promote and sustain eosinophilic inflammation via IL-5-driven eos maturation of precursors.

Role of insulin receptor substrate-2 in interleukin-9-dependent proliferation.

Interleukin-9 (IL-9) stimulation results in JAK, STAT and IRS1/2 phosphorylation. The role of IRS adaptor proteins in IL-9 signaling is not clear. We show that IL-9 induces IRS2 phosphorylation and association with phosphatidylinositol-3 kinase (PI 3-K) p85 subunit in TS1 cells and BaF/9R cells, which proliferate upon IL-9 stimulation. We observed a PI 3-K-dependent phosphorylation of protein kinase B (PKB) in TS1 cells, but not in BaF/9R, nor in other IL-9-dependent cell lines. Finally, 32D cells that were transfected with the IL-9 receptor but lack IRS expression survived in the presence of IL-9. Ectopic IRS1 expression allowed for IL-9-induced proliferation, in the absence of significant PKB phosphorylation.

Linkage analysis of chromosome 12 markers in Italian families with atopic asthmatic children.

We investigated 116 Italian atopic families (560 individuals) for linkage with 13 DNA markers on chromosome 12. All the subjects were phenotyped for asthma, total serum IgE, bronchial hyperresponsiveness, skin-prick positivity to common aeroallergens, and atopy. A relative location map of the markers was prepared from Centre d'Etude du Polymorphisme Humain families. Affected sib pair multipoint linkage methods were used to perform the statistical analyses. We report suggestive linkage for asthma with markers on chromosome 12. The region of interest centers around marker D12S390 (maximum logarithm of odds [mlod] = 2.81; p = 0.003). These results provide additional support that asthma susceptibility factors are located on chromosome 12q.

IL-9 expression by human eosinophils: regulation by IL-1beta and TNF-alpha.

BACKGROUND: IL-9 is a pleiotropic cytokine that exhibits biologic activity on cells of diverse hemopoietic lineage. IL-9 stimulates the proliferation of activated T cells, enhances the production of IgE from B cells, and promotes the proliferation and differentiation of mast cells and hematopoietic progenitors. OBJECTIVE: In this study we evaluated the expression of IL-9 messenger (m)RNA and protein by human peripheral blood eosinophils. We also investigated the role of IL-1beta and TNF-alpha in the release of IL-9 from human peripheral blood eosinophils. METHODS: RT-PCR, in situ hybridization, and immunocytochemistry were used to investigate the presence of IL-9 mRNA and protein in human peripheral blood eosinophils from asthmatic patients and normal control subjects. Furthermore, biologic assay was used to investigate the release of IL-9 protein from IL-1beta- or TNF-alpha-stimulated eosinophils in vitro. RESULTS: RT-PCR analysis showed the presence of IL-9 mRNA in human peripheral blood eosinophil RNA preparations from subjects with atopic asthma, as well as in the eosinophil-differentiated HL-60 cell line. By using in situ hybridization, a significant difference (P <.01) in IL-9 mRNA expression was detected in human peripheral blood eosinophils freshly isolated from asthmatic subjects compared with those isolated from normal control subjects. Furthermore, the percentage of IL-9 immunoreactive eosinophils from asthmatic patients was increased compared with that found in normal control subjects (P <.01). We also demonstrate that cultured human peripheral blood eosinophils from asthmatic subjects synthesize and release IL-9 protein, which is upregulated on stimulation with TNF-alpha and IL-1beta. CONCLUSION: Human eosinophils express biologically active IL-9, which suggests that these cells may influence the recruitment and activation of effector cells linked to the pathogenesis of allergic disease. These observations provide further evidence for the role of eosinophils in regulating airway immune responses.

Interleukin-9 upregulates mucus expression in the airways.

Interleukin (IL)-9 has recently been shown to play an important role in allergic disease because its expression is strongly associated with the degree of airway responsiveness and the asthmatic-like phenotype. IL-9 is a pleiotropic cytokine that is active on many cell types involved in the allergic immune response. Mucus hypersecretion is a clinical feature of chronic airway diseases; however, the mechanisms underlying the induction of mucin are poorly understood. In this report, we show that IL-9 regulates the expression of a subset of mucin genes in lung cells both in vivo and in vitro. In vivo, the constitutive expression of IL-9 in transgenic mice results in elevated MUC2 and MUC5AC gene expression in airway epithelial cells and periodic acid-Schiff-positive staining (reflecting mucous glycogenates). Similar results were observed in C57BL/6J mice after IL-9 intratracheal instillation. In contrast, instillation of the T helper 1-associated cytokine interferon gamma failed to induce mucin production. In vitro, our studies showed that IL-9 also induces expression of MUC2 and MUC5AC in human primary lung cultures and in the human muccoepidermoid NCI-H292 cell line, indicating a direct effect of IL-9 on inducing mucin expression in these cells. Altogether, these results suggest that upregulation of mucin by IL-9 might contribute to the pathogenesis of human inflammatory airway disorders, such as asthma. These data extend the role of the biologic processes that IL-9 has on regulating the many clinical features of asthma and further supports the IL-9 pathway as a key mediator of the asthmatic response.

IL-9 and its receptor in allergic and nonallergic lung disease: increased expression in asthma.

BACKGROUND: Bronchial asthma is a chronic inflammatory disease associated with genetic components. Recently IL-9 has been reported as a candidate gene for asthma and to be associated with bronchial hyperresponsiveness and elevated levels of total serum IgE. OBJECTIVE: To investigate the contribution of IL-9 to the pathogenesis of asthma, we examined the expression of IL-9 and its receptor (IL-9R) in bronchial tissue from subjects with atopic asthma (n = 10), chronic bronchitis (n = 11), and sarcoidosis (n = 9) and from atopic (n = 7) and nonatopic (n = 10) healthy control subjects. METHODS: Bronchial biopsy specimens were examined for the presence of IL-9 and IL-9R protein and messenger RNA (mRNA) by immunocytochemistry and in situ hybridization, respectively. To phenotype the cells expressing IL-9 in asthmatic tissue, combined in situ hybridization and immunocytochemistry was also performed. RESULTS: There was a highly significant difference (P <.001) in the expression of IL-9 mRNA in asthmatic airways (20.6 +/- 4.0 cells/mm of basement membrane) compared with chronic bronchitis (5.6 +/- 4.4), sarcoidosis (2.5 +/- 1.8), atopic control subjects (7.7 +/- 2.2), and healthy control subjects (2.7 +/- 2.3). The number of IL-9 immunoreactive cells was also greater in asthmatic patients compared with the other groups (P <.05). Although the level of IL-9R mRNA expression did not differ in any of the groups (P >.05), IL-9R immunoreactivity was significantly higher in asthmatic compared with control subjects. Furthermore, IL-9 mRNA expression levels were also significantly correlated with FEV(1) (P <.05) and the airway responsiveness to methacholine producing a 20% fall in FEV(1) (P <. 01). The cells expressing IL-9 mRNA in asthmatic tissue were CD3(+) lymphocytes (68%), major basic protein(+) eosinophils (16%), and elastase(+) neutrophils (8%). CONCLUSION: The results of this study demonstrate the potential of IL-9 to be a marker for atopic asthma and furthermore suggest an important role for this cytokine in the pathophysiologic mechanisms of this disease.

Interleukin-9-induced expression of M-Ras/R-Ras3 oncogene in T-helper clones.

In an attempt to gain insight into the molecular mechanisms involved in interleukin-9 (IL-9) activities, representational difference analysis (RDA) was used to identify messages that are induced by IL-9 in a murine T-helper-cell clone. One of the isolated genes encodes for the newly described M-Ras or R-Ras3, which is part of the Ras gene superfamily. M-Ras expression was found to be induced by IL-9 but not IL-2 or IL-4 in various murine T-helper-cell clones, and this induction seems to be dependent on the JAK/STAT pathway. Contrasting with the potent upregulation of M-Ras expression, M-Ras was not activated by IL-9 at the level of guanosine triphosphate/guanosine diphosphate (GTP/GDP) binding. However, IL-3 increased GTP binding to M-Ras, suggesting that M-Ras induction might represent a new mechanism of cooperativity between cytokines such as IL-3 and IL-9. Constitutively activated M-Ras mutants induced activation of Elk transcription factor by triggering the MAP kinase pathway and allowed for IL-3-independent proliferation of BaF3 cells. Taken together, these results show that cytokines such as IL-9 can regulate the expression of a member of the RAS family possibly involved in growth-factor signal transduction.

IL-9 induces chemokine expression in lung epithelial cells and baseline airway eosinophilia in transgenic mice.

Recent data have identified IL-9 as a key cytokine in determining susceptibility to asthma. These data are supported by the finding that allergen-exposed IL-9-transgenic mice exhibit many features that are characteristic of human asthma (airway eosinophilia, elevated serum IgE and bronchial hyperresponsiveness) as compared to the background strain. A striking feature of these animals is a robust peribronchial and perivascular eosinophilia after allergen challenge, suggesting that IL-9 is a potent factor in regulating this process. In an attempt to gain insights into the molecular mechanism governing IL-9 modulation of lung eosinophilia, we investigated the ability of this cytokine to induce the expression of CC-type chemokines in the lung because of their effect on stimulating eosinophil chemotaxis. Here we show that IL-9-transgenic mice in contrast to their congenic controls exhibit baseline lung eosinophilia that is associated with the up-regulation of CC-chemokine expression in the airway. This effect appears to be through a direct action of IL-9 because the addition of recombinant IL-9 to primary epithelial cultures and cell lines induced the expression of these chemokines in vitro. These data support a mechanism for IL-9 in regulating the expression of eosinophil chemotactic factors in lung epithelial cells.

IL-9 pathway in asthma: new therapeutic targets for allergic inflammatory disorders.

BACKGROUND: Asthma is a complex heritable inflammatory disorder of the airways associated with clinical signs of allergic inflammation and bronchial hyperresponsiveness (BHR). The incidence of asthma continues to rise in industrialized countries despite advances in the identification of cellular and molecular mediators that are associated with the disease. Because of its importance in human health, additional research and alternative therapeutic strategies are justified to create more effective treatments for this debilitating disease. OBJECTIVE: Studies use recombinant inbred mice to demonstrate that BHR in mouse models of asthma is associated with a genetic alteration at the IL-9 locus, where IL-9 expression in lung is strongly associated with bronchial responsiveness. We have investigated the ability of intratracheal instilled IL-9 to induce asthmatic-like responses in naive C57BL/6 (B6) mice, which express very low levels of IL-9. METHODS: IL-9 or vehicle was intratracheal instilled in naive B6 mice for 10 days. Mice were analyzed for effects on BHR, lung eosinophilia, and serum total IgE levels. RESULTS: Phenotypic effects of B6 mice instilled with IL-9 were increased eosinophils in the bronchoalveolar lavage and significantly elevated serum total IgE. Moreover, IL-9 was found to induce IL-5Ralpha in vivo and in vitro, suggesting a potential mechanism for the novel actions described for IL-9 on eosinophils. CONCLUSION: Increased levels of IL-9 in the airway of naive B6 mice induced lung eosinophilia and serum total IgE levels, which are 2 clinical features of asthma. These data support a central role for the IL-9 pathway in the complex pathogenesis of allergic inflammation.

Interleukin-9 promotes allergen-induced eosinophilic inflammation and airway hyperresponsiveness in transgenic mice.

Human atopic asthma is a complex heritable inflammatory disorder of the airways associated with clinical signs of allergic inflammation and airway hyperresponsiveness. Recent studies demonstrate that the degree of airway responsiveness is strongly associated with interleukin (IL)-9 expression in murine lung. To investigate the contribution of IL-9 to airway hyperresponsiveness, and to explore directly its relationship to airway inflammation, we studied transgenic mice overexpressing IL-9. In this report we show that IL-9 transgenic mice (FVB/N-TG5), in comparison with FVB/NJ mice, display significantly enhanced eosinophilic airway inflammation, elevated serum total immunoglobulin E, and airway hyperresponsiveness following lung challenge with a natural antigen (Aspergillus fumigatus). These data support a central role for IL-9 in the complex pathogenesis of allergic inflammation.

Asthma and bronchial hyperresponsiveness linked to the XY long arm pseudoautosomal region.

We examined the long arm XY pseudoautosomal region for linkage to asthma, serum IgE, and bronchial hyperresponsiveness. In 57 Caucasian families multipoint nonparametric analyses provide evidence for linkage between DXYS154 and bronchial hyperresponsiveness (P = 0.000057) or asthma (P = 0.00065). This genomic region is approximately 320 kb in size and contains the interleukin-9 receptor gene. These results suggest that a gene controlling asthma and bronchial hyperresponsiveness maybe located in this region and that the interleukin-9 receptor is a potential candidate.

Molecular analysis of human interleukin-9 receptor transcripts in peripheral blood mononuclear cells. Identification of a splice variant encoding for a nonfunctional cell surface receptor.

Genetic studies on mouse models of asthma have identified interleukin-9 (IL9) as a determining factor in controlling bronchial hyperresponsiveness, a hallmark of the disease. Recently, the human IL9 receptor (hIL9R) gene locus has also been implicated in determining susceptibility to bronchial hyperresponsiveness and asthma. In order to evaluate the structure and function of the encoded product, we analyzed receptor transcripts derived from peripheral blood mononuclear cells of 50 unrelated donors. Sequence analysis of the entire coding region identified a splice variant that contains an in frame deletion of a single residue at codon 173 (DeltaQ). This variant could be permanently expressed in a cytokine-dependent murine T-cell line but lacked the ability to induce proliferation in response to human IL9. In situ analyses of cells expressing the wild-type and DeltaQ receptors found both forms to be expressed at the cell surface, but the DeltaQ receptor was unable to bind hIL9 and could not be recognized by N-terminal specific antibodies. These findings demonstrate that hIL9RDeltaQ presents an altered structure and function and suggests its potential role in down-regulating IL9 signaling in effector cells and associated biological processes.

Linkage analysis of susceptibility to ozone-induced lung inflammation in inbred mice.

Exposures to the common air pollutant ozone (O3) cause decrements in pulmonary function and induce airway inflammation that is characterized by infiltration of polymorphonuclear neutrophils (PMNs; refs 1-4). Because of the impact that O3 may have on public health, it is critical to identify susceptibility factors. Highly reproducible, significant inter-individual variations in human pulmonary function responses to O3 support the hypothesis that genetic background is an important determinant. Initial analysis of PMN responses to O3 exposure in segregant populations derived from inflammation-prone (susceptible) C57BL/6J (B6) and inflammation-resistant C3H/HeJ (C3) inbred mice indicated that susceptibility was controlled by a locus we termed Inf2 (ref. 7). Subsequent analyses with recombinant inbred strains suggested that a more complex interaction of genes is involved. In this report, we identify a quantitative trait locus (QTL) for O3 susceptibility on chromosome 17. Candidate genes for the locus include Tnf, the gene encoding the pro-inflammatory cytokine tumour necrosis factor-alpha (Tnf). Antibody neutralization of the protein product of this putative candidate gene significantly protected against O3 injury in susceptible mice. These results strongly support linkage of O3 susceptibility to a QTL on chromosome 17 and Tnf as a candidate gene.